CN117815216A

CN117815216A - Anti-photoaging and/or dermis pigmentation inhibitor

Info

Publication number: CN117815216A
Application number: CN202410125329.4A
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: 2024-04-05
Also published as: JP7439059B2; JP2024037928A; CN113784753A; JPWO2020213743A1; WO2020213743A1

Abstract

The subject of the present invention is to provide a method, a device, and a medicament for preventing and/or improving photoaging and/or dermis pigmentation, a screening method of an anti-photoaging and/or dermis pigmentation inhibitor, an evaluation method of an anti-photoaging and/or dermis pigmentation inhibition cosmetic treatment, and an evaluation method of photoaging and/or dermis pigmentation degree. The solution is to prevent and/or improve photoaging and/or dermal pigmentation by adjusting the M1/M2 balance. By using the M1/M2 balance as an index, the photoaging and/or dermal pigmentation degree can be objectively evaluated, and a pharmaceutical agent and a cosmetic treatment for preventing and/or improving photoaging and/or dermal pigmentation can be searched for based on such a method.

Description

Anti-photoaging and/or dermis pigmentation inhibitor

The present application is a divisional application of patent application having a filing date of 20/4/2020 and a filing number of 202080029442.2 and entitled "method, apparatus, and screening method of anti-photoaging and/or dermis pigmentation inhibitor, evaluation method of anti-photoaging and/or dermis pigmentation inhibition cosmetic treatment, and evaluation method of photoaging and/or dermis pigmentation degree" for preventing and/or improving photoaging and/or dermis pigmentation.

Technical Field

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

Background

Aging phenomena of human skin are broadly classified into "natural aging" and "photoaging". Photoaging is a phenomenon specifically observed at the exposed site, and is skin-specific. The skin fibrous tissue is damaged by the generation of active oxygen and the damage of cellular DNA induced by the influence of UV and the like, and is considered to be the cause of appearance of the phenotype such as wrinkles and sagging. For example, a phenomenon such as a decrease in collagen fibers composed of collagen and a denaturation of elastic fibers composed of elastin is observed by skin photoaging. In addition, melanocytes are destroyed and melanin pigment is formed in large amounts, which causes spots and the like.

Furthermore, pigmentation represented by spots and darkness 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 rapid cycle, and therefore 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 basal membrane, and the like. The cycle of dermal cell renewal is very slow compared to epidermis, so such melanin tends to accumulate without being expelled. For this reason, improvement of pigmentation of dermis is very difficult.

As a cosmetic method of preventing photoaging, for example, patent document 1 discloses a prevention or inhibitor 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 amaranthus belonging to the family amaranthaceae and having a collagen synthesis promoting effect and a collagen peptide derived from an animal.

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

In addition, it has been suggested that inflammation is a cause of skin photoaging, and anti-inflammatory agents have been developed in large quantities. Patent document 3 discloses an anti-aging cosmetic composition containing a compound that induces activation of autophagy as adiponectin expression increases. There is also a suggestion that phagocytosis by macrophages is used to improve dermal pigmentation, and patent document 15 discloses a dermal plaque preventing/improving agent which attracts macrophages to fibroblasts which ingest melanin falling into the dermis to phagocytize the macrophages.

Prior art literature

Patent literature

Patent document 1: japanese patent No. 5657723

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

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

Patent document 4: japanese patent application laid-open No. 2014-504629

Patent document 5: japanese patent application laid-open 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 application laid-open No. 2018-140953

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

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

Patent document 12: japanese patent laid-open No. 09-187248

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

Patent document 14: japanese patent laid-open No. 09-227367

Patent document 15: japanese patent laid-open 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 No. 2007-063292

Non-patent literature

Non-patent document 1: journal of the American College of Cardiology, vol.62, no.20,2013, november 12,2013:1890-901

Non-patent document 2: experimental & Molecular Medicine (2014) 46, e70; 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 ofInvestigative Dermatology,2009April;129 (4) 1016-25.Epub 2008Oct 9.

Non-patent document 5: stem Cell Research & Therapy (2018) 9:88, https:// doi.org/10.1186/s13287-018-0821-5

Non-patent document 6: journal of the European Academy of Dermatology and Venereology 2011European Academy of Dermatology and Venereology,2012,26,1577-1580

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 subject of the present invention is to provide a method and a device for preventing and/or improving photoaging and/or dermis pigmentation, and a method for screening an anti-photoaging and/or dermis pigmentation inhibitor, a method for evaluating an anti-photoaging and/or dermis pigmentation inhibition cosmetic treatment, and a method for evaluating photoaging and/or dermis pigmentation degree.

Means for solving the problems

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

Thus, the following invention was completed:

(1) Cosmetic method for preventing and/or improving 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 adjusting the M1/M2 balance comprises a step of applying weak physical stimulation to the skin, wherein the step is performed by applying physical stimulation to the skin of the subject, for example, with a cycle at a frequency of 60Hz or less, the cycle comprising the steps of:

(a) Stretching the skin of the subject to a stretch of 0.1% to 50.0%; and

(b) Recovering from the extended state;

and/or the number of the groups of groups,

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

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

wherein the stretch ratio is calculated by the following formula,

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

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

the device comprises:

stimulus generating unit for generating physical stimulus, and

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

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

(a) Stretching the skin to a stretch of 0.1% -50.0%; and

(b) Recovering from the extended state;

and/or the number of the groups of groups,

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

(b-1) recovering 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 anti-photoaging and/or dermis pigmentation 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; and

the candidate agent is evaluated as having an anti-photoaging and/or dermal pigmentation inhibiting effect when the M1/M2 balance in the biological sample to which the agent is administered is improved as compared to before the agent is administered.

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

performing cosmetic treatment on the skin sample;

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

when the M1/M2 balance in the skin sample subjected to the above-mentioned cosmetic treatment is improved as compared with that before the treatment is performed, the above-mentioned treatment is evaluated as having an anti-photoaging and/or dermis pigmentation inhibiting effect.

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

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

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

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

a step of evaluating the photoaging and/or dermis pigmentation inhibition degree of the skin based on the calculation result obtained in the calculation step; and

and displaying the evaluation result obtained in the evaluation step.

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

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

a data input unit for inputting data on M1/M2 balance of the skin of the subject;

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

an evaluation unit configured to evaluate photoaging and/or dermis pigmentation degree 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 dermis pigmentation inhibitor comprising an agent for determining the M1/M2 balance.

(10) An anti-photoaging and/or dermis pigmentation inhibitor for preventing and/or improving photoaging and/or dermis pigmentation by modulating the M1/M2 balance.

(11) The anti-photoaging and/or dermis pigmentation inhibitor according to (10), comprising: a compound represented by the following general formula (1) or a salt thereof, an extract of Hypericum perforatum, an extract of thyme, an extract of phellodendron amurense, and/or an extract of Eucalyptus globulus.

{ in which R ₁ And R is ₂ The 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=0 to 3) }

(12) An M1/M2 balance adjusting/improving agent comprising: a compound represented by the following general formula (1) or a salt thereof, an extract of Hypericum perforatum, an extract of thyme, an extract of phellodendron amurense, and/or an extract of Eucalyptus globulus.

(13) A composition comprising the 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 according to 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 ameliorated by adjusting the M1/M2 balance. Further, by using the M1/M2 balance as an index, the photoaging and/or dermal pigmentation degree can be objectively evaluated, and based on such a method, it is possible to search for a pharmaceutical agent and a cosmetic treatment for preventing and/or improving photoaging and/or dermal pigmentation.

Drawings

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

FIG. 1b is a photomicrograph showing macrophages (CD 11b: red) and M1 macrophages (CD 86: green) in skin tissue of a young person and an elderly person.

FIG. 1c is a photomicrograph showing macrophages (CD 11b: red) and M2 macrophages (CD 206: green) in skin tissue of the young and the elderly.

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

The left panel of FIG. 1e is a photomicrograph showing co-staining of M1 macrophages (CD 86: red) or M2 macrophages (CD 206: red) and procollagen (green) in skin tissue of both the young and the elderly. The rectangles present in the 4 panels of the left image are the images with the characteristic parts enlarged. The right panel of FIG. 1e is a schematic representation of M1, M2, fibroblast, collagen production/destruction relationships.

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

FIG. 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-1 beta, 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 levels of mRNA for the cell surface markers (M1: CD86, M2: CD 206) of M1 and M2 macrophages. Correction was performed using the mRNA expression level of GAPDH, and the result was shown as relative value (%) where M0 was 100%.

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

FIG. 3b shows the amount of pro-collagen (μg/well) in fibroblasts to which the supernatant of M1 and M2 macrophages was added according to experiment 3.

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

FIG. 3d is a micrograph showing beta-galactosidase (beta-Gal) in fibroblasts to which the supernatant of M1 and M2 macrophages was added according to experiment 3.

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

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

FIG. 4a is a photomicrograph showing β -Gal in low and old fibroblasts added with supernatant of M1 and M2 macrophages according to experiment 4.

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

FIG. 4c shows the amount of pro-collagen (μg/well) in low and old fibroblasts to which supernatant of M1 and M2 macrophages was added according to experiment 4.

Fig. 4d is a micrograph showing collagen (red) and nuclei stained by DAPI (blue) in low-and old-age 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 fibroblasts derived from neonatal foreskin added with the supernatant of M1 and M2 macrophages according to experiment 5. The upper left photograph shows fibroblasts cultured without the addition of the supernatant of macrophages.

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 parts shown in triangles show the double stained parts of CD68 and CD86, or CD68 and CD 206.

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

Fig. 8 is a graph showing the ratio (%) of the number of p21 positive cells to the total number of cells in each layer (left: upper layer=epidermal cell layer, right: lower layer=fibroblast layer) of the 3D model prepared according to experiment 6.

Fig. 9 is a graph showing the number of M1 and M2 macrophages, as a whole, in an ex vivo (ex vivo) skin model irradiated with a solar simulator according to experiment 7. The relative value (%) of the cell number of each macrophage irradiated with the sample was shown in the right bar (irradiation (+)) assuming that the cell number of each macrophage was 100% (left bar: irradiation (-)).

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

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

FIG. 12 shows the application of stretching stimulus to an ex vivo skin model according to experiment 8 (stretching) and non-application of stretching stimulusComparison of the number of M1 and M2 macrophages with the total number of macrophages in the population (control). The left graph shows every 1mm ² Macrophage number of skin sample (individual cells/1 mm) ² ) Is a diagram of (a). The right graph shows the ratio (%) of each macrophage (M1, M2) to the total number of macrophages.

Fig. 13 shows the results of the screening performed in experiment 9. Bars are graphs showing the relative values (%) when the control (cont) was 100% based on the CD86 expression level (CD 86/GAPDH value) obtained when the respective concentrations of the extracts of hypericum perforatum (0.1%), thyme (0.1%) and tranexamic acid formamide (0.03%, 0.06%) were added.

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

Fig. 15 shows the results of the screening performed in experiment 10. Bars are graphs showing the relative value (%) of the CD86 expression level (CD 86/GAPDH value) when the control (cont) was 100% when the phellodendron extract (0.01%) and the eucalyptus extract (0.1%) were added at each concentration.

FIG. 16a is a graph showing the relative values (%) of the mRNA expression amounts of collagenolytic enzymes (MMP-1, MMP-2) and inflammatory cytokines (IL-1β) in fibroblasts to which the supernatant of M1 and M2 macrophages was added according to experiment 11, where M1 was 100%.

FIG. 16b is a graph showing the relative values (%) of the amounts of mRNA expressed by the respective collagen production/maturation factors (COL 1A1, COL1A2, HSP47, and ADAMTS-2) in the fibroblasts to which the supernatants of M1 and M2 macrophages had been added according to experiment 11, with the result of M1 being 100%.

Fig. 17a shows the ages of the subjects from which the samples in experiment 12 were taken, for the low and high age groups.

FIG. 17b is a graph (individual cells/mm) showing the number of M1, M2 macrophages in skin tissue (50 μm immediately below the basement membrane) of the underage and senior subjects in experiment 12 ² )。

FIG. 17c is a graph (individual cells/mm) showing the number of M1 and M2 macrophages in skin tissue (200 μm immediately below the basement membrane) of the underage and senior subjects in experiment 12 ² )。

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

Fig. 18a shows how fibroblasts, M1 macrophages, and M2 macrophages look after 24 hours from melanin addition in experiment 13. The cells that engulf melanin have a circular shape as shown in the right figure.

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

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

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

Detailed Description

Macrophages are known to be cells that are locally present in various tissues in the body and elicit an immune response to foreign bodies, pathogens, and also participate in inflammation. Macrophages differentiate from M0 macrophages in an undifferentiated state (hereinafter sometimes abbreviated as M0) into M1 and M2. M1 macrophages (hereinafter, abbreviated as M1 in some cases) are known as inflammatory types, and M2 macrophages (hereinafter, abbreviated as M2 in some cases) are known as repair types (anti-inflammatory types). Imbalance in the balance between M1 macrophages and M2 macrophages is 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, it is not clear about the relationship of skin photoaging, pigmentation and M1/M2 balance.

The present inventors have found that the balance between M1 macrophages and M2 macrophages (M1/M2 balance) is disturbed specifically at the site where skin exposed to light and pigmentation occur, and that the regulation of M1/M2 balance is particularly important in the prevention and improvement of photoaging and pigmentation. Furthermore, the present inventors have found that a satisfactory effect can be obtained by applying a specific physical stimulus 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 in which pigmentation has occurred, if physical stimulation is applied at a specific stretching rate at a frequency of 60Hz or less, for example, 1Hz or less and 10Hz or less, the M1/M2 balance is adjusted and improved. In addition, the present inventors have screened various substances using the M1/M2 balance as an index of skin photoaging and pigmentation, and as a result, have found that the extracts of hypericum perforatum, the compounds of the present invention represented by tranexamic acid formamide, thyme extract, phellodendron amurense, and eucalyptus extract have an effect of adjusting/improving the M1/M2 balance, and function as an anti-photoaging agent and an anti-pigmentation agent. The extract of Hypericum perforatum has been reported to have a type I collagen production promoting effect, a hair-growing effect, and the like (patent documents 9 and 10). The compounds of the present invention represented by tranexamic acid formamide are reported to have skin roughness improving effect, whitening effect, antiallergic effect, and the like (patent documents 11, 14, and the like). Thyme extract has been reported to have antiallergic effect, antibacterial effect, and the like (patent documents 12 and 13). Cortex Phellodendri is also used as antidiarrheal, gastric drug, oral drug for dyspepsia and anorexia, or external drug for traumatic injury and sprain, and has also been reported to have antibacterial, antioxidant, skin barrier function improving effect, and pigment cell activating effect (patent documents 16 and 17). Eucalyptus extract has been reported for antibacterial and antioxidant effects (patent document 18). However, the effect of these substances on the regulation/improvement of the M1/M2 balance was first discovered by the inventors.

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 sometimes is not treatment by a doctor or a medical practitioner.

In the present specification, pigmentation refers to pigmentation of melanin or the like in dermis and epidermis. The present invention is effective in both dermis and epidermis, and is particularly expected to be a countermeasure against pigmentation of dermis in the sense that the improvement method is limited except for phagocytosis by melanocytes. By the application of the formulation of the present invention, spots, darkness, dark circles, etc. caused by pigmentation are ameliorated. Further, the present invention is effective for decoloring tattoos, and the like which are hardly erased when a dye is added by injecting the dye into the dermis layer.

M1 macrophages can be assayed using markers such as CD86, CD80, iNOS, and the like. M2 macrophages can be assayed using markers such as CD206, CD163, agr1, and the like. Examples of markers for the whole macrophages including M1 and M2 include CD11b and CD 68. Additionally or alternatively, it can be determined by quantifying M1-specific cytokines such as IL-1beta, TNF-alpha, 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 refer to a ratio of the number of M1 macrophages to the number of M2 macrophages, or may refer to 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. Since the ratio of M1 is high and the ratio of M2 is low in skin in a photoaged state and the ratio of M2 is high in melanophagocytosis, the adjustment/improvement of the M1/M2 balance may be to increase the ratio of M2 to M1 (the number of M2/the number of M1 and/or the mRNA amount of the M2 marker/the mRNA amount of the M1 marker). The rise may be, for example, a rise having a statistically significant difference (e.g., student's t test) that sets the significance level to 5%, and/or may be, for example, a rise 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, 100% or more. Or the adjustment/improvement of the M1/M2 balance may be such that the ratio of M2 to M1 (the number of M2/the number of M1, and/or the amount of mRNA of the M2 marker/the amount of mRNA of the M1 marker) falls within a certain range, for example, about 4/6 to about 9/1, about 5/5 to about 8/2, about 5/5 to about 7/3, or the like, may be close to the above range, may be maintained within the above range, or may be maintained in a steady state centering around the above range.

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

The elongation is calculated by the above formula 1. 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%. Any range of elongation such as 0.1% to 1.0%, 0.1% to 5.0%, 0.1% to 10.0%, 0.1% to 20.0%, 0.1% to 30.0%, 1.0% to 5.0%, 1.0% to 10.0%, 1.0% to 20.0%, 1.0% to 30.0%, 1.0% to 50.0%, 10.0% to 20.0%, 10.0% to 30.0% may be used.

The stretching speed means a speed (%/s) up to the maximum stretching rate (%) in 1 cycle. The recovery speed means a speed (%/s) at which the vehicle returns from the maximum stretch ratio to the non-stretch state. The stretching speed/recovering speed may be any speed such as 0.010%/s to 40%/s, 0.05%/s to 30%/s, 0.10%/s to 20%/s, 0.2%/s to 15%/s, 0.3%/s to 10%/s, etc. The stretch rate may be the same as the recovery rate or may be different.

The adjustment/improvement of the M1/M2 balance includes a step of applying weak physical stimulation to the skin, and the apparatus of the present invention is an apparatus for performing a step of applying weak physical stimulation to the skin, wherein the step is performed, for example, by a cycle at a frequency of 60Hz or less, and the cycle includes the steps of: (a-1) pressing the skin of the subject by 1 μm to 1000 μm; and (b-1) recovering the skin of the subject from the pressed state.

The term "pressing the skin to a depth of 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 μm to 1000 μm, 10 μm to 300 μm, 10 μm to 100 μm, etc. from the outermost surface of the skin.

The frequency means the number of cycles per 1 second when the cycle from the start of extension or compression to the non-extension or non-compression state is set to 1 cycle. In cycle 1, it may involve maintaining an extended or depressed state for a certain period of time and/or stopping in a non-extended or non-depressed state. For example, in 1 cycle, it may further comprise: (a') maintaining an extended or depressed state after (a) and before (b), or after (a-1) and before (b-1) 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') after (b) and before (a) of the next cycle, or (a-1) after and before (b-1), is stopped in a non-stretched or non-depressed state for 0 to 30 seconds, 0 to 20 seconds, 0 to 10 seconds, 1 to 20 seconds, 1 to 10 seconds. 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 10Hz. Any frequency range such as 0.001Hz to 60Hz, 0.01Hz to 60Hz, 0.001Hz to 10Hz, 0.01Hz to 10Hz, 0.1Hz to 60Hz, 0.1Hz to 10Hz, 0.5Hz to 60Hz, 0.5Hz to 50Hz, 0.5Hz to 10Hz, 0.5Hz to 5Hz, 0.5Hz to 1Hz, 0.001Hz to 0.01Hz, 0.001Hz to 0.1Hz, 0.001Hz to 1Hz, 0.01Hz to 1Hz, 0.1Hz to 1Hz, 1Hz to 60Hz, 1Hz to 10Hz, 1Hz to 5Hz, etc. may be used.

Examples of the commercially available beauty device, massage equipment, and the like include those using electromagnetic waves having a frequency of about 0.3 to 300MHz, such as RF waves, and those using ultrasonic waves having a frequency of about 1 to 7 MHz. The frequencies used by the method/apparatus of the present invention are extremely low compared to these frequencies. If the frequency of applying a strong force to the skin as in the conventional beauty device or the like, there is a risk of giving adverse effects such as redness, indentation, injury, pain, inflammation to 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 the stimulation is too strong if the stretch rate and frequency are too high, so by adjusting these values to appropriate values, gentle stimulation of the skin is more satisfactory.

In addition, conventionally, it is common knowledge of those skilled in the art that, in the cosmetic devices using a member such as a motor, only a frequency exceeding 60Hz can be selected based on a mechanical member of the motor, which is generally used in the field. When the frequency of 60Hz or less, for example, the frequency of 60Hz or less, 10Hz or less, or 1Hz or less, which is the limit of the cosmetic devices in the related art, is used, it is necessary to create a special machine. Further, even at such low frequencies, there is a fixed concept of "too weak" in exerting the effects of the present invention, and so far, little research has been conducted. However, the present inventors have tried to actually apply physical stimulation to the skin using a very low frequency in view of conventional technical knowledge, and as a result, surprisingly, even a gentle stimulation at such a low frequency exerts a good effect.

Although devices with low intermediate frequency are also commercially available in EMS equipment and the like, they are designed to act particularly in deep layers of muscle, subcutaneous fat and the like, and the influence on the skin surface layer as in the present invention is not clear. In addition, such a device may be accompanied by a crisp and tingling stimulus when a current flows even at a low frequency, unlike the present invention in which a gentle stimulus is applied to the skin. On the other hand, the present invention may be a cosmetic method by a simple method of directly applying stretching stimulus to skin without applying energy such as ultrasonic waves, electric current, and magnetic field. Further, the stretching stimulus having such a frequency is mild, and the adjustment/improvement effect of the M1/M2 balance is exerted as described in examples. Thus, 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 adversely affecting the skin.

The physical stimulus may be a physical stimulus generated by an instrument such as a beauty device, an experimental apparatus, a massage using a human hand or an instrument, or a movement of the face, or may be a physical stimulus realized by contact or non-contact. In one aspect, the mechanically generated physical stimulus may be applied to the skin using an instrument having a stimulus generating section that generates the physical stimulus and a stimulus applying section that applies the physical stimulus by contact or non-contact. Physical stimulation may be achieved by, for example, stretching, pressing, beating, rubbing, attracting the skin, and/or non-contact, for example, by applying shock waves to the skin with ultrasound, air pressure, water pressure, or the like, thereby displacing the skin. As the movement of the face, cheek swelling, eye opening, and the like can be performed. The massage may be performed by the subject itself or by an operator such as a beauty member using an implement such as a hand or a roller. However, the physical stimulus of the present invention is not limited to this range.

Examples of the device of the present invention include a cosmetic device having a skin contact portion that comes into contact with the skin of a user to apply the physical stimulus of the present invention. For example, the device may be composed of a grip portion and a skin stretching portion or a skin pressing portion. For example, the device shown in fig. 14 is designed such that the skin is stretched at a specific frequency and a specific stretching rate by contacting the skin with the skin contact portion.

Further, for example, the device of the present invention includes a power source that generates an electrical signal, a stimulation generating unit that converts the electrical signal from the power source into physical stimulation to generate physical stimulation, and a skin stimulating unit that receives the physical stimulation generated by the stimulation generating unit and applies the physical stimulation to the skin of the user.

For example, the device shown in fig. 14 includes a grip portion, a power source, a control portion for controlling physical stimulation, a stimulation generating portion, and a skin contacting portion including a skin stimulating portion and a skin fixing portion. The design is as follows: the user holds the grip portion to put the skin contact portion on the skin, fixes the skin by the skin fixing portion, and by operating the control portion, the electric signal from the power supply is converted into physical stimulus by the stimulus generating portion, the physical stimulus is transmitted to the skin stimulating portion, and the skin is fixed to the skin fixing portion while being stretched at a specific frequency and stretching rate by the skin stimulating portion. For example, the stimulus generating section may be a section driven by a motor or the like to convert an electric signal into a physical stimulus. The skin stimulation unit shown in fig. 14 applies an expansion stimulation to the skin, and may apply a pressing stimulation to the skin, for example.

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

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

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

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

Further, the number of these arbitrary cycles is 1 set, and the number of sets may be 1 to 100 sets, 2 to 50 sets, or 3 to 10 sets, for example.

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

The time interval between cycles or groups is not limited either. For example, the stretching or pressing stimulation may be performed in 1 or more groups alone, or 1 or more groups may be performed 1 time per day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 time per 1, 2, 3, 4 weeks, or the like, continuously or intermittently, periodically or aperiodically.

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

Alternatively, the modulation/improvement of the M1/M2 balance may be the administration of an M1/M2 balance modulating/improving agent or a composition containing the same. The inventors have found that the compounds of the present invention, extracts of Hypericum perforatum, thyme, phellodendron amurense, and/or Eucalyptus globulus as such M1/M2 balance adjusting/improving agents. Accordingly, the present invention also provides an M1/M2 balance adjusting/improving agent, an anti-photoaging agent, a pigmentation inhibitor, and a composition containing the same, which are composed of the compound of the present invention, an extract of hypericum perforatum, an extract of thyme, an extract of phellodendron amurense, and/or an extract of eucalyptus, or which contain the compound of the present invention, an extract of hypericum perforatum, an extract of thyme, an extract of phellodendron amurense, and/or an extract of eucalyptus as an active ingredient.

In the present specification, "the compound of the present invention" refers to 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) cyclohexane carboxamide (tranexamic acid carboxamide (C) ₉ H ₁₉ C ₁ N ₂ O)) or a salt thereof. The above-mentioned compounds can be synthesized by a method described in patent document 14, for example, and commercially available products can be used.

Hypericum perforatum (Hypericum erectum) is a perennial plant of Hypericum genus of Guttiferae. The extract of Hypericum perforatum used in the present invention is preferably an extract of aerial parts of Hypericum perforatum, but it contains an active ingredient in seeds, roots and the like, and thus any of 1 or 2 or more extracts thereof may be used. The extract of Hypericum perforatum can also be commercially available.

Thyme (Thymus) is a perennial plant of the genus thyme of the family Labiatae. In the present invention, various thymes such as ordinary thyme (t.vulgaris), lemon thyme (T.x citrus), asian thyme (t.serpyllum) and the like can be used. The thyme extract used in the present invention is preferably a whole plant extract of thyme, but any of 1 or 2 or more extracts of the above can be used because the extract contains an active ingredient in seeds, flowers, roots, etc. The extract of thyme can also be commercially available.

Cortex Phellodendri is crude drug obtained by drying bark of cortex Phellodendri (Phellodendron amurense) or cortex Phellodendri (Phellodendron chinense) of Rutaceae. The cortex Phellodendri or its extract can be prepared by conventional method, or commercially available product 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 eucalyptus leaves, but any of 1 or 2 or more of them may be used because the eucalyptus extract contains an active ingredient in seeds, flowers, bark, roots, etc. Eucalyptus extract can be prepared by conventional method, or commercially available product can be used.

In the case of using the extract, the extraction method is not particularly limited, but is preferably an extraction method using a solvent. In the extraction, plant bodies may be used as they are, but they may be pulverized into particles or powder for extraction, and extraction of the active ingredient may be performed under mild conditions in a short period of time with high extraction efficiency. The extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the kind of solvent, and the like. Typically, the temperature is set in a 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 solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, or standing may be performed without stirring, and ultrasonic waves may be applied.

The type of the solvent is not particularly limited, but is preferably water, a lower alcohol such as hydrous ethanol or ethanol, an organic solvent such as hexane or a 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 for extraction treatment. By adding the enzyme, the cell tissue of the plant can be disintegrated, whereby the extraction efficiency can be further improved. As the enzyme, a tissue maceration enzyme is preferably used. Examples of such enzymes include pectinase, cellulase, hemicellulase, alpha-amylase, and phytase. These enzymes may be used alone or in combination of at least 2 kinds.

By such 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 used after conventional purification treatments such as sterilization, washing, filtration, decolorization, deodorization, etc. In addition, the aqueous dispersion may be used after being concentrated or diluted as needed. The solvent may be used after all of it is volatilized to form a solid (dried product), or the dried product may be used after being redissolved in an arbitrary solvent.

In addition, since the extract contains the same active ingredient as the extract in the squeeze liquid obtained by squeezing the raw material plant, the extract may be replaced with the squeeze liquid.

However, the M1/M2 balance adjusting/improving agent and the composition are not limited to the above-described substances, and any known substances for adjusting/improving the M1/M2 balance, such as those described in patent documents 4 to 7, may be used. The route of administration may be any one selected from, for example, transdermal administration, oral administration, subcutaneous administration, transmucosal administration, intramuscular administration, etc., but photoaging is a phenomenon specifically observed in the skin at a portion where light is contacted, and therefore transdermal administration which can be administered at a specific position of the skin is also sometimes preferable. In addition, it is sometimes preferable that pigmentation occurring in the epidermis or dermis is applied transdermally so as to reach the epidermis or dermis from the skin. The adjustment/improvement of the M1/M2 balance may be, for example, differentiation induction into M2 macrophages, or any other method of adjusting/improving the M1/M2 balance may be used.

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

The preparation of the present invention may be formulated into a composition comprising the above-mentioned active ingredient in combination with 1 or more other ingredients such as excipients, carriers and/or diluents. The composition and the form thereof are arbitrary and may be appropriately selected depending on the conditions of the active ingredient, the use, and the like. The composition can be prepared by a conventional method in a prescription appropriately combined with an excipient, carrier, diluent, and/or the like and other ingredients depending on the dosage form thereof.

The preparation of the present invention can be used for 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 present invention can be mixed with various foods, drinks and feeds to be taken by humans and animals.

When the present invention is applied to external skin preparations such as cosmetics, drugs, quasi drugs, etc., the mixing amount (dry mass) of the plant substance or its extract can be appropriately determined according to the kind, purpose, form, use method, etc. For example, the compound of the present invention, the extract of Hypericum perforatum, the extract of thyme, the extract of Berberis corktree, and/or the extract of Eucalyptus globulus can be blended in the total amount of the cosmetic (in the case of the extract or crude drug, the dry mass conversion) in each of 0.00001% to 50%.

In addition to the above components, components used in 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 needed within a range that does not impair the effects of the present invention.

The external preparation for skin of the present invention can be used as a cosmetic, quasi-drug, etc. to be applied to the skin, and is particularly suitable for cosmetic application, and the formulation is not limited as long as it can be applied to the skin, and any formulation such as a solution, a solubilizing, an emulsifying, a powder dispersing, a water-oil two-layer, a water-oil-powder three-layer, an ointment, a cosmetic, a gel, an aerosol, etc. can be applied.

When the preparation of the present invention is used as a cosmetic, it can be used in the form of a lotion, an emulsion, a foundation, a lipstick, a cleansing cream, a massage cream, a mask, 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 in the formulation and composition of the present invention is not limited to the above-mentioned dosage form and form. Furthermore, 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, etc.

The subjects to which the methods, devices, formulations and compositions of the present invention are applied may be subjects who objectively or subjectively identify skin photoaging and/or pigmentation (e.g., dermal pigmentation), or may be subjects who wish to prevent skin photoaging and/or pigmentation. For example, it may be an object determined that the M1/M2 balance is broken down. In embodiment 1, the skin photoaging degree and/or the pigmentation degree (for example, dermal pigmentation degree) may be determined to be high by using the M1/M2 balance in the skin as an index. Alternatively, the skin may be subject to a phenotype in which the photoaging specificity of the skin is concerned, such as spots, wrinkles, sagging, or the like, or subject to skin or pigmentation such as spots, darkness, marks of birthmarks, tattoos, or the like. Spots, wrinkles, sagging, dullness, marks of birthdays, tattoos, etc. can be determined by visual judgment using well-known indicators.

In addition, by the present invention, there is also provided a screening method of an anti-photoaging agent, 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 agent is administered is improved as compared with that before administration of the agent, the agent is evaluated as having an anti-photoaging and/or pigmentation inhibitory effect. Also provided is a method of evaluating anti-photoaging and/or pigmentation inhibiting cosmetic treatments comprising the steps of: performing cosmetic treatment on the skin sample; measuring the M1/M2 balance in the skin sample before and after performing the cosmetic treatment; when the M1/M2 balance in the skin sample subjected to the above-mentioned cosmetic treatment is improved as compared with that before the treatment is performed, the above-mentioned treatment is evaluated as having an anti-photoaging and/or pigmentation inhibiting effect. The method of the present invention enables screening of whether or not a candidate drug or cosmetic treatment has an anti-photoaging and/or pigmentation inhibiting effect, and enables product development and new skin care. That is, by the present invention, an anti-photoaging agent and/or a pigmentation inhibitor and an anti-photoaging and/or pigmentation inhibiting cosmetic treatment for preventing and/or improving photoaging and/or pigmentation by adjusting the M1/M2 balance are provided. The pigmentation may be dermal pigmentation.

The biological sample may be any sample such as a skin sample and an immune cell sample. The skin sample may be a collected skin sample, for example, a skin sample collected from an animal such as a human being in an isolated state, or may be cultured skin cells, for example, in an in vitro state such as single-layer or multi-layer cultured cells, cultured keratinocytes or cultured fibroblasts. Alternatively, an artificial skin sample such as a 3D skin model may be used. The immune cell sample may be an immune cell present in the collected skin, an immune cell in blood after collecting blood infiltrated into the skin, or a cultured immune cell. The 3D skin model is not limited, but may be produced by the methods described in patent document 8 and non-patent documents 10 to 12, for example, or may be produced by a method that is changed to facilitate measurement of the M1/M2 balance. The biological sample is not limited as long as the M1/M2 balance can be measured.

For example, a screening method for an anti-photoaging and/or pigmentation inhibitor can be carried out by differentiating immune cells such as THP-1 into M0, adding a reagent, culturing for a certain period of time, and then collecting the culture supernatant, and quantifying the amount of M2-specific cytokines such as IL-10 by an assay such as ELISA. In such a screening method, cells differentiated into M2 by adding IL-4, IL-13, or the like can be used as positive controls, and 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 production ability, if IL-10 in the supernatant increases after M0 differentiation compared with a control (control) cultured in a medium alone, it can be said that differentiation into M2 is performed, and an agent can be regarded as an "improver" in which the presence or absence of an agent in the presence of IL-4, IL-13 or the like can be compared to evaluate whether or not a difference in the amount of IL-10 has occurred.

In the present specification, the candidate drug means a drug which is investigated for the effect of anti-photoaging and/or pigmentation inhibition, and includes a drug in a development stage in addition to a drug already sold as a product, and may be a drug selected for use as a cosmetic in development of a cosmetic.

The cosmetic treatment is not particularly limited, and any treatment that is considered to be effective in suppressing photoaging and/or pigmentation, such as application of a cosmetic containing, for example, the preparation of the present invention or other components, is included. The term "cosmetic" refers to, for example, a cosmetic liquid, an emulsion, a cosmetic liquid, a cream, a foundation, etc., and is not limited thereto, but means to include all substances applied to the skin, for example, sunscreens, etc., even if the improvement of the skin condition is not a direct object. Alternatively, the cosmetic treatment may be, for example, physical stimulation such as stretching stimulation, pressing stimulation, massage, etc. applied to the skin. Cosmetic treatments may be one treatment or may be continuous treatments from days to weeks. Cosmetic treatments may be performed by individuals, or may be performed in beauty parlors, cosmetic shops, beauty salons, and the like.

The present invention also provides a screening kit for an anti-photoaging and/or pigmentation inhibitor, which comprises an antibody for detecting any marker such as CD86, CD206, CD163, etc., for example, for measuring the number of M1 and M2, and a reagent for measuring the amount of mRNA of these markers, for example, a reagent for measuring the M1/M2 balance. The invention further provides the following methods, systems and devices.

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

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

A system for evaluating photoaging and/or pigmentation degree of skin of a subject, having: a database part for storing data about a preset reference value of M1/M2 balance of skin; a data input unit for inputting data on M1/M2 balance of the skin of the subject; a calculation unit for comparing the reference value stored in the database unit with the data of M1/M2 balance of the skin of the subject inputted through the data input unit, and calculating the reference value; an evaluation unit configured to evaluate 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 of the evaluation 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 concerning M1/M2 balance of the subject's skin, the device comprising a calculation unit for calculating photoaging and/or pigmentation degree of the subject if data concerning M1/M2 balance of the subject's skin is inputted, the calculation unit comprising a neural network which has been learned by performing a machine learning process using teacher data so as to calculate the estimated photoaging and/or pigmentation degree when data concerning M1/M2 balance of the skin is inputted.

By the methods, systems, devices of the present invention, objective photoaging and/or pigmentation levels based on M/M2 balance can be determined.

Examples

The present invention will be described in further detail by examples. In addition, the present invention is not limited thereto.

Experiment 1: tissue staining

The skin of the young person (20 to 30) and the aged person (60 to 70) with photoaging, which are derived from the white race, shown in fig. 1a, was frozen into a slice, and after slicing, stained with the marker of 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 positive for the antibody doublets of (1) to (3) up to 200 μm immediately below the epidermis were counted as macrophages (FIG. 1 d). In addition, in order to study the relationship between M1, 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 fig. 1b, c, the graph of the count results is shown in fig. 1d, and the results of double staining with M1, M2 macrophage antibodies and anti-procollagen antibodies are shown in fig. 1e to the left. As shown in fig. 1b, c, d, M1 macrophages were observed in a large amount for the aged persons who had developed photoaging, while M2 macrophages were reduced. More specifically, as shown in fig. 1b, M1 macrophages are present only around blood vessels in the elderly, while they are scattered throughout tissues in the elderly. As shown in fig. 1c, M2 macrophages are present in the whole tissue for the low-age population, while the number of M2 macrophages is reduced for the high-age population. Further, as shown in FIG. 1d, the total number of macrophages (the number of M1+M2) did not change between the low-age and the high-age persons, and only the M1/M2 balance changed. For the young, the ratio of M2 to M1 (number of M2/number of M1) is in the range of about 5/5 to about 7/3, whereas for the young, the ratio of M2 to M1 is drastically reduced, and the M1/M2 balance is greatly collapsed, which is significantly different from the value of the young.

Further, as shown in the upper left 2 photographs of fig. 1e, the positions of M1 macrophages and procollagen were shifted regardless of the low-age and high-age persons, and the positions of M2 macrophages and procollagen were observed to be identical as shown in the lower left 2 photographs. Therefore, as shown in the schematic diagram of the right drawing of fig. 1e, it is suggested that M1 may act on fibroblasts to promote collagen destruction and M2 may promote collagen production.

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

According to the method described in non-patent document 1, differentiation is induced into M1 and M2 macrophages using THP-1, which is an established cell line of human origin. 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 further added to stimulate for 24 hours to differentiate into macrophages. Further, 100ng/mL LPS (sigma) and 20ng/mL IFNγ (R & D) were added to stimulate for 24 hours when differentiating into M1, and 20ng/mL IL-4 (R & D) and 20ng/mL IL13 (R & D) were added to stimulate for 24 hours when differentiating into M2. When observed with a microscope, the change in morphology was confirmed as shown in FIG. 2 b.

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

As shown in FIG. 2c, it was shown that macrophages differentiated by the method described in experiment 2 produced inflammatory cytokines (IL-1 beta, TNF-alpha) characteristic of M1 and anti-inflammatory cytokines (IL-10) characteristic of M2, respectively. Further, these differentiation-induced macrophages showed an increase in CD86, CD206 expression as a surface marker of M1, M2 macrophages, respectively. From these results, it was confirmed that differentiation induction was successful. Thus, M1, M2 macrophages and undifferentiated M0 macrophages differentiated by the method of experiment 2 were used in the following experiments 3, 4.

Experiment 3: experiment of addition of M1 and M2 macrophage supernatant to fibroblast

As shown in FIG. 3a, THP-1 was differentiated into M1 and M2 by the same method as in experiment 2 or in an undifferentiated state of M0, the supernatant was removed, washed 1 time with PBS, and then cultured in a medium for 48 hours. These supernatants containing secretions 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 addition of the supernatant, the fibroblasts were cultured for 72 hours and the amount of pre-collagen 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, after nuclei of cells were stained with DAPI using β -gal as an aging index, β -gal in the cells was stained with Senescence Detection Kit (aging test kit, abcam) (fig. 3 d), and the numbers of β -gal positive cells and DAPI positive cells were counted (fig. 3 e).

Further, in order to investigate the contribution degree of M1 macrophages and M2 macrophages to melanin production, supernatants of the respective macrophages were added to fibroblasts and cultured in the same manner as described above. Then, fibroblasts were collected and mRNA was collected, and real-time PCR was performed using probes for HGF, ET1, bFGF, IL-1alpha, SCF, and clusterin to quantify the respective mRNA expression amounts (FIG. 3 f).

The results are shown in FIGS. 3b to 3 f. FIG. 3b shows the amount of pre-collagen after 72 hours of culture by adding the supernatant of each macrophage (M1, M2) to the fibroblasts. FIG. 3c shows the local presence of collagen and hyaluronic acid in fibroblasts after 72 hours of culture with addition of supernatants of macrophages (M1, M2). As can be seen from fig. 3b, c, M1 significantly inhibited collagen production. FIG. 3d shows intracellular β -gal in fibroblasts after 72 hours of culture with addition of supernatants of macrophages (M1, M2). From FIG. 3d, it is suggested that for M1, beta-gal positive cells increase, promoting aging, on the other hand, M2 inhibits 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 graph of FIG. 3e, M2 not only inhibits cell aging, but also promotes cell proliferation. The left panel of FIG. 3e 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, while M2 has an aging inhibiting and cell proliferation effect. Further, as shown in FIG. 3f, it is also known that mRNA expression of a melanogenesis-related factor acts in a direction to increase melanogenesis by M1, and acts in a direction to inhibit melanogenesis by M2. As reported in non-patent document 6, it is known that fibroblasts secrete factors such as SCF and HGF by optical stimulation such as UV, and cell death is caused. Further, as reported in non-patent documents 7 to 9, fibroblasts secrete factors such as HGF, ET1, bFGF, SCF, clusterin, etc. by light stimulation, and act directly or indirectly on melanocytes to produce melanin. Thus, it was suggested that cell death and melanogenesis caused by the light stimulation were caused by the breakdown of the balance of M1/M2.

Experiment 4: experiments on the addition of M1 and M2 macrophage supernatant to low-and old-age fibroblasts

Next, in order to confirm whether the anti-aging effect of M2 macrophages is also useful for aged fibroblasts, that is, whether there is a rejuvenation effect, the effects of the macrophage supernatant of the fibroblasts due to the difference in age were investigated by adding M1, M2 macrophage supernatants to the low-age and old cells.

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

FIG. 4a shows a staining pattern of β -gal. FIG. 4b shows a graph obtained by plotting the results of the number of β -gal positive cells of the low-age-derived fibroblasts and the aged-derived fibroblasts and the total number of cells per well. As is clear from the lower panel of FIG. 4a, even in the case of aged cells, the beta-gal positive cells were significantly reduced by adding M2 supernatant, and aging was inhibited. It is also clear from fig. 4b that aging is promoted by M1 and aging is suppressed by M2 regardless of age. This is supported by the conclusion that cells added with M1 supernatant produced significantly lower amounts of collagen than M2 supernatant, regardless of age, as shown in figures 4c, d.

Experiment 5: co-culture experiments of M1 macrophage and M2 macrophage and fibroblast from neonatal foreskin

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

The results are shown in fig. 5. For the neonatal prepuce-derived germ cells, collagen was also reduced if M1 supernatant was added, on the other hand, collagen was largely observed if M2 was added. That is, even in the case of the low-age cells, if the M1/M2 balance is disrupted, collagen is affected.

Experiment 6: effects of breakdown of M1/M2 balance using 3D skin models

To study the effect of M1/M2 macrophages on epidermal cells and fibroblasts, three 3D skin models formed of a 3-layer structure as described in the following table were produced.

TABLE 1

The 3D skin model was made as followsIs carried out. Human dermal fibroblasts (0.2X10) were seeded in cell culture inserts (. Phi.12 mm, average pore size of porous membranes: 0.4 μm) ⁶ And (c) were cultured for 1 week using 200. Mu.M ascorbic acid-2-magnesium phosphate (APM), 10% FBS-DMEM, and 1 exchange medium for 2 days. On the above, a 0.5% type i collagen-10% fbs-DMEM solution containing human dermal fibroblasts was dispensed into a control model, and the above solutions of M1 macrophages and M2 macrophages differentiated by the method of experiment 2, to which 30,000 cells were further added, were dispensed into an M1 model and an M2 model, respectively, to prepare collagen gels on human dermal fibroblasts, and cultured for 1 to 5 days.

Further, epidermal keratinocytes dispersed in Humedia-KG2 (Kyoto) medium were formed to be 5X 10 ⁵ The cells/well were inoculated onto the collagen gel, and the culture medium in which Humedia-KG2 and 10% FBS-DMEM were mixed at 1:1 and 200. Mu.M APM was added to the same liquid level as the inside was added to the outside of the insert and cultured for 3 days.

Then, the medium in the insert or glass ring was removed, 200. Mu.M APM, 10. Mu. M N-hydroxy-2- [ [ (4-methoxyphenyl) sulfonyl ] 3-picolyl) amino ] -3-methylbutanamide hydrochloride (CGS 27023A (MMP inhibitor)), 10. Mu.MBIPBIPU (heparanase inhibitor) was added to the medium for skin model (prepared by mixing 10% FBS-DMEM and Humedia-KG2 EGF (-) at 1:1 to Ca1.8mM) on the outside until the bottom surface of the insert was high, and the inside of the insert was subjected to gas-liquid interface culture in a state of being exposed to air. 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 presence of M1 and M2 macrophages was confirmed by staining 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 (FIG. 6). Then, the cells stained with anti-p 21 antibody (abcam) and DAPI (VECTOR) were used as the total number of cells in each of the epidermal cell layer and the fibroblast layer, and the number of cells also stained with the anti-p 21 antibody was counted as the number of p21 positive cells. The ratio of the number of p21 positive cells to the total number of cells was determined by the following formula.

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, p21 positive cells increased in the M1 model (M1) and decreased in the M2 model (M2) relative to the control model (cont) in both the upper epidermal cell layer and the lower fibroblast layer, which are in contact with the intermediate layer. This trend is consistent with experiment 3, which used a monolayer culture. Thus, M1 macrophages are also suggested to have aging and cell death promoting effects in 3D skin models that are closer to human skin, and on the other hand, M2 macrophages have aging and cell death inhibiting effects.

Experiment 7: experiment on irradiation with sunlight on ex vivo model of human skin

Native skin (skin collected from 38 year old female) manufactured by Genoskin was cultured with the culture broth attached after arrival for 1 day. The next day, an optical filter was placed in a 1000W solar simulator manufactured by Oriel Co., ltd, and only UVA and UVB were subjected to 11.5J/cm ² Irradiation, the culture was continued with the attached culture medium (irradiation (+). As a control, a sample not subjected to irradiation (-)) was used. Samples were collected 5 days after irradiation, and M1 macrophages, M2 macrophages, and total macrophages were stained as in experiment 1, and the numbers were counted and plotted.

The results are shown in fig. 9. If the solar simulator is illuminated, the M1 number is most increased. On the other hand, the increase in the number of M2 is very small compared with the case of M1. That is, it was found that the balance of M1/M2 was broken down by the optical stimulus, and the ratio of M1 was increased.

Experiment 8: stretching stimulus experiment on ex vivo model using human skin

Next, a method of adjusting or improving the M1/M2 balance was studied.

Sample: nativeSkin (skin after collection from 38 years old women) (6-hole size, diameter about 2 to 2.5 cm) manufactured by Genoskin Co., ltd.

Stretching conditions: an stretching tool having grip portions for gripping both ends of skin in the hole as shown in fig. 11 was produced, and the grip portions were stretched to stretch the skin. The hole to which the tissue piece was added was horizontally placed, and the skin was stretched from both ends by operating the grip part, and stretching was performed at a rate of 10% to a stretching rate of 10% as shown in fig. 10, and the sample was returned to the original non-stretched state at a return rate of 10% per second. This was set to 1 cycle, and the total of 90 cycles was performed over 30 minutes. The 90 cycles were set to 1 group, and a suspension time of 30 minutes to 1 hour was set between the groups, and a total of 3 groups (total 270 cycles) was performed over 3 hours. As a control, a sample (control) not subjected to stretching stimulation was used.

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

The results are shown in fig. 12. For the samples to which the stretching stimulus was applied, the total number of macrophages did not change much from the number of M1, but the number of M2 was very increased, compared to the control. That is, it is known that the balance of M1/M2 is improved by the stretching stimulus.

Experiment 9: screening of anti-photoaging and/or pigmentation agents for preventing and/or improving photoaging and/or pigmentation by modulating or improving the M1/M2 balance

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

As screening-target agents, a total of 19 ingredients including an extract of Hypericum perforatum, an extract of thyme, and tranexamic acid formamide (N-methyl-trans-4- (aminomethyl) cyclohexane formamide) were studied. The herba Hyperici Erecti extract is obtained from aerial parts of herba Hyperici Erecti purchased from herba Hyperici Erecti コ. Thyme extract is an extract of whole plants of thyme in asia purchased from the flourishing industry. The tranexamic acid carboxamide is synthesized by the method described in patent document 14. The Hypericum perforatum extract was dissolved in 50% ethanol, thyme extract was dissolved in butanediol, and tranexamic acid formamide was dissolved in PBS.

THP-1 cells identical to experiment 2 were used as they are in an undifferentiated state of M0 and cultured overnight at 37 ℃. The screening agents were added to the culture medium so that the amount of the herba Hyperici perforati extract was 0.1%, the amount of the thyme extract was 0.1%, and the amounts of the tranexamic acid formamide were 0.06% and 0.03%, respectively, and the culture medium was incubated at 37℃for two months with the same amount of the solvent. The cells were collected to extract RNA, and the expression levels of CD86 and GAPDH were quantified by real-time PCR to determine CD86/GAPDH. Agents that have reduced CD86/GAPDH values reproducibly compared to the control (cont) were explored as inhibitors of M1 differentiation.

The results are shown in fig. 13. From fig. 13, it is shown that CD86/GAPDH values are significantly reduced if the extracts of hypericum perforatum, thyme, and tranexamic acid formamide are added, and have an M1 differentiation inhibitory effect, suggesting that they can be used as an M1/M2 balance regulator.

Experiment 10: screening of anti-photoaging agents to prevent and/or improve photoaging and/or pigmentation by modulating or improving the M1/M2 balance

For screening more drugs, a study was made on 8 kinds of total components including phellodendron extract and eucalyptus extract by the same method as in experiment 9. The cortex Phellodendri extract is dry extract of cortex Phellodendri of Rutaceae, and the Eucalyptus extract is extract of folium Eucalypti Globueli. The cortex Phellodendri extract was dissolved in butanediol to give 0.01%, and the Eucalyptus extract was dissolved in 50% ethanol to give 0.1%. Their solvents were used in the control.

By the same method as in experiment 9, the addition of the drug, the recovery of cells, the extraction of RNA, and the measurement of CD86/GAPDH were performed, and the drug having a reduced CD86/GAPDH value compared with the control (cont) was searched for as an M1 differentiation inhibitor. The results are shown in fig. 15. Fig. 15 shows that in addition to the agents of experiment 9, phellodendron and eucalyptus extracts can be used as the M1/M2 balance adjusting/improving agent.

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

In order to examine the contribution degree of M1 macrophages and M2 macrophages to collagen degradation, except for experiment 3, the supernatant of each macrophage was added to fibroblasts and cultured in the same manner as in experiment 3, and mRNA was recovered after 72 hours. As an index of collagen degradation, real-time PCR was performed using probes for MMP-1, MMP-2, and IL-1β (Taqman probe manufactured by Applied Bioosystems Co., ltd.) to quantify the respective mRNA expression amounts (FIG. 16 a).

Further, in order to investigate the contribution degree of M1 macrophages and M2 macrophages to collagen production/maturation, the supernatants of the respective macrophages were added to fibroblasts in the same manner as in experiment 3, and mRNA was recovered after 72 hours. As an index of collagen production/maturation, real-time PCR was performed using COL1A1, COL1A2, HSP47, and ADAMTS-2 probes (Taqman probes manufactured by Applied Bioosystems Co., ltd.) to quantify the respective mRNA expression amounts (FIG. 16 b).

From fig. 16a, b, it was confirmed that M1 contributes to collagen degradation, while M2 contributes to collagen production/maturation.

Experiment 12: collagen breakdown and production in ex vivo models of human skin using young and elderly people

The skin of the sideburns of the aged (20 to 30) and the elderly (60 to 80) shown in fig. 17a was frozen and sliced in the same manner as in experiment 1, and positive cells were counted by the same method as in experiment 1 using an anti-procollagen antibody (millipore), an anti-fragmented collagen (adinogen), and CD68, CD11b, CD206, and CD86 antibodies, which stain the 3/4 side collagen-cleaved fragments.

The results are shown in fig. 17b, c, d. For the advanced age group, the proportion of M1 macrophages is high and the proportion of M2 macrophages is low compared to the young age group. However, regardless of age, there is a tendency that the number of macrophages showing 3/4 collagen positivity is large compared with fibroblasts showing 3/4 collagen positivity, and the number of M2 macrophages showing 3/4 collagen positivity is large compared with M1 macrophages showing 3/4 collagen positivity.

Experiment 13: in vitro (in vitro) experiments showing the uptake capacity of melanin in the dermis

M1 macrophages and M2 macrophages obtained by differentiating THP-1 cells were differentiated by the same method as in experiment 3. To each of the differentiated M1 macrophages, M2 macrophages, and fibroblasts (manufactured by kukukukukukuku corporation), a Melanin solution (Melanin-Bioreagent, synthetic, suitable for cell culture) was dissolved in PBS and added at a concentration of 0.02% W/V. After washing the cells with PBS 24 hours later, the cells were photographed with a microscope and recovered. The number of cells of the recovered cells was quantified by measuring the amount of melanin using alamar blue (Life Technologies) as follows.

Quantification of melanin:

after addition of Almarblue adjusted to 1:10 with the medium of the respective cells (macrophage: RPMI1640, fibroblast: 1 DMEM), the cells were cultured at 37℃for 30 minutes. Then, 100 ul/well of the supernatant was collected, and fluorescence was measured at excitation/emission of 544nm/590nm by using an Assent (Thermo Co.). After the measurement, the sample was washed with PBS, and incubated at room temperature for 3 hours after 1M NaOH was added, and the sample was completely dissolved. The cell solution was measured at OD475 using powercam HT (DS PHARMA BIOMEDICAL).

The results are shown in fig. 18a to c. In the figure, the unit "melanin/alamar blue" is a relative value ((2)/(1)) of the intensity of fluorescence ((1)) measured at 544nm/590nm for the supernatant and the intensity ((2)) measured at 475nm for the solution in which melanin was dissolved even after the cells were dissolved, and shows the melanin content per cell. From these figures, M2 phagocytoses very much melanin. This trend was also observed after 24 hours, but the cell culture was continued to be observed until after 5 days, and as a result the difference became more clear (fig. 18 c).

Experiment 14: ex vivo experiments showing the uptake capacity of melanin in dermis

Since it is known from experiment 13 that M2 macrophages engulf more melanin than fibroblasts and M1 macrophages, the dermis layer of human skin counted in experiment 13 was observed ex vivo using LSM880 (manufactured by chemical company). In contrast to the fact that M2 macrophages are darkened with melanin when both the elderly and the low-age are observed in the bright field, no melanin is present in the vicinity of M1 macrophages. From this, it was found that M2 macrophages take in more melanin than fibroblasts and M1 macrophages in dermis ex vivo, and the number thereof was counted and plotted (fig. 19).

According to fig. 19, the number of M2 macrophages that ingest melanin was greater than that of M1 macrophages for both old and young, and age differences were less observed in this trend. From this result, it is suggested that melanin taken into the dermis is M2 macrophages, and thus if the M1/M2 balance is adjusted by increasing the proportion of M2 macrophages, pigmentation in the dermis can be prevented and/or improved.

Since the increase in the proportion of M1 by photoaging (experiment 7) and the increase in the collagen content of skin (experiments 11 and 12) caused by the high proportion of M2 tend to increase the pigment phagocytosis (experiments 13 and 14), it was suggested that the compounds of the present invention such as tranexamic acid formamide, thyme extract, cortex Phellodendri and eucalyptus extract, which were screened as inhibitors of M1 differentiation, and the physical stimulation of the present invention having an effect of improving the balance of M1/M2 have a high photoaging and/or pigmentation inhibiting effect.

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

Industrial applicability

By the present invention, prevention and/or improvement of photoaging and/or dermal pigmentation, evaluation of photoaging and/or dermal pigmentation degree, anti-photoaging and/or dermal pigmentation inhibitor and exploration of anti-photoaging and/or dermal pigmentation inhibition cosmetic treatment can be performed.

Claims

1. An agent for anti-photoaging and/or dermal pigmentation inhibition for preventing and/or ameliorating photoaging and/or dermal pigmentation by increasing the ratio of M2 macrophages relative to M1 macrophages.

2. The agent for anti-photoaging and/or dermal pigmentation inhibition according to claim 1, comprising tranexamic acid formamide, an extract of hypericum perforatum, an extract of thyme, an extract of phellodendron amurense, and/or an extract of eucalyptus.

3. An agent for increasing the ratio of M2 macrophages relative to M1 macrophages comprising tranexamic acid formamide, an extract of hypericum perforatum, an extract of thyme, phellodendron amurense, and/or an extract of eucalyptus.

4. The agent according to any one of claims 1 to 3, wherein the ratio of M2 macrophages to M1 macrophages is the number of M2 macrophages/the number of M1 macrophages, or the amount of M2 macrophages/the amount of M1 macrophages.