CN113784753A

CN113784753A - Method and device for preventing and/or ameliorating photoaging and/or dermal pigmentation, photoaging-resistant and/or dermal pigmentation inhibitor, screening method for photoaging-resistant and/or dermal pigmentation inhibitor, evaluation method for photoaging-resistant and/or dermal pigmentation-inhibiting cosmetic treatment, and evaluation method for photoaging-resistant and/or dermal pigmentation degree

Info

Publication number: CN113784753A
Application number: CN202080029442.2A
Authority: CN
Inventors: 堀场聪; 细井纯一; 高木雅哉; 松浦有宇子; 加治屋健太朗
Original assignee: Shiseido Co Ltd
Current assignee: Shiseido Co Ltd
Priority date: 2019-04-19
Filing date: 2020-04-20
Publication date: 2021-12-10
Also published as: JP7439059B2; JP2024037928A; CN117815216A; JPWO2020213743A1; WO2020213743A1

Abstract

The present invention addresses the problem of providing a method, a device, and a pharmaceutical agent for preventing and/or ameliorating photoaging and/or dermal pigmentation, a method for screening an inhibitor against photoaging and/or dermal pigmentation, a method for evaluating a cosmetic treatment for inhibiting photoaging and/or dermal pigmentation, and a method for evaluating the degree of photoaging and/or dermal pigmentation. The solution is that photoaging and/or dermal pigmentation can be prevented and/or improved by adjusting the M1/M2 balance. By using the M1/M2 balance as an index, photoaging and/or degree of dermal pigmentation can be objectively evaluated, and agents and cosmetic treatments for preventing and/or improving photoaging and/or dermal pigmentation can be searched based on such a method.

Description

Method and device for preventing and/or ameliorating photoaging and/or dermal pigmentation, photoaging-resistant and/or dermal pigmentation inhibitor, screening method for photoaging-resistant and/or dermal pigmentation inhibitor, evaluation method for photoaging-resistant and/or dermal pigmentation-inhibiting cosmetic treatment, and evaluation method for photoaging-resistant and/or dermal pigmentation degree

Technical Field

The present invention relates to a method and an apparatus for preventing or improving photoaging and/or dermal pigmentation by adjusting the balance of M1/M2, a photoaging and/or dermal pigmentation inhibitor, a screening method for a photoaging and/or dermal pigmentation inhibitor using the balance of M1/M2 as an index, an evaluation method for a cosmetic treatment for inhibiting photoaging and/or dermal pigmentation, an evaluation method for photoaging and/or dermal pigmentation degree, and the like.

Background

The aging phenomena of human skin are roughly divided into "natural aging" and "photoaging". Photoaging is a phenomenon specifically observed at the site of exposure and is skin-specific. In photoaging, the skin fibrous tissue is damaged by the induction of active oxygen generation, cellular DNA damage, and the like due to the influence of UV and the like, and it is considered that this is a cause of appearance of a phenotype such as wrinkles and sagging. For example, a phenomenon is observed in which collagen fibers composed of collagen decrease and elastic fibers composed of elastin denature due to skin photoaging. In addition, melanocytes are also destroyed, and melanin pigment is formed in a large amount, which causes spots and the like.

In addition, pigmentation, which is represented by spots and dark, occurs by accumulation of melanin produced by melanocytes located in the basal layer of the epidermis. Melanin is usually present in the epidermis and basal layer, but the epidermis is renewed at a relatively fast cycle, and thus such melanin is easily discharged. On the other hand, melanin is sometimes present in the dermis layer due to the fact that melanin falls into the dermis from the gap of the basement membrane, or the like. The cycle of renewal of dermal cells is very slow compared to the epidermis, and thus such melanin is often accumulated without being discharged. For this reason, improvement of the pigmentation of the dermis is very difficult.

As a cosmetic method for preventing photoaging, for example, patent document 1 discloses a prophylactic or inhibitory agent for preventing or inhibiting photoaging of skin by preventing inhibition of leukocyte elastase. Patent document 2 discloses a photoaging inhibitor composition characterized by containing a plant extract of the genus agalypha of the family amaranthaceae having a collagen synthesis-promoting effect and a collagen peptide derived from an animal.

As a cosmetic method for improving the pigmentation in the dermis, non-patent document 13 proposes to prevent the melanin from falling into the dermis by strengthening the basement membrane.

In addition, inflammation is also suggested to be a cause of the photoaging phenomenon of the skin, and anti-inflammatory agents have been extensively developed. Patent document 3 discloses an anti-aging cosmetic composition containing a compound that induces autophagy activation with an increase in adiponectin expression. It is also suggested that phagocytosis by macrophages is utilized to improve dermal pigmentation, and patent document 15 discloses a dermal spot preventing/improving agent that induces macrophages to fibroblasts that take up melanin that has fallen into the dermis and engulfs them.

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 5657723

Patent document 2: japanese patent laid-open publication No. 2017-203004

Patent document 3: japanese patent laid-open publication No. 2018-177805

Patent document 4: japanese Kokai publication Hei 2014-504629

Patent document 5: japanese patent laid-open publication No. 2015-140334

Patent document 6: japanese patent No. 6178088

Patent document 7: japanese patent No. 6273304

Patent document 8: japanese patent No. 5744409

Patent document 9: japanese patent laid-open publication No. 2018-140953

Patent document 10: japanese patent laid-open publication No. 2019-043855

Patent document 11: japanese patent laid-open publication No. 2013-053130

Patent document 12: japanese laid-open patent publication No. H09-187248

Patent document 13: japanese patent laid-open publication No. 2003-261455

Patent document 14: japanese laid-open patent publication No. H09-227367

Patent document 15: japanese patent laid-open publication No. 2018-072098

Patent document 16: japanese patent No. 6535146

Patent document 17: japanese patent laid-open publication No. 2019-031482

Patent document 18: japanese patent laid-open publication No. 2007 and 063192

Non-patent document

Non-patent document 1: journal of the American College of Cardiology, Vol.62, No.20,2013, November 12,2013:1890-

Non-patent document 2: experimental & Molecular Medicine (2014)46, e 70; doi:10.1038/emm.2013.135

Non-patent document 3: NATURE, VOL495,28MARCH 2013, pp524-530, doi:10.1038/NATURE11930

Non-patent document 4: journal of Investigative Dermatology,2009 April; 129(4), 1016-25, Epub 2008Oct 9.

Non-patent document 5: stem Cell Research & Therapy (2018)9:88, https:// doi. org/10.1186/s13287-018-

Non-patent document 6: journal of the European Academy of Dermatology and Venereology 2011European Academy of Dermatology and Venereology 2012,26,1577-

Non-patent document 7: british Journal of Dermatology,2005,153, pp733-739

Non-patent document 8: pigment Cell Melanoma Research, Vol.27, Issue 3, pp502-504

Non-patent document 9: ann Dermatol Vol.28, No.3, pp279-289,2016

Non-patent document 10: endocrinology,2011.152(10): pp3779-90

Non-patent document 11: british Journal of Dermatology,2005.153Suppl 2: pp37-46

Non-patent document 12: experimental Dermatology,2011.20(11): pp953-5

Non-patent document 13: fujifilm Research & Development (No.55-2010): pp33-37

Disclosure of Invention

Problems to be solved by the invention

The invention provides a method and a device for preventing and/or improving photoaging and/or dermal pigmentation, a photoaging and/or dermal pigmentation inhibitor, a screening method for the photoaging and/or dermal pigmentation inhibitor, an evaluation method for the photoaging and/or dermal pigmentation inhibition cosmetic treatment, and an evaluation method for the photoaging and/or dermal pigmentation degree.

Means for solving the problems

The present inventors have conducted intensive studies on the effect of inhibiting photoaging and dermal pigmentation, and as a result, found that an imbalance in the M1/M2 balance of macrophages is associated with photoaging and dermal pigmentation. Based on such findings, a photoaging-resistant and/or hypodermal pigmentation inhibitor for preventing and/or improving photoaging and/or hypodermal pigmentation, and an evaluation method of a cosmetic treatment for preventing photoaging and/or hypodermal pigmentation inhibition were established. By such methods, novel cosmetic methods, devices, and anti-photoaging and/or dermal pigmentation inhibitors that exert anti-photoaging and/or dermal pigmentation inhibitory effects have also been developed.

Thereby completing the following invention:

(1) cosmetic method for preventing and/or ameliorating photoaging and/or dermal pigmentation by adjusting the M1/M2 balance.

(2) The method according to (1), wherein the M1/M2 balance is adjusted such that the ratio of M2 to M1 is increased.

(3) The method according to (1) or (2), wherein the adjustment of the M1/M2 balance comprises a step of applying a weak physical stimulus to the skin, wherein the step is performed by applying a physical stimulus that cycles at a frequency of 60Hz or less to the skin of the subject, the cycle comprising the steps of:

(a) stretching the skin of the subject to an elongation of 0.1% to 50.0%; and

(b) recovering from the extended state;

and/or the presence of a gas in the gas,

(a-1) pressing the skin of the subject to 1 to 1000 μm; and

(b-1) restoring the skin of the subject from the pressed state;

wherein the elongation is calculated by the following formula,

(wherein the fixed points A and B are arbitrary positions on the epidermis or the substrate to which the epidermis is adhered, and a straight line passing through the fixed points A and B is parallel to the stretching direction)

(4) A cosmetic device for preventing and/or improving photoaging and/or dermal pigmentation by adjusting M1/M2 balance,

the device comprises:

a stimulus generating section for generating a physical stimulus, and

a stimulus applying section for applying the physical stimulus generated by the stimulus generating section to the skin,

the apparatus is an apparatus for performing a process of applying a weak physical stimulus to the skin, wherein the process is cycled at a frequency of, for example, 60Hz or less, the cycling comprising the steps of:

(a) stretching the skin to an elongation of 0.1% to 50.0%; and

(b) recovering from the extended state;

and/or the presence of a gas in the gas,

(a-1) pressing the skin of the subject to 1 to 1000 μm; and

(b-1) restoring the skin of the subject from the pressed state;

wherein the elongation is calculated by the above formula 1.

(5) A method of screening for an inhibitor of anti-photoaging and/or dermal pigmentation, the method comprising the steps of:

administering a candidate agent to a biological sample;

measuring the M1/M2 balance in the biological sample before and after administration of the candidate agent; and

when the M1/M2 balance in the biological sample to which the candidate agent was administered is improved as compared to that before administration of the agent, the agent is evaluated to have an anti-photoaging and/or dermal pigmentation inhibitory effect.

(6) A method for evaluating a cosmetic treatment for anti-photoaging and/or inhibition of dermal pigmentation, the method comprising the steps of:

applying a cosmetic treatment to the skin sample;

measuring the M1/M2 balance in skin samples before and after cosmetic treatment; and

when the balance of M1/M2 in the skin sample subjected to the cosmetic treatment was improved as compared to that before the treatment, the treatment was evaluated to have an anti-photoaging and/or dermal pigmentation inhibitory effect.

(7) A method for evaluating photoaging and/or dermal pigmentation, performed by 1 or more computers, the method having the steps of:

acquiring data on a reference value of a preset skin M1/M2 balance;

a step of acquiring data on the M1/M2 balance of the skin of the subject;

comparing the reference value with the data on the M1/M2 balance of the skin of the subject to calculate;

a step of evaluating the photoaging and/or the degree of inhibition of dermal pigmentation of the skin based on the calculation result obtained by the calculating step; and

and displaying the evaluation result obtained in the evaluation step.

(8) A system for evaluating photoaging and/or dermal pigmentation, the system having:

a database part for storing data of reference value of M1/M2 balance related to preset skin;

a data input section that inputs data on the M1/M2 balance of the skin of the subject;

a calculation unit for calculating by comparing the reference value stored in the database unit with the data on the M1/M2 balance of the skin of the subject inputted through the data input unit;

an evaluation unit for evaluating the photoaging and/or the degree of dermal pigmentation of the skin based on the calculation result obtained by the calculation unit; and

and a display unit for displaying the evaluation result obtained by the evaluation unit.

(9) A screening kit for an anti-photoaging and/or dermal pigmentation inhibitor comprising an agent for determining the M1/M2 balance.

(10) An anti-photoaging and/or dermal pigmentation inhibitor for use in preventing and/or ameliorating photoaging and/or dermal pigmentation by adjusting the M1/M2 balance.

(11) The anti-photoaging and/or dermal pigmentation inhibitor according to (10), comprising: a compound represented by the following general formula (1) or a salt thereof, a Hypericum erectum extract, a thyme extract, phellodendron amurense, and/or a eucalyptus extract.

{ formula (II) wherein R₁And R₂The same or different, each represents 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 general formula (2),

(wherein X represents a lower alkyl group, a lower alkoxy group, a hydroxyl group, an amino group, a halogen atom, and n is 0 to 3) }

(12) An M1/M2 balance adjustment/improvement agent comprising: a compound represented by the following general formula (1) or a salt thereof, a Hypericum erectum extract, a thyme extract, phellodendron amurense, and/or a eucalyptus extract.

(13) A composition comprising the pharmaceutical agent according to any one of (10) to (12).

(14) The method according to (1) or (2), wherein the M1/M2 balance is adjusted by administering the agent of any one of (10) to (12).

ADVANTAGEOUS EFFECTS OF INVENTION

By the methods, devices, and medicaments of the present invention, photoaging and/or dermal pigmentation can be prevented and/or improved by adjusting the M1/M2 balance. Further, by using the M1/M2 balance as an index, photoaging and/or degree of dermal pigmentation can be objectively evaluated, and based on such a method, a drug or a cosmetic treatment for preventing and/or improving photoaging and/or dermal pigmentation can be searched for.

Drawings

Fig. 1a shows the kind of marker of macrophage used in experiment 1, and the age, skin type, and sex of the subject.

FIG. 1b is a photomicrograph showing macrophages (CD11 b: red) and M1 macrophages (CD 86: green) in skin tissue of the younger and the elderly.

FIG. 1c is a photomicrograph showing macrophages (CD11 b: red) and M2 macrophages (CD 206: green) in skin tissue of the younger and the elderly.

FIG. 1d is a graph showing the number of M1, M2 macrophages and the total number of whole macrophages in skin tissue of an elderly person and a young person (individual cells/mm)²)。

FIG. 1e, left panel, is a micrograph showing co-staining of M1 macrophages (CD 86: red) or M2 macrophages (CD 206: red) and procollagen (green) in skin tissue of the elderly and the younger. Rectangles present in the 4 blocks in the left figure are enlarged views of characteristic portions. FIG. 1e, right panel, is a schematic representation of the relationship between M1, M2, fibroblasts, collagen production/destruction.

Figure 2a shows a summary of the method of experiment 2.

Figure 2b is a photomicrograph of M0, M1, and M2 macrophages induced by experiment 2.

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 by experiment 2. The lower panel of FIG. 2c is a graph showing the expression amounts of mRNA of cell surface markers (M1: CD86, M2: CD206) of M1 and M2 macrophages. The mRNA expression level of GAPDH was corrected and shown as a relative value (%) with respect to the case of M0 of 100%.

Figure 3a shows a summary of the method of experiment 3.

Figure 3b shows the amount of procollagen (μ g/well) in fibroblasts added with supernatants from M1 and M2 macrophages, as per experiment 3.

Fig. 3c is a micrograph showing collagen (red) and hyaluronic acid (green) in fibroblasts to which supernatants of M1 and M2 macrophages were added according to experiment 3.

Fig. 3d is a micrograph showing β -galactosidase (β -Gal) in fibroblasts to which supernatants of M1 and M2 macrophages were added according to experiment 3.

FIG. 3e is a graph showing the results of the aging-associated β -galactosidase (SA β -Gal) assay in fibroblasts to which supernatants of M1 and M2 macrophages were added according to experiment 3 (left panel: proportion (%) of SA β -Gal positive cells relative to the total number of DAPI positive cells), and the total number of DAPI positive cells per well (right panel).

FIG. 3f is a graph showing the mRNA expression levels of various melanogenesis-enhancing factors (HGF, ET1, bFGF, IL-1alpha, SCF) and melanogenesis-inhibiting factors (clusterin, DKK1) in fibroblasts to which supernatants of M1 and M2 macrophages were added according to experiment 3.

FIG. 4a is a micrograph showing β -Gal in both aged and aged fibroblasts supplemented with supernatant from M1 and M2 macrophages, according to experiment 4.

Fig. 4b is a graph showing the results of SA β -Gal assay (ratio (%) of SA β -Gal positive cells to the total number of DAPI positive cells) in the aged (upper) and aged (lower) fibroblasts to which the supernatants of M1 and M2 macrophages were added according to experiment 4 and the total number of DAPI positive cells per well.

Figure 4c shows the amount of procollagen (μ g/well) in aged and aged fibroblasts of supernatant supplemented with M1 and M2 macrophages, as per experiment 4.

Fig. 4d is a micrograph showing collagen (red) and nuclei stained by DAPI (blue) in aged and aged fibroblasts to which supernatants of M1 and M2 macrophages were added according to experiment 3.

FIG. 5 is a photomicrograph showing type I collagen (red) and macrophages (CD 68: green) in neonatal foreskin derived fibroblasts supplemented with supernatants of M1 and M2 macrophages, according to experiment 5. The upper left photograph shows fibroblasts cultured without the addition of macrophage supernatant.

FIG. 6 is a photomicrograph showing macrophages (CD 68: red) and M1 macrophages (CD 86: green) or M2 macrophages (CD 206: green) in a 3D model made according to experiment 6. The portions indicated by triangles show double-stained portions of CD68 and CD86, or CD68 and CD 206.

Fig. 7 is a micrograph showing p21 (red) and nuclei stained by DAPI (blue) in the 3D model made according to experiment 6.

Fig. 8 is a graph showing the ratio (%) of the number of p 21-positive cells to the total number of cells in each layer (left: upper layer: epidermal cell layer, right: lower layer: fibroblast cell layer) of the 3D model prepared according to experiment 6.

Fig. 9 is a graph showing the number of macrophages M1 and M2, and the number of macrophages in an ex vivo (ex vivo) skin model irradiated with a solar simulator in accordance with experiment 7. The number of cells of each macrophage when irradiation was not performed was defined as 100% (left bar: irradiation (-)), and the relative value (%) of the number of cells of each macrophage irradiated therewith was shown in the right bar (irradiation (+)).

Fig. 10 shows the setting of the extension stimulus performed in experiment 8.

Fig. 11 shows the device and skin sample used in experiment 8.

Fig. 12 shows the number of M1, M2 macrophages and the total number of whole macrophages in the case of the ex vivo skin model to which the stretching stimulus was applied (stretching) compared with the case of the non-application of the stretching stimulus (control) according to experiment 8. The left picture shows that every 1mm²Macrophage number (cells/1 mm) of skin sample²) The figure (a). The right panel is a graph showing the proportion (%) of each macrophage (M1, M2) to the total number of whole macrophages.

Fig. 13 shows the results of the screening performed in experiment 9. The bars are graphs in which the expression level of CD86 (CD86/GAPDH value) when each concentration of forsythia suspense extract (0.1%), thyme extract (0.1%), and tranexamic acid formamide (0.03%, 0.06%) was added is represented by the relative value (%) when the control (cont) was set to 100%.

FIG. 14 shows an example of the apparatus of the present invention.

Fig. 15 shows the results of the screening performed in experiment 10. The bars are graphs in which the expression level of CD86 (CD86/GAPDH value) when phellodendron amurense extract (0.01%) and eucalyptus extract (0.1%) were added at each concentration was represented by the relative value (%) when the control (cont) was set to 100%.

FIG. 16a is a graph showing the expression levels of mRNA for each of the collagenolytic enzymes (MMP-1 and MMP-2) and inflammatory cytokine (IL-1. beta.) in fibroblasts to which the supernatants of M1 and M2 macrophages were added in accordance with experiment 11, as relative values (%) with the result of M1 being 100%.

FIG. 16b is a graph showing the expression level of mRNA of each collagen production/maturation factor (COL1A1, COL1A2, HSP47 and ADAMTS-2) in fibroblasts to which the supernatants of M1 and M2 macrophages were added in accordance with experiment 11, as a relative value (%) when the result of M1 was set to 100%.

Figure 17a shows the age of each subject who had taken the sample in experiment 12 in separate low and high age groups.

FIG. 17b is a graph showing the number of M1, M2 macrophages in skin tissue (50 μ M immediately below the basement membrane) of the younger and older persons in experiment 12 (individual cells/mm)²)。

FIG. 17c is a graph showing the numbers of M1, M2 macrophages in skin tissue (200 μ M immediately below the basement membrane) of the younger and older persons in experiment 12 (individual cells/mm)²)。

FIG. 17d is a graph showing the numbers of CD68 negative cells, M1 and M2 macrophages showing 3/4 collagen positivity in skin tissues of the elderly and the younger in experiment 12 (cells/mm)²)。

Fig. 18a shows the appearance of fibroblasts, M1 macrophages, and M2 macrophages after 24 hours from melanin addition in experiment 13. Cells that have engulfed melanin form a circular shape as shown in the right figure.

Fig. 18b shows the amount of melanin (melanin/alamar blue) taken up per cell by fibroblasts, M1 macrophages, M2 macrophages 24 hours after addition of melanin in experiment 13.

Fig. 18c shows the appearance of M1 macrophages and M2 macrophages after 5 days from melanin addition in experiment 13.

Fig. 19 is a graph showing the number of melanin phagocytosed M1 macrophages and M2 macrophages in the dermis layer counted for each age according to experiment 14. The numbers of melanophagocytic M1 macrophages and M2 macrophages are shown as an average across all subjects under fig. 19.

Detailed Description

Macrophages are cells that are locally present in various tissues in the body and cause an immune response to foreign bodies, pathogens, and are also involved in inflammation. Macrophages differentiate from M0 macrophages in an undifferentiated state (hereinafter sometimes abbreviated as M0) into M1 type and M2 type. M1 macrophages (hereinafter sometimes abbreviated as M1) are known as inflammatory types, and M2 macrophages (hereinafter sometimes abbreviated as M2) are known as reparative types (anti-inflammatory types). Imbalance in the balance between M1 macrophages and M2 macrophages has been reported to be associated with diseases such as obesity, type 2 diabetes, and arteriosclerosis (patent documents 4 to 6, and non-patent documents 1 to 5). However, the relationship between skin photoaging, pigmentation and the M1/M2 balance is unclear.

The present inventors have found that the balance between these M1 macrophages and M2 macrophages (M1/M2 balance) is disturbed specifically at the site of skin exposed to light and the occurrence of pigmentation, and that adjusting M1/M2 balance is particularly important for preventing and improving photoaging and pigmentation. Further, the present inventors have found that a satisfactory effect is exhibited when a specific physical stimulus is applied to the skin of a subject using the M1/M2 balance as an index of skin photoaging and pigmentation. More specifically, it is found that even in the case of photoaged skin or skin with pigmentation, if physical stimulation is applied at a specific stretching rate at a frequency of 60Hz or less, such as 1Hz or less and 10Hz or less, the M1/M2 balance is adjusted/improved. Further, the present inventors screened various substances using the M1/M2 balance as an index of skin photoaging and pigmentation, and found that the hypericum erectum extract, the compound of the present invention represented by tranexamic acid formamide, the thyme extract, phellodendron amurense, and the eucalyptus extract have an effect of adjusting/improving the M1/M2 balance, and function as an anti-photoaging agent and an anti-pigmentation agent. Type I collagen production promoting action, hair growth promoting action, etc. have been reported in forsythia suspense extracts (patent documents 9 and 10). The compounds of the present invention represented by tranexamic acid carboxamide have been reported to have an effect of improving rough skin, an effect of whitening skin, an anti-allergic effect, and the like (patent documents 11, 14 and the like). Thyme extract has been reported to have antiallergic and antibacterial effects (patent documents 12 and 13). Phellodendron amurense is also used as an antidiarrheal, a gastric drug, an oral drug for dyspepsia and anorexia, or an external drug for traumatic injury and sprain, and has been reported to have antibacterial, antioxidant, skin barrier function improving effects, pigment cell activating effects, and the like (patent documents 16 and 17). Eucalyptus extracts have been reported to have antibacterial and antioxidant effects (patent document 18). However, it was found for the first time by the present inventors that these substances have the effect of adjusting/improving the M1/M2 balance.

Accordingly, the present invention provides methods, devices, and anti-photoaging and/or pigmentation inhibitors 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 performed by a doctor or a medical practitioner.

In the present specification, pigmentation means pigmentation of pigment such as melanin in the dermis and epidermis. The present invention is effective in both dermis and epidermis, and is expected to be a measure against dermal pigmentation, particularly, in the sense that improvement methods are limited in addition to phagocytosis by melanocyte phagocytosis. Through the use of the preparation of the present invention, spots, dullness, dark circles, and the like caused by pigmentation are improved. Further, it is also effective for decoloring tattoos, and the like, which are difficult to erase by injecting a dye into the dermis layer when the dye is added.

M1 macrophages can be measured using markers such as CD86, CD80, iNOS, and the like. M2 macrophages can be measured using markers such as CD206, CD163, Agr1, and the like. Examples of markers for the whole macrophage including M1 and M2 include CD11b and CD 68. In addition or alternatively, it can be determined by quantifying M1-specific cytokines such as IL-1beta, TNF-alpha, and M2-specific cytokines such as IL-10. However, the marker is not limited to the above-mentioned marker as long as it can measure the M1/M2 balance.

In the present specification, the M1/M2 balance may be a ratio of the number of M1 macrophages to the number of M2 macrophages, or may be a ratio of the amount of mRNA of a marker (e.g., CD86, CD80, iNOS, etc.) of M1 macrophages to the amount of mRNA of a marker (e.g., CD206, CD163, Agr1, etc.) of M2 macrophages. In the skin in a photoaged state, the ratio of M1 is high, the ratio of M2 is low, and melanophagocytosis is high, and M2 is used, so that the adjustment/improvement of the M1/M2 balance may be to increase the ratio of M2 to M1 (number of M2/number of M1, and/or mRNA amount of M2 marker/mRNA amount of M1 marker). The increase may be, for example, an increase having a statistically significant difference (e.g., student's t-test) in which the significance level is 5%, and/or may be, for example, an increase of 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more. Alternatively, the balance of M1/M2 may be adjusted/improved so that the ratio of M2 to M1 (number of M2/number of M1, and/or amount of mRNA of M2 marker/amount of mRNA of M1) is in a certain range, for example, from about 4/6 to about 9/1, from about 5/5 to about 8/2, from about 5/5 to about 7/3, or the like, and may be maintained in the range, or the steady state may be maintained around the range.

Adjustment/improvement of the M1/M2 balance may be achieved by applying physical stimuli such as stretching stimuli, pressing stimuli, massaging, etc. to the skin. The adjustment/improvement of the M1/M2 balance comprises a step of applying a weak physical stimulus to the skin, and the apparatus of the present invention is an apparatus for performing the step of applying a weak physical stimulus to the skin, wherein the step can be cycled at a frequency of, for example, 60Hz or less, and the cycle comprises the steps of: (a) stretching the skin to an elongation of 0.1% to 50.0%; and (b) recovering from the extended state.

The elongation is calculated by the above equation 1. The physical stimulation may be performed at an elongation of 0.001% to 80.0%, 0.01% to 60.0%, 0.1% to 50.0%, preferably 0.1% to 50.0%. For example, the elongation may be in any range of 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%.

The stretching speed is the speed (%/s) until the maximum stretching ratio (%) is reached in 1 cycle. The recovery rate is a rate (%/s) of recovery from the maximum stretching rate to the non-stretched state. The stretching rate/recovery rate may be any rate such as 0.010%/s to 40%/s, 0.05%/s to 30%/s, 0.10%/s to 20%/s, 0.2%/s to 15%/s, or 0.3%/s to 10%/s. The extension speed and the recovery speed may be the same or different.

The adjustment/improvement of the M1/M2 balance comprises a step of applying a weak physical stimulus to the skin, and the apparatus of the present invention is an apparatus for performing the step of applying a weak physical stimulus to the skin, wherein the step can be cycled at a frequency of, for example, 60Hz or less, and the cycle comprises the steps of: (a-1) pressing the skin of the subject to 1 to 1000 μm; and (b-1) restoring the skin of the subject from the compressed state.

The expression "pressing the skin 1 μm to 1000 μm" means pressing the skin to a depth of 1 μm to 1000 μm from the outermost surface of the skin. The depth of the pressing can be arbitrarily set to 1 to 1000 μm, 10 to 300 μm, 10 to 100 μm, or the like from the outermost surface of the skin.

The frequency is the number of cycles per 1 second when the cycle of returning to the non-stretched or non-pressed state from the start of stretching or pressing is 1 cycle. In 1 cycle, may include an extended or compressed state for a certain time and/or be suspended in a non-extended or non-compressed state. For example, in the 1 cycle, the method may further include: (a') after (a) and before (b), or after (a-1) and before (b-1), maintaining a stretched or pressed state for 0 seconds to 30 minutes, 1 second to 20 minutes, 5 seconds to 10 minutes, or 10 seconds to 5 minutes; and/or (b') stopping in a non-stretched or non-pressed state for 0 to 30 seconds, 0 to 20 seconds, 0 to 10 seconds, 1 to 20 seconds, 1 to 10 seconds after (b) and before (a) of the next cycle, or (a-1) and before (b-1). The frequency may be, for example, 0.0000001Hz to 10kHz, 0.000001Hz to 1kHz, 0.00001Hz to 100Hz, preferably 0.0001Hz to 60Hz, 0.0001Hz to 10 Hz. For example, a frequency in any range of 0.001Hz to 60Hz, 0.01Hz to 60Hz, 0.001Hz to 10Hz, 0.01Hz to 10Hz, 0.1Hz to 60Hz, 0.1Hz to 10Hz, 0.5Hz to 60Hz, 0.5Hz to 50Hz, 0.5Hz to 10Hz, 0.5Hz to 5Hz, 0.5Hz to 1Hz, 0.001Hz to 0.01Hz, 0.001Hz to 0.1Hz, 0.001Hz to 1Hz, 0.01Hz to 1Hz, 0.1Hz to 1Hz, 1Hz to 60Hz, 1Hz to 10Hz, 1Hz to 5Hz, and the like can be used.

Some commercially available beauty equipment, massage equipment, and the like use electromagnetic waves such as RF waves having a frequency of about 0.3 to 300MHz, and use ultrasonic waves having a frequency of about 1 to 7 MHz. The frequencies employed by the method/apparatus of the present invention are extremely low compared to these frequencies. If the skin is strongly stressed as in a conventional beauty apparatus or the like, there is a risk of adverse effects such as redness, indentation, injury, pain, and inflammation on the skin, but if the frequency of the present invention is adopted, the risk is low, and non-invasive physical stimulation can be performed. This is because the inventors found that if the stretching frequency and the frequency are too high, the stimulation is too strong, and thus it is more satisfactory to gently stimulate the skin by adjusting these values to appropriate values.

In addition, conventionally, it is common knowledge of those skilled in the art that only frequencies exceeding 60Hz can be selected based on mechanical components of a motor for cosmetic devices using components such as a motor generally used in this field. When the frequency of the present invention, which is a limit of the conventional beauty equipment, is not more than 60Hz, for example, not more than 60Hz, not more than 10Hz, or not more than 1Hz, it is necessary to manufacture a special machine. Further, even in the case of such a low frequency, there is a fixed concept of "too weak" in order to exhibit the effect of the present invention, and few studies have been made so far. However, the present inventors have tried to actually apply physical stimulation to the skin using a very low frequency in consideration of the conventional technical common knowledge, and as a result, surprisingly, even such a low frequency mild stimulation exerts a favorable effect.

Although devices of low or medium frequency are also commercially available in EMS devices and the like, they are designed to act particularly in deep layers of muscles, subcutaneous fat and the like, and the influence on the surface layer of skin as in the present invention is unclear. Further, such a device may be accompanied by a tingling and crisp stimulus when a current is passed even at a low frequency, and is different from the present invention in which a mild stimulus is applied to the skin. On the other hand, the present invention may be a cosmetic method performed by a simple method of directly applying a stretching stimulus to the skin without applying energy such as ultrasonic waves, electric current, or a magnetic field. Further, the stretching stimulation having such a frequency is a mild stimulation, and exerts the adjustment/improvement effect of the M1/M2 balance as described in the examples. Therefore, if the method/apparatus of the present invention is used, the prevention and/or improvement effect of photoaging and/or pigmentation can be expected without exerting an adverse effect on the skin.

The physical stimulation may be physical stimulation generated by an instrument such as a beauty device, an experimental apparatus, massage using a human hand or an instrument, or movement of the face, or may be physical stimulation realized by contact or non-contact. In one aspect, a mechanically generated physical stimulus may be applied to the skin using an instrument including a stimulus generating unit that generates a physical stimulus and a stimulus applying unit that applies the physical stimulus by contact or non-contact. The physical stimulation may be achieved by, for example, contact such as stretching, pressing, beating, rubbing, or sucking the skin, and/or may be achieved by, for example, non-contact such as applying a shock wave to the skin with ultrasonic waves, air pressure, or water pressure to displace the skin. As the movement of the face, it is possible to perform the bulging of the cheeks, the opening of the eyes, and the like. Examples of the massage include a massage using an instrument such as a hand or a roller by a subject such as the subject itself or a beauty member. However, the present invention is not limited to the above-described range as long as the physical stimulation is performed.

Examples of the device of the present invention include a cosmetic device including a skin contact portion that comes into contact with the skin of a user to apply the physical stimulation of the present invention. For example, the device may be a device including a grip portion and a skin stretching portion or a skin pressing portion. For example, the device shown on the left side of fig. 14 is designed to stretch the skin at a specific frequency and stretching rate by contacting the skin with the skin contact portion.

For example, the device of the present invention may include a power supply that generates an electrical signal, a stimulus generation unit that converts the electrical signal from the power supply into a physical stimulus to generate a physical stimulus, and a skin stimulation unit that receives the physical stimulus generated by the stimulus generation unit and applies the physical stimulus to the skin of the user.

For example, the device shown on the left side of fig. 14 includes a grip portion, a power source, a control portion for controlling physical stimulation, a stimulation generation portion, and a skin contact portion including a skin stimulation portion and a skin fixing portion. Designed in the following way: the user holds the grip portion and places the skin contact portion on the skin, fixes the skin by the skin fixing portion, and operates the control portion so that the electrical signal from the power source is converted into the physical stimulus by the stimulus generating portion, and the physical stimulus is transmitted to the skin stimulus portion, and the skin is fixed to the skin fixing portion and stretched at a specific frequency and stretching ratio by the skin stimulus portion. For example, the stimulus generating unit may be a part that is driven by a motor or the like to convert an electric signal into a physical stimulus. The skin stimulation portion shown on the left side of fig. 14 applies a stretching stimulus to the skin, and may apply a pressing stimulus to the skin, for example.

Alternatively, the device of the present invention may be a cosmetic device including a power source, a control unit for controlling physical stimulation, a stimulation generation unit, and a skin contact unit including a skin contact surface formed of a sheet material. As an example, a skin contact portion of such a cosmetic device is shown on the right of fig. 14. The sheet material may be a material that is capable of passing an electrical current and converting an electrical signal from a power source into a physical stimulus. Examples of such sheet materials include Dielectric Elastomer Actuators (DEA), conductive polymers, IPMC, PVC gel, mckinen type, and the like.

The power supply of the device of the invention can be an internal power supply or an external power supply, and can also be rechargeable. The device of the present invention may use data stored in a mobile phone, a cloud, or the like, or may be remotely operated by wireless.

The physical stimulation may be stimulation to stretch the skin by applying physical stimulation by contact or non-contact as described above. The physical stimulus may be applied in a parallel direction, i.e. transverse, with respect to the skin surface, in a perpendicular direction, i.e. longitudinal, with respect to the skin surface, or in any direction, such as an oblique direction, a twisting direction, etc.

The number of cycles of physical stimulation is not limited. For example, the cycle may be 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. For example, as described in the examples, 27 cycles may be sufficient.

Further, the arbitrary number of cycles is set to 1 group, and the group may be divided into arbitrary number of groups, for example, 1 to 100 groups, 2 to 50 groups, or 3 to 10 groups.

The time for performing the physical stimulation is not limited. For example, the cycle may be repeated with or without an off time, and the cycle may be performed for a fixed time period of 5 minutes to 3 hours, 10 minutes to 2 hours, or 30 minutes to 1 hour.

The cycle or the interval between the groups is not limited. For example, the stretching or compression stimulation may be performed individually for 1 or more groups, or may be performed continuously or intermittently at regular or irregular intervals, such as 1 or more times per day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 time for 1, 2, 3, 4 weeks, and the like.

However, the frequency, the stretching rate, the number of cycles, and the frequency are not limited to those described above as long as the skin is sufficiently stimulated to exhibit the anti-photoaging and/or pigmentation inhibitory effect. The waveform for performing the physical stimulation may be arbitrarily set to a rectangular wave, a sine wave, a triangular wave, a sawtooth wave, or the like.

Alternatively, the adjustment/improvement of the M1/M2 balance may be the administration of a M1/M2 balance adjustment/improvement agent or a composition containing the same. The present inventors have found that the compound of the present invention, hypericum erectum extract, thyme extract, phellodendron amurense, and/or eucalyptus extract function as such a M1/M2 balance-adjusting/improving agent. Accordingly, the present invention also provides M1/M2 balance adjustment/improvement agent, anti-photoaging agent, pigmentation inhibitor, and composition containing the compound of the present invention, hypericum erectum extract, thyme extract, phellodendron amurense, and/or eucalyptus extract, or the compound of the present invention, hypericum erectum extract, thyme extract, phellodendron amurense, and/or eucalyptus extract as an active ingredient.

The "compound of the present invention" in the present specification means a compound represented by the following general formula (1) or a salt thereof.

Particularly preferred compounds of the invention are N-methyl-trans-4- (aminomethyl) cyclohexanecarboxamide (tranexamic acid carboxamide (C)₉H₁₉C₁N₂O)) or a salt thereof. The above compound can be prepared, for example, by the method described in patent document 14As a synthetic product, a commercially available product can be used.

Hypericum erectum (Hypericum erectum) is a perennial plant of the genus Hypericum of the family Guttiferae. The extract of Hypericum erectum used in the present invention is preferably an extract of the aerial parts of Hypericum erectum, but any 1 or 2 or more extracts of the above may be used because the seed, root, etc. contain an active ingredient. The extract of Hypericum erectum may also be commercially available.

Thyme (Thymus) is a perennial plant of the genus Thymus of the family Labiatae. In the present invention, various thymes such as common thyme (t.vulgaris), lemon thyme (T.x citriodorus), asian thyme (t.serpyllum), and the like can be used. The thyme extract used in the present invention is preferably an extract of the whole plant of thyme, but since seeds, flowers, roots, and the like contain an active ingredient, any 1 or 2 or more kinds of these extracts may be used. Commercially available thyme extracts can also be used.

Phellodendron amurense is a crude drug obtained by drying the bark of Phellodendron amurense (Phellodendron amurense) or Phellodendron amurense (Phellodendron chinense) belonging to the family Rutaceae. Phellodendron amurense or extract thereof can be produced by a conventional method, and commercially available products can be used.

Eucalyptus is a tree of the genus Eucalyptus (Eucalyptus) of the family Myrtaceae. The eucalyptus extract used in the present invention is preferably an extract of leaves of eucalyptus, but since seeds, flowers, bark, roots, and the like contain an active ingredient, any 1 or 2 or more kinds of these extracts may be used. The eucalyptus extract can be produced by a conventional method, or a commercially available product can be used.

When an extract is used, the extraction method is not particularly limited, but an extraction method using a solvent is preferred. In the extraction, the plant material may be used as it is, but the extraction of the active ingredient can be carried out in a short time under mild conditions with high extraction efficiency by pulverizing the plant material into granules or powder. The extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the kind of the solvent, and the like. Usually, the temperature is set in the range from room temperature to the boiling point of the solvent. The extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of the solvent, the extraction temperature, and the like. Further, in the extraction, stirring may be performed, or the mixture may be left standing without stirring, or ultrasonic waves may be applied.

The type of the solvent is not particularly limited, but is preferably water, hydrous ethanol, lower alcohol such as ethanol, organic solvent such as hexane, or mixed solvent of these solvents such as hexane/ethanol. The extraction may be performed at normal temperature or under heating (for example, using a heated solvent such as warm water or hot water). In addition, an enzyme may be added to the solvent to perform the extraction treatment. By adding the enzyme, the cell tissue of the plant can be disintegrated, thereby further improving the extraction efficiency. As the enzyme, a cell tissue macerating enzyme is preferably used. Examples of such enzymes include pectinase, cellulase, hemicellulase, α -amylase, and phytase. These enzymes may be used alone in 1 kind, or may be used in combination in 2 or more kinds.

By such an extraction operation, the active ingredient is extracted and dissolved in the solvent. The solvent containing the extract may be used as it is, or may be subjected to conventional purification treatment such as sterilization, washing, filtration, decolorization, deodorization, etc. Further, it may be concentrated or diluted as necessary. Further, the solvent may be completely volatilized to prepare a solid (dried product) and then used, or the dried product may be redissolved in an arbitrary solvent and then used.

Furthermore, since the squeezed liquid obtained by squeezing the raw material plant also contains the same active ingredients as the extract, the squeezed liquid may be used instead of the extract.

However, the M1/M2 balance control/improvement agent and composition are not limited to the above-mentioned ones, and any of known M1/M2 balance control/improvement agents such as those described in patent documents 4 to 7 can be used. The route of administration may be arbitrarily selected, for example, transdermal administration, oral administration, subcutaneous administration, transmucosal administration, intramuscular administration, and the like, but photoaging is a phenomenon observed specifically in the skin at the portion exposed to light, and therefore transdermal administration capable of being administered at a specific position on the skin may be preferred. In addition, it is sometimes preferable that the pigmentation occurring in the epidermis or dermis is transdermally administered so that the pigmentation can reach the epidermis or dermis from the skin. The M1/M2 balance can be adjusted/improved by, for example, inducing differentiation into M2 macrophages, or by any other M1/M2 balance adjustment/improvement method.

For example, the agents or compositions of the present invention modulate/improve the M1/M2 balance, which results in the inhibition of photoaging and/or pigmentation of the skin. The M1/M2 balance adjustment/improvement agent, the light aging inhibitor, and the pigmentation inhibitor of the present invention (hereinafter, these may be collectively referred to as the "preparation of the present invention") may contain 1 of the above-mentioned active ingredients alone, or may contain 2 or more of them in any combination and ratio.

The preparation of the present invention may be prepared as a composition comprising the above-mentioned active ingredient in combination with 1 or 2 or more other ingredients such as an excipient, a carrier and/or a diluent. The composition and form of the composition are arbitrary and may be selected appropriately according to conditions such as the active ingredient and the use. The composition can be prepared by a conventional method according to the formulation thereof, in a suitable combination with excipients, carriers, diluents and the like, and other components.

The preparation of the present invention can be used in humans and animals by being mixed with cosmetics or the like, or can be administered to humans and animals as a pharmaceutical preparation. In addition, the compound can be mixed with various foods, drinks, and feeds and ingested by humans and animals.

When the present invention is applied to external preparations for skin such as cosmetics, pharmaceuticals, quasi drugs, etc., the amount of the plant or extract thereof to be blended (dry mass) can be determined appropriately according to the type, purpose, form, method of use, etc. of the plant or extract. For example, the compound of the present invention, an extract of hypericum erectum, an extract of thyme, an extract of cork tree, and/or an extract of eucalyptus may be blended in an amount of 0.00001% to 50% each (in the case of an extract or a crude drug, in terms of dry mass).

In addition to the above components, if necessary, components used for general skin external preparations such as cosmetics, medicines, quasi drugs, etc., for example, antioxidants, oil components, ultraviolet ray protection agents, surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients, etc., may be appropriately blended as necessary within a range not to impair the effects of the present invention.

The skin preparation for external use of the present invention can be applied to cosmetics, quasi drugs, etc. applied to the outer skin, and is particularly suitable for cosmetic applications, and any form of formulation can be applied, as long as it can be applied to the skin, including solution type, solubilization type, emulsification type, powder dispersion type, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc.

When the preparation of the present invention is used as a cosmetic, the preparation can be used in the form of a lotion, a milky lotion, a foundation, a lipstick, a lip stick, a cleansing cream, a massage cream, a pack, a hand cream, a hand powder, a bath lotion, a body cream, a bath cosmetic, or the like.

However, the form that can be used for the preparation and composition of the present invention is not limited to the above-mentioned dosage form and form. In addition, the formulations or compositions of the present invention may be used in conjunction with the devices or methods of the present invention or other devices or methods and the like.

The subject to whom the methods, devices, formulations and compositions of the present invention are applied may be a subject who has objectively or subjectively identified skin photoaging and/or pigmentation (e.g., dermal pigmentation), or who desires to prevent skin photoaging and/or pigmentation. For example, the object may be determined to be an M1/M2 balance crash. In embodiment 1, the skin may be judged to be high in photoaging and/or pigmentation (e.g., dermal pigmentation) using the M1/M2 balance in the skin as an index. Alternatively, the skin may be concerned about a photo-aging-specific phenotype of the skin, for example, spots, wrinkles, sagging, or the like, or may be concerned about epidermis, pigmentation, for example, spots, darkness, birthmarks, tattoo marks, or the like. The spots, wrinkles, sagging, dull marks, birthmarks, tattoo marks, and the like can be determined by visual judgment using a known index.

Further, according to the present invention, there is also provided a screening method of a light aging inhibitor, comprising the steps of: administering a candidate agent to a biological sample; measuring the M1/M2 balance in the biological sample before and after administration of the candidate agent; when the M1/M2 balance in the biological sample to which the candidate drug has been administered is improved as compared to that before the drug is administered, the drug is evaluated to have an anti-photoaging and/or pigmentation-inhibiting effect. Also provided is a method of evaluating a cosmetic treatment against photoaging and/or pigmentation inhibition comprising the steps of: applying a cosmetic treatment to the skin sample; measuring the M1/M2 balance in skin samples before and after cosmetic treatment; when the balance of M1/M2 in the skin sample subjected to the cosmetic treatment was improved as compared to that before the treatment, the treatment was evaluated to have an anti-photoaging and/or pigmentation inhibitory effect. The method of the present invention enables screening of candidate agents or cosmetic treatments for effects of preventing photoaging and/or inhibiting pigmentation, and enables product development and new skin care. That is, by the present invention, there are provided a photoaging resistant agent and/or a pigmentation inhibitor and a photoaging resistant and/or pigmentation suppressing cosmetic treatment for preventing and/or improving photoaging and/or pigmentation by adjusting the M1/M2 balance. The pigmentation may be a dermal pigmentation.

The biological sample may be any sample 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 isolated state after collection from an animal such as a human, or may be a skin sample in an in vitro state in which skin cells are cultured, for example, single-layer or multi-layer cultured cells, cultured 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 infiltrated into the skin, cultured immune cells, or the like. The 3D skin model may be created by, for example, the method described in patent document 8 and non-patent documents 10 to 12, or may be created by changing the method so as to easily measure the M1/M2 balance, although not limited thereto. The biological sample is not limited as long as the M1/M2 balance can be measured.

For example, screening for a photoaging-and/or pigmentation-resistant inhibitor can be carried out by differentiating immune cells such as THP-1 into M0, adding a drug, culturing for a certain period of time, collecting the culture supernatant, and quantifying the amount of a cytokine or the like specific to M2 such as IL-10 by an ELISA or the like assay. In such screening method, as a positive control, cells differentiated into M2 by adding IL-4, IL-13 or the like can be used, and further, experiments in which candidate agents were added to IL-4 and IL-13 can be compared. That is, since M2 has a high IL-10-producing ability, if IL-10 in the supernatant is increased after differentiation at M0 as compared with a control (control) cultured only with a medium, it can be said that the differentiation to M2 is achieved, and such a drug can be referred to as an "improving agent" in which the presence or absence of the drug in the presence of IL-4, IL-13, or the like can be compared to evaluate whether or not the amount of IL-10 is different.

In the present specification, the candidate drug refers to a drug for which the anti-photoaging and/or pigmentation inhibitory effect is examined, and includes a drug at the development stage in addition to a drug already sold as a product, and may be a drug selected as a cosmetic drug in the development of cosmetics.

The cosmetic treatment is not particularly limited, and may include any treatment considered to be effective for the inhibition of photoaging and/or pigmentation, such as the application of a cosmetic preparation containing the preparation of the present invention or other ingredients. The cosmetic is a cosmetic applied to the skin, and includes, for example, a lotion, an emulsion, a lotion, a cream, a foundation, and the like, but is not limited thereto, and means all substances applied to the skin, for example, a sunscreen agent, and the like, even if the improvement of the skin condition is not an immediate purpose. Alternatively, the cosmetic treatment may be, for example, applying a physical stimulus such as a stretching stimulus, a pressing stimulus, a massage, or the like to the skin. The cosmetic treatment may be a single treatment or may be a continuous treatment from several days to several weeks. The beauty treatment may be performed by an individual person, or may be performed in a beauty room, a sales shop for cosmetics, a beauty salon, or the like.

The present invention also provides a screening kit for an anti-photoaging and/or pigmentation inhibitor, which comprises a drug for measuring the M1/M2 balance, such as an antibody for detecting any marker, e.g., CD86, CD206, CD163, and the like, which can measure the number of M1 or M2, and a drug for measuring the mRNA amount of the marker. The invention further provides the following method, system and device.

A method of assessing photoaging and/or pigmentation of the skin of a subject, performed by 1 or more computers, having the steps of: acquiring data on a reference value of a preset skin M1/M2 balance; a step of acquiring data on the M1/M2 balance of the skin of the subject; comparing the reference value with the data on the M1/M2 balance of the skin of the subject to calculate; a step of evaluating the photoaging and/or pigmentation degree of the skin based on the result calculated by the calculating step; and a step of displaying a result obtained by the evaluation in the evaluation step.

A method of assessing photoaging and/or pigmentation of the skin of a subject, comprising the steps of: a step of analyzing, by an analysis section of the server, a relationship between the M1/M2 balance of the skin and the photoaging and/or the degree of pigmentation by mechanical learning using a method performed using a neural network using teacher data based on data on the M1/M2 balance of the skin and the photoaging and/or the degree of pigmentation stored in a storage section; receiving, by a receiving unit of the server, data of an M1/M2 balance of a skin of a subject; and a step of evaluating, by an evaluation unit of the server, the photoaging and/or pigmentation degree of the skin of the subject based on the analyzed relationship by inputting the received M1/M2 balance of the skin of the subject.

A system for evaluating photoaging and/or pigmentation of a subject's skin, having: a database part for storing data of reference value of M1/M2 balance related to preset skin; a data input section that inputs data on the M1/M2 balance of the skin of the subject; a calculation unit for calculating by comparing the reference value stored in the database unit with the data on the M1/M2 balance of the skin of the subject inputted through the data input unit; an evaluation unit for evaluating the photoaging and/or pigmentation degree of the skin based on the calculation result obtained by the calculation unit; and a display unit for displaying the result evaluated by the evaluation unit.

A photoaging and/or pigmentation degree calculating device for calculating photoaging and/or pigmentation degree of a subject's skin by using data on M1/M2 balance of the subject's skin, the device comprising a calculating unit for calculating photoaging and/or pigmentation degree of the subject if data on M1/M2 balance of the subject's skin is input, the calculating unit comprising a learned neural network to which a mechanical learning process using teacher data is applied, so that when data on M1/M2 balance of the skin is input, the estimated photoaging and/or pigmentation degree is calculated.

By the method, system and device, objective photoaging and/or pigmentation based on M/M2 balance can be determined.

Examples

The present invention will be described in further detail by examples. The present invention is not limited to this.

Experiment 1: tissue staining

Skin of eye tips derived from white races of the younger age (20 to 30 years) and the older age (60 to 70 years) shown in fig. 1a were prepared into frozen sections, and the sections were cut and stained with the markers for macrophages shown below.

(1) Staining of M1 macrophages: double staining was performed with goat-derived anti-human CD86 antibody (R & D) and rabbit-derived anti-human CD11b antibody (abcam) (fig. 1 b).

(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) (fig. 1 c).

(3) Staining of total macrophages: double staining was performed with mouse-derived anti-human CD68 antibody (abcam) and rabbit-derived anti-human CD11b antibody (abcam).

Cells that were doubly positive for antibodies (1) to (3) up to 200 μm just below the epidermis were counted as macrophages (fig. 1 d). In addition, to investigate the relationship between M1 and M2 macrophages and collagen production, double staining was performed with goat-derived anti-human CD86 antibody (R & D) and rat-derived anti-procollagen antibody (Millipore), or mouse-derived anti-human CD206 antibody (BD) and rat-derived anti-procollagen antibody (Millipore) (FIG. 1e, left).

The tissue staining of (1) to (3) is shown in FIGS. 1b and c, the counting results are shown in FIG. 1d, and the double staining results with M1, M2 macrophage antibody and anti-procollagen antibody are shown in the left of FIG. 1 e. As shown in fig. 1b, c, and d, M1 macrophages were observed in large numbers in aged people with photoaging, while M2 macrophages were reduced. More specifically, as shown in fig. 1b, M1 macrophages are present only in the vicinity of blood vessels for the younger age, while they are scattered throughout the tissues for the older age. As shown in fig. 1c, M2 macrophages were present in the entire tissue for the younger age, while the number decreased for the older age. Further, as shown in fig. 1d, the total number of macrophages (number of M1 + number of M2) did not change between the younger and the older, and only the M1/M2 balance changed. For the younger age, the ratio of M2 to M1 (number of M2/number of M1) ranged from about 5/5 to about 7/3, while for the older age the ratio of M2 to M1 decreased dramatically, with the M1/M2 balance collapsing considerably, which is significantly different from the value for the younger age.

Furthermore, as shown in the top left 2 photographs of fig. 1e, M1 macrophages and procollagen shifted in position in both the younger and older adults, while the positions of M2 macrophages and procollagen were observed to match each other as shown in the bottom left 2 photographs. Therefore, it is suggested that M1 may act on fibroblasts to promote collagen destruction and M2 may act on fibroblasts to promote collagen production, as shown in the schematic diagram of the right panel of FIG. 1 e.

Experiment 2: m1 and M2 differentiation stimulation experiments of THP-1

Differentiation into M1 and M2 macrophages was induced by the method described in non-patent document 1 using THP-1, which is an established cell line derived from human. Specifically, THP-1 was cultured by adding 1mM sodium pyruvate (Nakalai), 2mM L-glutamine (Nakalai) and 10% FBS to RPMI1640(Nakalai) by the method shown in FIG. 2 a. Then, 100nM PMA (abcam) was added thereto and stimulated for 24 hours to differentiate into macrophages. The cells were further stimulated for 24 hours with the addition of 100ng/mL LPS (sigma) and 20ng/mL IFN γ (R & D) for differentiation into M1, and for 24 hours with the addition of 20ng/mL IL-4(R & D) and 20ng/mL IL13(R & D) for differentiation into M2. Upon observation with a microscope, morphological changes were observed as shown in FIG. 2 b.

In addition, mRNA of each cell was extracted in the differentiated or undifferentiated state, and the expression level was quantified by real-time PCR using a TaqMan gene expression measurement system using probes for IL-1beta, TNF-alpha, and IL-10 (Applied Biosystems) (FIG. 2c, upper panel). Further, PCR was performed in the same manner using the CD86 antibody (R & D) and the CD206 antibody (BD) as in experiment 1, and the expression level was quantified (fig. 2c, lower panel). The respective values were corrected for the mRNA expression level of GAPDH.

As shown in FIG. 2c, it was revealed that macrophages differentiated by the method described in experiment 2 produced inflammatory cytokines (IL-1beta, TNF-alpha) characteristic to M1 and anti-inflammatory cytokines (IL-10) characteristic to M2, respectively. Further, these differentiation-induced macrophages showed increased expression of CD86, CD206 as surface markers of M1, M2 macrophages, respectively. From these results, it was confirmed that differentiation induction was successful. Therefore, M1, M2 macrophages and undifferentiated M0 macrophages differentiated by the method of experiment 2 were used in the following

experiments

3 and 4.

Experiment 3: experiment for adding M1 and M2 macrophage supernatant to fibroblast

As shown in FIG. 3a, after differentiating THP-1 into M1 and M2 or in an undifferentiated state of M0 in the same manner as in experiment 2, the supernatant was removed, washed 1 time with PBS, and then cultured for 48 hours with the addition of a medium. These supernatants containing secretion of M1, M2 such as inflammatory/anti-inflammatory cytokines were added to neonatal-derived fibroblasts. As a control (control), cells supplemented with RPMI1640(Nakalai) were used. After the addition of the supernatant, fibroblasts were cultured for 72 hours, and the amount of procollagen in the supernatant was quantified using PIP ELISA kit (TAKARA) (fig. 3 b). Cell fractions were stained with rabbit-derived anti-human collagen antibody (CEDERLANE) and biotinylated hyaluronic acid binding protein (HOKUDO) (fig. 3 c). Further, using β -gal as an aging index, nuclei of cells were stained with DAPI, and then β -gal in the cells were stained using the Senescence Detection Kit (abcam) (fig. 3d), and the numbers of β -gal positive cells and DAPI positive cells were counted (fig. 3 e).

Furthermore, in order to examine the degree of contribution of M1 macrophages and M2 macrophages to melanin production, supernatants from the respective macrophages were added to fibroblasts and cultured in the same manner as described above. Then, fibroblasts were recovered and mRNA was recovered, and the amount of mRNA expression was quantified by real-time PCR using probes for HGF, ET1, bFGF, IL-1alpha, SCF, and clusterin (FIG. 3 f).

The results are shown in FIGS. 3b to 3 f. FIG. 3b shows the amount of procollagen after 72 hours of culture with fibroblast cells added to the supernatant of each macrophage (M1, M2). Fig. 3c shows the local presence of collagen and hyaluronic acid in fibroblasts after 72 hours of culture with the addition of the supernatant of each macrophage (M1, M2). As can be seen from fig. 3b and c, M1 significantly inhibited collagen production. FIG. 3d shows intracellular β -gal in fibroblasts after 72 hours of culture with addition of supernatant of each macrophage (M1, M2). It is suggested from fig. 3d that for M1, β -gal positive cells were increased, promoting aging, on the other hand, M2 inhibited aging. FIG. 3e shows a graph 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. As shown in the right panel of fig. 3e, M2 not only inhibited cell aging, but also promoted cell proliferation. FIG. 3e, left panel, shows the ratio (%) of the number of β -gal positive cells to the total number of DAPI positive cells, suggesting that M1 has an aging and cell death promoting effect, and that M2 has an aging inhibiting and cell proliferation effect. Further, as shown in fig. 3f, it is also known that mRNA expression of the melanogenesis-related factor acts in a direction to increase melanogenesis by M1, and acts in a direction to suppress melanogenesis by M2. As reported in non-patent document 6, it is known that fibroblasts are stimulated by light such as UV to secrete factors such as SCF and HGF, thereby causing cell death. Furthermore, as reported in non-patent documents 7 to 9, fibroblasts are stimulated by light to secrete factors such as HGF, ET1, bFGF, SCF, clusterin, and the like, and directly or indirectly act on melanocytes to produce melanin. Thus, it was suggested that cell death and melanin production by light stimulation were caused by the breakdown of the balance of M1/M2.

Experiment 4: experiment for adding M1 and M2 macrophage supernatant to aged and aged fibroblasts

Next, in order to confirm whether the anti-aging effect of M2 macrophages is useful for aged fibroblasts, that is, whether the anti-aging effect is effective for rejuvenation, differences in the effects of macrophage supernatants due to age differences of fibroblasts were examined by adding M1 and M2 macrophage supernatants to aged and aged cells.

Specifically, each macrophage supernatant collected by the same method as in experiment 3 was added to neonatal foreskin-derived fibroblasts (fibroblasts derived from a low age group) and 68-year-old derived fibroblasts (fibroblasts derived from an old age group), and cultured for 72 hours. Then, β -gal and DAPI staining were performed in the same manner as in experiment 3, and the numbers of β -gal positive cells and DAPI positive cells were counted (fig. 4a and 4 b). Further, as in experiment 3, the supernatant of M1 and M2 was added to fibroblasts derived from old and young people, the fibroblasts were cultured for 72 hours, and the amount of procollagen in the supernatant was quantified using PIP ELISA kit (TAKARA) (fig. 4c), and stained with rabbit-derived anti-human collagen antibody (CEDERLANE) and DAPI (fig. 4 d).

FIG. 4a shows the staining pattern of β -gal. FIG. 4b shows a graph obtained by plotting the results of the number of β -gal positive cells of fibroblasts derived from old age and the total number of cells per well. As can be seen from the lower graph of fig. 4a, even for aged cells, the addition of M2 supernatant β -gal positive cells significantly decreased and aging was suppressed. Also, as is clear from fig. 4b, aging was promoted by M1 and was suppressed by M2 regardless of age. This is also supported by the results that cells added with M1 supernatant had significantly lower collagen production compared to M2 supernatant regardless of age, as shown in fig. 4c and d.

Experiment 5: co-culture experiment of M1 macrophage, M2 macrophage and neonatal foreskin-derived fibroblast

The amounts of collagen produced when the foreskin-derived germ cells of newborns were cultured with RPMI1640(Nakalai) and the same number of M1 or M2 macrophages (prepared by the method of experiment 2) and when the foreskin-derived fibroblasts of newborns and half of the M1 or M2 macrophages were cultured with RPMI were compared by staining with rabbit-derived anti-human collagen antibody CEDERLANE. Macrophages were also visualized by staining with mouse-derived anti-human CD68 antibody (abcam).

The results are shown in fig. 5. For neonatal foreskin-derived germ cells, collagen was also reduced if M1 supernatant was added, on the other hand, collagen was observed in large amounts if M2 was added. That is, even in the case of the low-aged cells, it was found that collagen was affected if the M1/M2 balance was disrupted.

Experiment 6: effect of M1/M2 balanced collapse Using 3D skin model

In order to investigate the effect of M1/M2 macrophages on endothelial cells and fibroblasts, three 3D skin models formed of a 3-layer structure as described in the following table were prepared.

TABLE 1

The 3D skin model is created as follows. Human dermal fibroblasts (0.2X 10 μm) were seeded in cell culture inserts (. phi.12 mm, average pore size of porous membrane: 0.4 μm)⁶One), 200 μ M magnesium ascorbate-2-phosphate (APM), 10% FBS-DMEM, 2 days with 1 exchange of medium for 1 week. On top of that, the control model was injected with a solution containing human dermal fibroblasts0.5% type I collagen-10% FBS-DMEM solution of cells was dispensed into M1 model and M2 model, and the above solutions, which were differentiated by the method of experiment 2, of M1 macrophage and M2 macrophage, added to 30,000 cells, respectively, were prepared into collagen gel on human dermal fibroblasts, and cultured for 1 to 5 days.

The epidermal keratinocytes dispersed in Humedia-KG2(クラボウ) medium were further treated to become 5X 10⁵One well was seeded on collagen gel, and cultured for 3 days by adding culture medium prepared by mixing Humedia-KG2 and 10% FBS-DMEM at 1:1 and adding 200. mu.M APM to the insert until the same liquid level as the inner side.

Then, the insert or the medium in the vitreous ring was removed, 200. mu.M APM, 10. mu. M N-hydroxy-2- [ [ (4-methoxyphenyl) sulfonyl ] 3-picolyl) amino ] -3-methylbutanamide hydrochloride (CGS27023A (MMP inhibitor)), and 10. mu.M BIPBIPU (heparanase inhibitor) were added to the skin model medium (prepared by mixing 10% FBS-DMEM and Humedia-KG2 EGF (-) at 1:1 to Ca 1.8mM) on the outside, and the inside of the insert was cultured in an air-liquid interface state until the height of the bottom surface of the insert was reached. The medium was exchanged 1 time for 2 to 3 days and cultured for 2 weeks.

After the cultured skin model was fixed with 4% PFA/pbs (nakarai), the skin model was stained with anti-CD 206 antibody (abcam), anti-CD 68 antibody (abcam), and anti-CD 86 antibody (abcam) in the same manner as in experiment 1, and the presence of M1 and M2 macrophages was confirmed (fig. 6). Then, the cells were stained with anti-p 21 antibody (abcam) and DAPI (vector), and the number of cells stained with DAPI in each of the epidermal cell layer and the fibroblast cell layer was counted as the total number of cells, and the number of cells also stained with anti-p 21 antibody was counted as the number of p 21-positive cells. The ratio of the number of p 21-positive cells to the total number of cells was determined by the following equation.

The ratio of the number of p21 positive cells to the total number of cells (number of p21 positive cells/total number of cells) × 100 (%)

The results are shown in fig. 7 and 8. As shown in these figures, in both the upper epidermal cell layer and the lower fibroblast layer adjacent to the middle layer, p 21-positive cells increased in the M1 model (M1) and decreased in the M2 model (M2) relative to the control model (cont). This tendency is consistent with experiment 3 using monolayer cultures. Therefore, in a 3D skin model closer to human skin, it was also suggested that M1 macrophages have an aging and cell death promoting effect, and on the other hand, M2 macrophages have an aging and cell death suppressing effect.

Experiment 7: solar light irradiation experiment using in-vitro model of human skin

Nativeskin (skin collected from a 38-year-old female) manufactured by Genoskin was cultured for 1 day in a culture medium attached thereto. The next day, an optical filter was placed in a 1000W solar simulator manufactured by Oriel corporation, and UVA and UVB were applied only at 11.5J/cm²Irradiation, and culture was continued with the attached culture medium (irradiation (+)). As a control, a sample (irradiation (-)) that was not irradiated was used. Samples were collected after 5 days of irradiation, and M1 macrophages, M2 macrophages, and total macrophages were stained and counted as shown in experiment 1.

The results are shown in fig. 9. The number of M1 is the most increased if the solar simulator is illuminated. On the other hand, the increase in the number of M2 was very small compared to the case of M1. That is, it was found that the balance of M1/M2 was lost and the ratio of M1 was increased by the light stimulation.

Experiment 8: stretching stimulation experiment using in vitro model of human skin

Next, studies were made on methods for adjusting or improving the M1/M2 balance.

Sample preparation: nativeskin (skin collected from a 38-year-old female) (6-well size, about 2 to 2.5cm in diameter) manufactured by Genoskin was used.

Stretching conditions are as follows: a stretching tool having grip portions for gripping both ends of the skin in the hole as shown in fig. 11 was produced, and the skin was stretched by stretching the grip portions. The holes with the tissue pieces added thereto were horizontally arranged, the skin was stretched from both ends by operating the grip portions, stretching was performed at a rate of 10% per second to a stretching ratio of 10% as shown in fig. 10, and the sample was returned to the original non-stretched state at a recovery rate of 10% per second. This was set to 1 cycle, and 90 cycles in total were performed over 30 minutes. The 90 cycles were set as 1 group, and a pause time of 30 minutes to 1 hour was set between the groups to perform a total of 3 groups over 3 hours (total of 270 cycles). As a control, a sample (control) not subjected to the stretching stimulus was used.

The observation method comprises the following steps: m1 macrophages, M2 macrophages, and total macrophages were stained and counted in the same manner as in experiment 1, except that the total macrophages were stained with only rabbit-derived anti-human CD11b antibody (abcam) for skin samples with or without the application of the stretching stimulus. The ratio (%) of each macrophage (M1, M2) to the total number of macrophages was obtained and plotted.

The results are shown in fig. 12. The total number of macrophages for the samples with applied extensional stimulation did not vary much from the number of M1, but the number of M2 was very increased compared to the control. That is, it was found that the balance of M1/M2 was improved by the extension stimulus.

Experiment 9: screening for anti-photoaging and/or pigmentation Agents for preventing and/or improving photoaging and/or pigmentation by adjusting or improving the M1/M2 balance

Screening for agents that modulate or improve the M1/M2 balance was performed.

As the drug to be screened, 19 kinds of total components including a forsythia suspense extract, a thyme extract, and tranexamic acid carboxamide (N-methyl-trans-4- (aminomethyl) cyclohexanecarboxamide) were studied. The Hypericum erectum extract is obtained from Hypericum erectum aerial parts purchased from YI WAN ファルコス. The thyme extract is an extract of the whole plant of thyme asia, purchased from murraya. Tranexamic acid formamide is synthesized by the method described in patent document 14. The extract of Hypericum erectum is dissolved in 50% ethanol, the extract of Thymus vulgaris is dissolved in butanediol, and tranexamic acid formamide is dissolved in PBS.

The same THP-1 cells as in experiment 2 were used as they were in an undifferentiated state of M0 and cultured overnight at 37 ℃. The above-mentioned screening drugs were added to the medium so that the content of the extract of Hypericum erectum was 0.1%, the content of the extract of Thymus vulgaris was 0.1%, and the content of the formamide was 0.06% and 0.03%, respectively, and the same amount of the solvent was added to the control, followed by culturing at 37 ℃ for two nights. The cells were recovered to extract RNA, and the expression levels of CD86 and GAPDH were quantified by real-time PCR to determine CD 86/GAPDH. An agent having a reproducibly decreased CD86/GAPDH value as compared with the control (cont) was searched for as an M1 differentiation inhibitor.

The results are shown in fig. 13. Fig. 13 shows that when hypericum erectum extract, thyme extract, and tranexamic acid formamide were added, CD86/GAPDH values were significantly reduced, and M1 differentiation inhibition was exhibited, suggesting that the compound could be used as a M1/M2 balance control/improving agent.

Experiment 10: screening of photoaging-resistant agents for preventing and/or improving photoaging and/or pigmentation by adjusting or improving the M1/M2 balance

In order to screen more drugs, 8 kinds of total ingredients including phellodendron amurense extract and eucalyptus extract were investigated in the same manner as in experiment 9. The phellodendron amurense extract is dried bark of phellodendron amurense belonging to Rutaceae, and the eucalyptus extract is extract of eucalyptus leaves. Phellodendron amurense extract was dissolved in butylene glycol to a concentration of 0.01%, and eucalyptus extract was dissolved in 50% ethanol to a concentration of 0.1%. Their solvents were used in the control.

The addition of a drug, the recovery of cells, the extraction of RNA, and the measurement of CD86/GAPDH were carried out in the same manner as in experiment 9, and a drug with a reproducibly decreased CD86/GAPDH value as compared with the control (cont) was searched for as an M1 differentiation inhibitor. The results are shown in fig. 15. Fig. 15 shows that phellodendron amurense or eucalyptus extract can be used as a balance adjusting/improving agent of M1/M2 in addition to the drug of experiment 9.

Experiment 11: effect of M1, M2 macrophage supernatant on collagen

In order to examine the degree of contribution of M1 macrophages and M2 macrophages to collagen degradation, except for experiment 3, supernatants of the respective macrophages were added to fibroblasts and cultured in the same manner as experiment 3, and mRNA was recovered after 72 hours. As an index of collagen degradation, real-time PCR was carried out using probes for MMP-1, MMP-2, and IL-1. beta. (Taqman probe, Applied biosystems) to quantify the respective mRNA expression levels (FIG. 16 a).

Furthermore, in order to examine the degree of contribution of M1 macrophages and M2 macrophages to collagen production/maturation, supernatants from the respective macrophages were added to fibroblasts and cultured in the same manner as in experiment 3, and mRNA was recovered after 72 hours. As an indicator of collagen production/maturation, real-time PCR was performed using probes for COL1A1, COL1A2, HSP47, and ADAMTS-2 (Taqman probe manufactured by Applied biosystems) to quantify the mRNA expression levels of the respective probes (FIG. 16 b).

From fig. 16a, b it was confirmed that M1 contributes to collagen breakdown, on the other hand, M2 contributes to collagen production/maturation.

Experiment 12: collagen breakdown and production in vitro model using human skin of young and elderly persons

Skin of white human-derived temple of the younger age (20 to 30 years) and the older age (60 to 80 years) shown in fig. 17a was prepared as a frozen section in the same manner as in experiment 1, and after slicing, positive cells were counted in the same manner as in experiment 1 using anti-procollagen antibody (millipore), anti-fragmented collagen (AdipoGen) obtained by staining the collagen cleavage fragment on the 3/4 side, and CD68, CD11b, CD206, and CD86 antibodies similar to experiment 1.

The results are shown in FIGS. 17b, c, and d. The proportion of M1 macrophages was high and the proportion of M2 macrophages was low for the older group compared to the younger group. However, at all ages, the number of macrophages showing 3/4 collagen positivity was larger than that of fibroblasts showing 3/4 collagen positivity, and the number of M2 macrophages showing 3/4 collagen positivity was further larger than that of M1 macrophages showing 3/4 collagen positivity.

Experiment 13: in vitro (in vitro) experiment showing melanin uptake capacity in dermis

M1 macrophages and M2 macrophages obtained by differentiating THP-1 cells were differentiated in the same manner as in experiment 3. A Melanin solution (Sigma, Melanin-Bioreagent, Synthetic, suitable for cell culture) was dissolved in PBS to adjust the concentration to 0.02% W/V, and added to each of the differentiated M1 macrophages, M2 macrophages, and fibroblasts (クラボウ Co.). After 24 hours, the cells were washed with PBS and then photographed with a microscope, and the cells were collected. The number of cells of the recovered cells was quantified by measuring melanin content with alamar blue (Life Technologies) as described below.

Quantification of melanin:

after adding Almarblue adjusted to 1:10 in a medium of each cell (macrophage: RPMI1640, fibroblast: 1DMEM), the cells were cultured at 37 ℃ for 30 minutes. Then, 100. mu.l/well of the supernatant was collected, and fluorescence was measured at excitation/emission: 544nm/590nm by using an Ascent (Thermo). After measurement, the cells were washed with PBS, added with 1M NaOH, and incubated at room temperature for 3 hours to completely dissolve. The cell solution was measured as OD475 using POWERCAN HT (DS PHARMA BIOMEDICAL).

The results are shown in FIGS. 18a to c. The unit "melanin/alamar blue" in the figure is a relative value ((2)/(1)) of intensity of fluorescence ((1)) measured at 544nm/590nm for the supernatant and intensity ((2)) measured at absorbance 475nm for a solution in which melanin was also dissolved in the supernatant, obtained by staining the cultured cells with alamar blue, and shows the melanin content per cell. From these figures, it is clear that M2 engulfs very much melanin. This tendency was also observed after 24 hours, but the difference became more clear as the culture of the cells was continued until after 5 days (fig. 18 c).

Experiment 14: ex vivo experiments showing the ability to uptake melanin in the dermis

Since experiment 13 revealed that M2 macrophages engulf melanin more than fibroblasts and M1 macrophages, the dermis layer of human skin counted in experiment 13 was observed ex vivo using LSM880 (manufactured by カールツァイス). It was found that M2 macrophages were stained black with melanin when both aged and aged were observed in the bright field, whereas melanin was not present in the vicinity of M1 macrophages. From this, it was found ex vivo that M2 macrophages take up melanin more than fibroblasts and M1 macrophages in the dermis, and the number of the macrophages was counted and plotted (fig. 19).

According to fig. 19, the number of M2 macrophages was greater than the number of M1 macrophages for both old and young people, and age differences were less observed in this tendency. This result suggests that melanin uptake into the dermis is M2 macrophage, and thus if the M1/M2 balance is adjusted by increasing the ratio of M2 macrophages, pigmentation in the dermis can be prevented and/or improved.

The increase in the ratio of M1 by photoaging (experiment 7) and the increase in the amount of skin collagen with a high ratio of M2 (experiments 11 and 12) and the increase in phagocytosis (experiments 13 and 14) are shown, and therefore, the physical stimulation-based anti-photoaging and/or pigmentation-inhibiting effects of the compounds of the present invention, such as tranexamic acid carboxamide, which has been screened as an M1 differentiation inhibitor, thyme extract, phellodendron amurense and eucalyptus extract, and the invention having an M1/M2 balance-improving effect are shown to be high.

From the above results, it was suggested that imbalance of the balance of M1/M2 occurs by light stimulation, thereby causing photoaging in the skin exposed to light, or pigmentation in the dermis, but that photoaging and/or pigmentation in the dermis can be prevented and/or improved by adjusting or improving the balance of M1/M2. The adjustment or improvement of the balance of M1/M2 is achieved by applying a stretching stimulus or a pressing stimulus, and applying a compound of the present invention such as tranexamic acid carboxamide, or a component having an M1 differentiation inhibitory action such as an extract of hypericum erectum, an extract of thyme, phellodendron amurense, and/or an extract of eucalyptus, as an M1/M2 balance adjustment/improvement agent, and further, the prevention and/or improvement of photoaging and/or dermal pigmentation is expected.

Industrial applicability

By means of the present invention, it is possible to carry out the search for the prevention and/or improvement of photoaging and/or dermal pigmentation, the evaluation of photoaging and/or dermal pigmentation degree, the anti-photoaging and/or dermal pigmentation inhibitor and the anti-photoaging and/or dermal pigmentation inhibiting cosmetic treatment.

Claims

1. Cosmetic method for preventing and/or ameliorating photoaging and/or dermal pigmentation by adjusting the M1/M2 balance.

2. The method of claim 1, wherein adjusting the M1/M2 balance is increasing the ratio of M2 to M1.

3. The method according to claim 1 or 2, wherein adjusting the M1/M2 balance comprises a procedure of applying a weak physical stimulus to the skin, wherein the procedure is achieved by applying a physical stimulus to the skin of the subject that cycles at a frequency of 60Hz or less, said cycle comprising the steps of:

(a) stretching the skin of the subject to an elongation of 0.1% to 50.0%; and

(b) recovering from the extended state;

and/or the presence of a gas in the gas,

(a-1) pressing the skin of the subject by 1 to 1000 μm; and

(b-1) restoring the skin of the subject from the compressed state;

wherein the elongation is calculated by the following formula,

wherein the fixed points A and B are arbitrary positions on the epidermis or the substrate to which the epidermis is bonded, and a straight line passing through the fixed points A and B is parallel to the stretching direction.

4. A cosmetic device for preventing and/or improving photoaging and/or dermal pigmentation by adjusting M1/M2 balance,

the device is provided with:

a stimulus generating section for generating a physical stimulus, and

the apparatus is an apparatus for performing a process of applying a weak physical stimulus to the skin, wherein the process is circulated at a frequency of, for example, 60Hz or less, and the circulation includes the steps of:

(a) stretching the skin to an elongation of 0.1% to 50.0%; and

(b) recovering from the extended state;

and/or the presence of a gas in the gas,

(a-1) pressing the skin of the subject by 1 to 1000 μm; and

(b-1) restoring the skin of the subject from the compressed state;

wherein the elongation is calculated by the above formula 1.

5. A method of screening for an inhibitor of anti-photoaging and/or dermal pigmentation, the method comprising the steps of:

administering a candidate agent to a biological sample;

when the M1/M2 balance in the biological sample to which the candidate agent has been administered is improved as compared to that before administration of the agent, the agent is evaluated as having anti-photoaging and/or dermal pigmentation inhibitory effects.

6. A method for evaluating a cosmetic treatment for anti-photoaging and/or inhibition of dermal pigmentation, the method comprising the steps of:

applying a cosmetic treatment to the skin sample;

in the case where the balance of M1/M2 in the skin sample on which the cosmetic treatment was performed was improved compared to that before the treatment, the treatment was evaluated as having anti-photoaging and/or dermal pigmentation inhibitory effects.

7. A method for evaluating photoaging and/or dermal pigmentation, performed by 1 or more computers, the method having the steps of:

acquiring data on a reference value of a preset skin M1/M2 balance;

a step of acquiring data on the M1/M2 balance of the skin of the subject;

a step of calculating by comparing the data on the M1/M2 balance of the skin of the subject with reference to the reference value;

a step of evaluating the photoaging and/or dermal pigmentation suppression degree of the skin based on the calculation result obtained by the calculating step; and

and a step of displaying the evaluation result obtained in the evaluation step.

8. A system for evaluating photoaging and/or dermal pigmentation, the system having:

a calculation unit that calculates the M1/M2 balance data of the skin of the subject inputted through the data input unit by referring to the reference value stored in the database unit;

an evaluation section that evaluates photoaging and/or dermal pigmentation degree of the skin based on a calculation result obtained by the calculation section; and

9. A screening kit for an anti-photoaging and/or dermal pigmentation inhibitor comprising an agent for determining the M1/M2 balance.

10. An anti-photoaging and/or dermal pigmentation inhibitor for use in preventing and/or ameliorating photoaging and/or dermal pigmentation by adjusting the M1/M2 balance.

11. The anti-photoaging and/or dermal pigmentation inhibitor according to claim 10, comprising: a compound represented by the following general formula (1) or a salt thereof, a Hypericum erectum extract, a thyme extract, phellodendron amurense, and/or a eucalyptus extract,

in the general formula (1), R₁And R₂The same or different, each represents 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 general formula (2),

in the general formula (2), X represents a lower alkyl group, a lower alkoxy group, a hydroxyl group, an amino group or a halogen atom, and n is 0 to 3.

12. An M1/M2 balance adjustment/improvement agent comprising: a compound represented by the following general formula (1) or a salt thereof, a Hypericum erectum extract, a thyme extract, phellodendron amurense, and/or a eucalyptus extract,

13. A composition comprising the agent of any one of claims 10-12.

14. The method of claim 1 or 2, wherein adjusting the M1/M2 balance is achieved by administering the agent of any one of claims 10-12.