WO2023063118A1

WO2023063118A1 - Inducer or activator of m2 or m2-like macrophages, method for inducing or activating m2 or m2-like macrophages, composition for preventing and/or ameliorating dermal pigmentation, and method for preventing and/or ameliorating dermal pigmentation

Info

Publication number: WO2023063118A1
Application number: PCT/JP2022/036772
Authority: WO
Inventors: 聡堀場
Original assignee: 株式会社資生堂
Priority date: 2021-10-14
Filing date: 2022-09-30
Publication date: 2023-04-20
Also published as: CN118043048A

Abstract

The present invention provides: an inducer or activator for M2 or M2-like macrophages, comprising as an active ingredient nicotinamide or a pharmaceutically acceptable salt thereof; and a method for inducing or activating M2 or M2-like macrophages that uses said inducer or activator.

Description

An agent for inducing or activating M2 or M2-like macrophages, a method for inducing or activating M2 or M2-like macrophages, a composition for preventing and/or improving dermal pigmentation, and preventing and/or dermal pigmentation how to improve

The present invention provides an agent for inducing or activating M2 or M2-like macrophages, a method for inducing or activating M2 or M2-like macrophages, a composition for preventing and/or improving dermal pigmentation, and dermal pigmentation. It relates to preventive and/or ameliorative methods.

The aging phenomenon of human skin can be broadly divided into "natural aging" and "photoaging". Photoaging is a phenomenon that is recognized site-specifically to light exposure, and is skin-specific. In photoaging, the production of active oxygen and damage to DNA in cells are induced by the effects of UV rays and the like, which is thought to be the cause of phenotypes such as wrinkles and sagging due to damage to dermal fibrous tissue. For example, photoaging of the skin causes phenomena such as reduction of collagen fibers and degeneration of elastin elastic fibers. In addition, melanocytes are also damaged, and a large amount of melanin pigment is produced, which causes spots and the like.

In addition, pigmentation such as spots and dullness is caused by the accumulation of melanin produced by melanocytes in the basal layer of the epidermis. Melanin is normally present in the epidermis and basal layer, but since the epidermis turns over in a relatively early cycle, such melanin is easily excreted. On the other hand, melanin may be present in the dermis layer for reasons such as melanin dropping into the dermis through gaps in the basement membrane. Since the turnover cycle of dermal cells is much slower than that of the epidermis, such melanin is often accumulated without being excreted. For these reasons, amelioration of dermal pigmentation is very difficult.

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

As a cosmetic method for improving pigmentation in the dermis, Non-Patent Document 6 proposes strengthening the basement membrane to prevent melanin from sinking into the dermis.

In addition, it has been suggested that inflammation plays a role in the photoaging phenomenon of the skin, and many anti-inflammatory agents have been developed. Patent Document 3 discloses an anti-aging cosmetic composition containing a compound that induces autophagy activation along with increased adiponectin expression. It has also been suggested that phagocytosis by macrophages is used to improve dermal pigmentation. In Patent Document 8, dermal blemishes are caused by attracting macrophages to fibroblasts that have taken up melanin that has fallen into the dermis and phagocytizing them. It discloses preventive/improving agents.

　Macrophages are cells that are localized in various tissues in the body, trigger immune responses against foreign substances and pathogens, and are known to be involved in inflammation. Macrophages are differentiated into M1 type and M2 type from undifferentiated M0 macrophages (hereinafter sometimes abbreviated as M0). M1 macrophages (hereinafter sometimes abbreviated as M1) are known as inflammatory type, and M2 macrophages (hereinafter sometimes abbreviated as M2) are known as repair type (anti-inflammatory type). 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 4-6, Non-Patent Documents 1-5).

Previously, the present inventors discovered that the balance of these M1 macrophages and M2 macrophages (M1/M2 balance) is specifically disturbed in areas of the skin exposed to light and where pigmentation occurs. We have found that regulation, especially increasing the ratio of M2 macrophages to M1 macrophages, is particularly important in preventing and improving photoaging and pigmentation in the dermis (Patent Document 11). Therefore, the present inventors have searched for substances that can prevent and improve photoaging and/or dermal pigmentation using the M1/M2 balance as an index. It was also found that the eucalyptus extract has that effect.

Japanese Patent No. 5657723 Japanese Unexamined Patent Application Publication No. 2017-203004 JP 2018-177805 A Japanese translation of PCT publication No. 2014-504629 JP 2015-140334 A Japanese Patent No. 6178088 Japanese Patent No. 6273304 JP 2018-072098 A Japanese Patent No. 4781842 WO2012/057123 WO2020/213743 JP-A-3-20207

An object of the present invention is to provide a new agent or method capable of inducing or activating M2 or M2-like macrophages, which has the effect of preventing and/or improving photoaging and/or dermal pigmentation.

The present inventors have extensively searched for an agent for preventing and/or improving photoaging and/or dermal pigmentation, and found that nicotinamide or a mixture of nicotinamide and succinicin extract reduces macrophages to M2. or can be induced or activated into M2-like macrophors. Based on such discoveries, the present invention has been developed. That is, the present invention includes the following aspects.

[1] An agent for inducing or activating M2 or M2-like macrophages, comprising nicotinamide or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] The inducing or activating agent according to item 1, which prevents and/or improves pigmentation in the dermis via the M2 or M2-like macrophages.
[3] The inducing or activating agent according to item 1 or 2, which suppresses glycolysis of macrophages.
[4] The inducing or activating agent according to any one of items 1 to 3, which promotes aerobic respiration of macrophages.
[5] The inducing or activating agent according to any one of Items 1 to 4, further comprising a cakushinin extract.

[6] A composition comprising nicotinic acid amide or a pharmaceutically acceptable salt thereof and a cinnamon extract.

[7] A method for inducing or activating M2 or M2-like macrophages in a subject in need thereof, comprising:
A method comprising applying nicotinamide or a pharmaceutically acceptable salt thereof to said subject to induce or activate M2 or M2-like macrophages.
[8] The method of item 7, wherein pigmentation in the dermis of the subject is prevented and/or improved via the M2 or M2-like macrophages.
[9] The method according to item 7 or 8, wherein glycolysis of macrophages is suppressed.
[10] The method according to any one of items 7 to 9, which promotes aerobic respiration of macrophages.
[11] The method according to any one of items 7 to 10, further comprising a cakushinin extract.
[12] A method for preventing and/or improving dermal pigmentation in a subject in need thereof, comprising:
A method comprising applying to said subject a composition comprising nicotinamide or a pharmaceutically acceptable salt thereof and a cinnamon extract.

[13] Use of nicotinamide or a pharmaceutically acceptable salt thereof for the manufacture of a composition for inducing or activating M2 or M2-like macrophages.
[14] The use according to item 13, which prevents and/or improves pigmentation in the dermis of a subject via the M2 or M2-like macrophages.
[15] Use according to item 13 or 14, which suppresses glycolysis of macrophages.
[16] Use according to any one of items 13 to 15, which promotes aerobic respiration of macrophages.
[17] Use according to any one of items 13 to 16, further comprising a cakushinin extract.

[18] Use of nicotinic acid amide or a pharmaceutically acceptable salt thereof and cinnamon extract for the manufacture of a composition for preventing and/or improving pigmentation in the dermis.

By applying the present invention, it becomes possible to induce or activate M2 or M2-like macrophages, which have the effect of preventing and/or improving photoaging and/or dermal pigmentation.

FIG. 1a shows a schematic of the induction method from THP-1 to M0, M1 and M2 macrophages. FIG. 1b is a photomicrograph of M0, M1 and M2 macrophages induced from THP-1. The upper diagram of FIG. 1c is a graph showing gene expression levels of cytokines (IL-1beta, TNF-alpha, IL-10) produced by M0, M1 and M2 macrophages induced from THP-1. The lower figure in FIG. 1c is a graph showing the mRNA expression levels of cell surface markers (M1: CD86, M2: CD206) of M1 and M2 macrophages. Corrected by the expression level of GAPDH mRNA, it is shown as a relative value (%) with the case of M0 as 100%. FIG. 2 shows the expression levels (marker/GAPDH value) of each marker of M1 and M2 when each concentration of Hakushinin extract (0.3 ppm, 1.0 ppm, 3.0 ppm) was added, compared with the control (Hakushinin hydrated It is a graph showing a relative value (%) when no decomposition: control) is taken as 100%. Fig. 3a shows that 30 ppm of unhydrolyzed Hakushinin (control) or 1.0 ppm or 3.0 ppm of Hakushinin hydrolyzate was added to immature (M0) THP-1 cells 30 days after addition of melanin. Shows how it looks after a minute. FIG. 3b is an enlarged view of FIG. 3a. Black arrows indicate macrophages taking up melanin. Figure 4 shows the intracellular activities of M0 macrophages, M1 macrophages and M2 macrophages in experiment 4 analyzed using a flux analyzer: (A) oxygen consumption rate (OCR), (B) extracellular acidification rate (ECAR). ) and (C) OCR/ECAR results. FIG. 5 shows the results of analysis using a flux analyzer in Experiment 5 on the effect of the addition of the Hakushinin extract on the intracellular activity of M0 macrophages 48 hours after addition. (A) Normalized OCR, (B) Basal OCR (OCR value at the third point of A measurement). FIG. 6 shows the results of analysis using a flux analyzer in Experiment 5 of the effect of the addition of the Hakushinin extract on the intracellular activity of M0 macrophages 48 hours after addition. (A) Normalized OCR, (B) OCR of mitochondrial respiration (difference value between 3rd and 12th point of A measurement). FIG. 7 shows the results of analysis using a flux analyzer in Experiment 6 of the influence of typical unsaturated fatty acids contained in the extract of Hakushinin alone on the intracellular activity 48 hours after the addition of M0 macrophages. (A) Basal OCR, (B) OCR of mitochondrial respiration. FIG. 8 shows the results of analysis using a flux analyzer in Experiment 7 of the influence of each component contained in the succinicin extract on the intracellular activity of M0 macrophages 24 hours after addition. (A) Basal OCR, (B) OCR of mitochondrial respiration. FIG. 9 shows a schematic of the method of Experiment 8. FIG. 10 shows the results of analysis using a flux analyzer in Experiment 8 on the influence of the extract of Chinese cucumber or each component contained in the extract on the intracellular activity during differentiation of M1 macrophages. (A) Basal OCR, (B) OCR of mitochondrial respiration, (C) OCR/ECAR. FIG. 11 shows the results of analysis of the intracellular activity of M0 macrophages 24 hours after addition of nicotinamide in Experiment 9 using a flux analyzer. (A) Schematic of experiment 9, (B) Basal OCR, (C) OCR of mitochondrial respiration, (D) OCR/ECAR, (E) normalized ECAR. FIG. 12 outlines the method of Experiment 10. FIG. 13 shows the results of analyzing the intracellular activity 24 hours after addition of nicotinamide to M1-differentiating macrophages in Experiment 10 using a flux analyzer. (A) Basal OCR, (B) OCR of mitochondrial respiration, (C) OCR/ECAR, (D) normalized ECAR. FIG. 14 outlines the method of Experiment 11. FIG. 15 shows the results of analyzing the intracellular activity 24 hours after addition of nicotinamide to M2-differentiating macrophages in Experiment 11 using a flux analyzer. (A) Basal OCR, (B) OCR of mitochondrial respiration, (C) OCR/ECAR, (D) normalized ECAR. FIG. 16 shows the results of analysis of intracellular activity 24 hours after addition of succinicin extract, nicotinic acid amide, or a mixture thereof (Mix) to M0 macrophages in experiment 12 using a flux analyzer. (A) Schematic of experiment 12, (B) OCR of mitochondrial respiration. Fig. 17 shows the result of analyzing the intracellular activity of macrophages during M1 differentiation 24 hours after addition of cinnamon extract, nicotinamide, or a mixture thereof (Mix) using a flux analyzer in Experiment 13. indicates (A) Schematic of experiment 13, (B) OCR of mitochondrial respiration, (C) OCR/ECAR. Fig. 18 shows the results of analysis of intracellular activity using a flux analyzer 24 hours after addition of cinnamon extract, nicotinamide, or a mixture thereof (Mix) to macrophages during M2 differentiation in Experiment 14. indicates (A) Schematic of experiment 14, (B) OCR of mitochondrial respiration.

　Macrophages are cells that are localized in various tissues in the body, trigger immune responses against foreign substances and pathogens, and are known to be involved in inflammation. Macrophages are differentiated into M1 type and M2 type from undifferentiated M0 macrophages (hereinafter sometimes abbreviated as M0). M1 macrophages (hereinafter sometimes abbreviated as M1) are known as inflammatory type, and M2 macrophages (hereinafter sometimes abbreviated as M2) are known as repair type (anti-inflammatory type).

The present inventors have previously discovered that the balance between these M1 macrophages and M2 macrophages (M1/M2 balance) is specifically disturbed in areas of skin exposed to light and where pigmentation occurs, and the M1/M2 balance is adjusted. is particularly important in preventing and improving photoaging and pigmentation in the dermis (Patent Document 11). Therefore, the present inventors used the M1/M2 balance as an index to search for substances that can prevent and improve photoaging and/or dermal pigmentation. or can be activated, leading to the development of the present invention. Hakushinin has been reported to have the effect of acting on fibroblasts to proliferate fibroblasts, promoting the production of collagen and hyaluronic acid, and suppressing the production of melanin (Patent Document 9 and 10). In addition, nicotinic acid amide is known to have a blood circulation-promoting action, a rough skin-improving action, a whitening action, and the like (for example, Patent Document 12). However, it has now been found for the first time by the present inventors that succinicin and nicotinamide can induce or activate repair-type M2 macrophages or M2-like macrophages, thereby adjusting/improving the M1/M2 balance. .

In one embodiment, the present invention provides an agent for inducing or activating M2 or M2-like macrophages, comprising nicotinamide or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, the agent for inducing or activating M2 or M2-like macrophages according to the present invention can prevent and/or improve pigmentation in the dermis through M2 or M2-like macrophages induced or activated by the agent. .

In one embodiment, the present invention provides a composition comprising nicotinamide or a pharmaceutically acceptable salt thereof and a cinnamon extract.

Also, in one embodiment, the present invention provides a method of inducing or activating M2 or M2-like macrophages in a subject in need thereof, comprising:
A method is provided comprising applying nicotinamide or a pharmaceutically acceptable salt thereof to said subject to induce or activate M2 or M2-like macrophages.

Also, in one embodiment, the present invention provides a method for preventing and/or improving dermal pigmentation in a subject in need thereof, comprising:
A method is provided comprising applying to said subject a composition comprising nicotinamide or a pharmaceutically acceptable salt thereof and a cinnamon extract.

The present invention also provides use of nicotinamide or a pharmaceutically acceptable salt thereof for producing a composition for inducing or activating M2 or M2-like macrophages.

The present invention also provides use of nicotinamide or a pharmaceutically acceptable salt thereof and succinicin extract for the manufacture of a composition for preventing and/or improving pigmentation in the dermis.

As used herein, pigmentation refers to pigmentation in the dermis and epidermis, and includes, for example, pigmentation such as melanin due to photoaging, as well as artificially injected pigments (e.g., tattoos and tattoos). . Although the present invention is effective for both the dermis and epidermis, it is highly expected as a countermeasure against dermal pigmentation in the sense that improvement methods are limited other than phagocytosis by melanophages. By applying the present invention, spots, dullness, dark circles, etc. due to pigmentation are improved. In addition, since the pigment is injected into the dermis layer, it is effective for decolorizing tattoos, etc., which are difficult to remove once the pigment is put in.

As used herein, "induction or activation of M2 or M2-like macrophages" refers to differentiation of macrophages (e.g., M0 macrophages or M1 macrophages) into M2 macrophages, or macrophages (e.g., M0 macrophages, M1 macrophages or M2 macrophages) were compared to intracellular activities possessed by M2 macrophages (e.g., promotion of aerobic respiration (mitochondrial respiration) and/or suppression of glycolysis (preferably, intracellular activity possessed by M1 macrophages). (case)) to induce or activate. Therefore, "induction or activation of M2 or M2-like macrophages" may be to increase the absolute number of M2 or M2-like macrophages, the number of M1 or M1-like macrophages relative to the number of M2 or M2-like macrophages , that is, to decrease the ratio (M1/M2 balance). As used herein, the term "M2-like macrophages" refers to the intracellular activity of M2 macrophages (e.g., promotion of aerobic respiration (mitochondrial respiration) and/or suppression of glycolysis (preferably, intracellular activity of M1 macrophages). A macrophage that exhibits )) when compared to and does not necessarily match the marker of M2 macrophages.

As used herein, M1-like macrophages refer to intracellular activities of M1 macrophages (e.g., suppression of aerobic respiration (mitochondrial respiration) and/or promotion of glycolysis (preferably, intracellular activities of M2 macrophages). When compared)), it refers to the macrophages shown and does not necessarily match the markers of M1 macrophages.

M1 macrophages can be measured using markers such as CD86, CD80, and iNOS as indicators. M2 macrophages can be measured using markers such as CD206, CD163, Agr1, and the like, for example. Overall macrophage markers, including M1 and M2, include CD11b, CD68, and the like. Additionally or alternatively, M1 and M2 macrophages may be measured 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 markers as long as the marker can measure the M1/M2 balance.

As used herein, the M1/M2 balance may refer to the ratio of the number of M1 macrophages and/or M1-like macrophages to the number of M2 macrophages and/or M2-like macrophages as described above. , may refer to the ratio of the mRNA amount of M1 macrophage markers (eg, CD86, CD80, iNOS, etc.) to the mRNA amount of M2 macrophage markers (eg, CD206, CD163, Agr1, etc.). In photoaged skin, the ratio of M1 is high and the ratio of M2 is low, and it is M2 that has high melanophagocytosis. number/number of M1 and/or mRNA amount of M2 marker/mRNA amount of M1 marker). The increase may be, for example, an increase with statistical significance (e.g., Student's t-test) with a significance level of 5%, and/or e.g., 1% or more, 5% or more, 10% or more, The increase may be 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. Alternatively, the adjustment / improvement of the M1 / M2 balance is performed by adjusting the ratio of M2 to M1 (number of M2 / number of M1 and / or mRNA amount of M2 marker / mRNA amount of M1 marker) within a certain range, for example, about It may be 4/6 to about 9/1, about 5/5 to about 8/2, about 5/5 to about 7/3, etc., or it may be close to the above range. It may be maintaining within a range, or maintaining constancy around the above range.

As used herein, the M1/M2 balance may refer to the balance between the intracellular activity of M1 macrophages and the intracellular activity of M2 macrophages, for example, the intracellular activity mainly used by M1 macrophages (e.g. state of glycolysis) and/or an index of intracellular activity (for example, state of aerobic respiration (mitochondrial respiration)) mainly used by M2 macrophages. Intracellular activity can be assessed, for example, by measuring the cellular oxygen consumption rate (OCR value), extracellular acidification rate (ECAR value), or OCR/ECAR value. As used herein, "increasing the ratio of M2 to M1" may refer to increasing intracellular mitochondrial activity primarily used by M2 macrophages, including, for example, M1 macrophages and M2 macrophages. aerobic respiration (mitochondrial respiration) is elevated in the population (e.g., aerobic respiration (mitochondrial respiration) predominates in a population comprising M1 and M2 macrophages) and/or in a population comprising M1 and M2 macrophages It may refer to a decrease in glycolytic activity, which is anaerobic respiration, e.g., elevated OCR values in a population comprising M1 and M2 macrophages and/or comprising M1 and M2 macrophages. It may mean that ECAR values are decreased in a population and/or OCR/ECAR values are increased in a population comprising M1 and M2 macrophages. Intracellular activity can be measured by using, for example, an extracellular flux analyzer XFe24 (for 24well) manufactured by Agilent Technologies (formerly Seahorse Bioscience), which is the main energy metabolism pathway of cells, glycolysis, and aerobic respiration by mitochondria. It is possible to non-invasively and highly sensitively measure the state of cells over time, but this device is an example, and intracellular activity can be measured using any device and method for evaluation. Therefore, it is not limited to the use of the device.

In photoaged skin, the ratio of M1 is high and the ratio of M2 is low, and since it is M2 that is more melanophagocytic, the adjustment/improvement of the M1/M2 balance is likely to increase the intracellular activity of M2 relative to M1. The ratio may be, for example, increasing OCR values in a population comprising M1 and M2 macrophages and/or decreasing ECAR values in a population comprising M1 and M2 macrophages. As used herein, "increase or decrease" may be, for example, an increase or decrease with a statistically significant difference (e.g., Student's t-test) with a significance level of 5%, and / or, for example, 1% or more, 5% or more, 10% or more, 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 increase or It may be a decrease.

Hakushinin (Hakushinin) is a crude drug obtained by drying the seeds of Platycladus orientalis Franco, Thuja orientalis L., and Biota orientalis Endl. of the Cupressaceae family. Although it is preferable to use seed kernels (endosperm portion) as the hakushinin used in the present invention, whole seeds can also be used because active ingredients are contained in whole seeds. Hakushinin may be produced by a conventional method, or a commercially available product may be used.

The method for preparing the extract that can be used in the present invention can be produced with reference to known methods (eg, Patent Documents 9 and 10), but is not limited to these. For example, as a method for preparing a cinnamon extract that can be used in the present invention, a method of subjecting a plant extract obtained by extracting seeds of a plant to alkali treatment (hereinafter referred to as the first method); There is a method (hereinafter referred to as the second method) of pulverizing the seeds of A. and maturing them for at least one week at a temperature of 10-35°C and a relative humidity of 20-90%.

The extraction solvent used in the first method and the second method may be any volatile solvent that is commonly used for extraction, particularly alcohols such as methanol and ethanol, hydrous alcohols, acetone, ethyl acetate, Polar/nonpolar organic solvents such as hexane and ether can be used alone or in combination, but acetone, ethyl acetate, and hexane are particularly preferred because they are inexpensive and can be easily concentrated under reduced pressure. In addition, extraction with supercritical carbon dioxide gas can also be used. On the other hand, an extraction solvent containing a large amount of water is not preferable because extraction of the active ingredient is suppressed.

For extraction, extract for a certain period of time with a solvent that is 1 to 100 times, preferably 1 to 30 times, the mass of the seeds. The seeds to be used can be appropriately pulverized as necessary, but may be used unpulverized if clogging becomes a problem during filtration. It is also possible to repeatedly extract with a small amount of solvent, and a reflux apparatus such as a Soxhlet may be used. In the case of extraction with supercritical carbon dioxide gas, extraction can be performed under general conditions of around 40° C. and 20 to 40 MPa.

In the first method, after removing the extraction solvent from these extracts, the extracts are hydrolyzed by alkaline treatment. Alkaline treatment is carried out by adding 0.2 to 2 times the volume of concentrated alkaline aqueous solution to the extract after removal of the solvent, thoroughly stirring and mixing, and aging for about 30 minutes to several days. The temperature during mixing is preferably room temperature to 70°C, more preferably 30 to 60°C. It is desirable to appropriately stir during aging so that the hydrophilic component and the lipophilic component do not separate. Examples of the concentrated alkaline aqueous solution include aqueous solutions of NaOH, KOH, etc. having a concentration of 1 to 10N.

As described above, if the extract that has been hydrolyzed by alkali treatment is further neutralized with an acid to near neutrality, the oily substance will separate. This oily substance has a very high anti-dermal pigmentation effect and is highly safe for the skin. In order to promote recovery of the oily substance, alcohol, acetone, etc. may be added as appropriate after the alkali treatment, and further operations such as decolorization, deodorization, desalting, and distillation may be added thereafter, if necessary.

According to the second method of preparing the Chinese cinnamon extract, the seeds are moderately pulverized or pressed in the pre-extraction stage, and then aged at around room temperature for a certain period of time. Regarding the aging period, there is no particular limitation as long as the desired effect can be obtained, but if the aging period is as short as one week or less, it will be difficult to obtain a sufficient whitening effect, and on the other hand if it lasts for several years, it will rot and deteriorate. The problem of oxidative change of smell occurs, which is not preferable. In addition, it is also possible to suppress offensive odor during aging by adding an antioxidant or performing nitrogen substitution, etc., as necessary.

Extraction is performed using the extraction solvent and extraction method described above from the seeds that have matured in this way. After extraction, if necessary, operations such as solvent removal, decolorization, deodorization, and distillation are performed. The extract obtained by pulverizing and ripening such a specific seed has the same anti-dermal pigmentation effect as the above-mentioned alkali-treated extract, but it is highly safe for the skin. show gender.

In the present invention, a plant-based, photoaging and/or safe anti-dermal pigmentation effect consisting of an extract prepared by the first method or the second method from the seeds of plants as shown above. An agent for preventing and/or improving dermal pigmentation can be provided, and an external preparation for skin containing the present agent can be provided.

Nicotinic acid amide (CAS No. 98-92-0) is an amide compound of nicotinic acid, and is known to have effects such as promoting blood circulation, improving rough skin, and whitening. However, it has now been found by the inventors for the first time that nicotinamide can modulate the M1/M2 balance, eg induce or activate M2 or M2-like macrophages. Nicotinamide or a pharmaceutically acceptable salt used in the present invention may be an extract from a natural product or a product synthesized by a known method. For example, specifically, those listed in the 17th revision of the Japanese Pharmacopoeia can be used. Pharmaceutically acceptable salts are inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, e.g. acetates, tartrates, citrates, fumarates, maleic acid salts, organic acid salts such as toluenesulfonates and methanesulfonates; metal salts such as sodium salts, potassium salts, calcium salts, aluminum salts such as triethylamine salts, guanidine salts, ammonium salts, hydrazine salts, and quinine salts; , salts with bases such as cinchonine salts, and the like.

The agent of the present invention may be used in combination with any substance known to adjust/improve the M1/M2 balance, such as the substances described in Patent Documents 4 to 7. The route of administration can also be arbitrarily selected, for example, transdermal administration, oral administration, subcutaneous administration, transmucosal administration, intramuscular administration, etc., and can be administered to a specific site on the skin to prevent and/or improve dermal pigmentation. Transdermal administration may be preferred. For pigmentation occurring in the epidermis and dermis, transdermal administration may be preferred so that the epidermis and dermis can be reached through the skin. Further, adjustment/improvement of the M1/M2 balance may be, for example, induction of differentiation into M2 macrophages, or any other M1/M2 balance adjustment/improvement method.

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

The agent of the present invention can also be a composition in which the above active ingredients are combined with one or more other ingredients such as excipients, carriers and/or diluents. The composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as the active ingredient and application. The composition can be produced by a conventional method in a formulation in which other ingredients such as excipients, carriers and/or diluents are appropriately combined according to the dosage form.

The agent of the present invention can be used for humans and animals by being blended into cosmetics or the like, or can be administered to humans and animals as a pharmaceutical formulation. In addition, it may be added to various foods, drinks, and feeds to be ingested by humans and animals.

When the present invention is applied to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the blending amount (dry mass) of the plant body or its extract depends on the type, purpose, form, method of use, etc. , can be determined accordingly. For example, 0.00001% to 50% of nicotinic acid amide or a pharmaceutically acceptable salt and/or a cinnamon extract (on a dry weight basis in the case of extracts or herbal medicines) can be blended in the total amount of the cosmetic.

In addition to the above ingredients, if necessary, ingredients that are usually used in external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, and UV protection agents, to the extent that they do not impair the effects of the present invention. , surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as necessary.

The external preparation for skin of the present invention can be applied as cosmetics, quasi-drugs, etc., which are applied to the outer skin, particularly preferably as cosmetics, and its dosage form is not limited as long as it can be applied to the skin. Arbitrary formulations such as solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, and aerosol are applicable.

When the agent of the present invention is used as cosmetics, lotion, emulsion, foundation, lipstick, lip balm, cleansing cream, massage cream, mask, hand cream, hand powder, body shampoo, body lotion, body cream, bath cosmetics, etc. You may use it as a form.

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

The subject to which the method, device, agent and composition of the present invention are applied is a subject in which skin photoaging and / or pigmentation (e.g., dermal pigmentation) is objectively or subjectively observed, even if it is a subject with pigmentation. It can also be a subject that one wishes to prevent. For example, it may be a subject determined to have an M1/M2 balance. In one embodiment, the subject may be determined to have a high degree of pigmentation (for example, degree of dermal pigmentation) using the M1/M2 balance in the skin as an index. Alternatively, the subject may be concerned with phenotypes specific to photoaging of the skin, such as spots, wrinkles, sagging, etc., or epidermis and pigmentation, such as spots, dullness, bruises, tattoo marks, etc. may be a target of concern. Spots, wrinkles, sagging, dullness, bruises, tattoo marks, etc. can be determined by visual judgment or using known indices.

Cosmetic treatments include, but are not particularly limited to, any treatment that is believed to be effective in inhibiting photoaging and/or pigmentation, such as applying cosmetics containing the agent of the present invention or other ingredients. The term “cosmetics” refers to cosmetics that are applied to the skin, such as lotions, milky lotions, serums, creams, foundations, etc., but not limited to these, and is not intended to directly improve skin conditions. However, it is intended to include anything that is applied to the skin, including, for example, sunscreens and the like. Alternatively, the cosmetic treatment may be applying physical stimuli to the skin, such as stretching stimuli, pressing stimuli, massages, and the like. A cosmetic treatment may be a one-time treatment or an ongoing treatment over a period of days to weeks. The cosmetic treatment may be performed privately, or may be performed at a beauty salon, a cosmetics store, a beauty salon, or the like.

Next, the present invention will be described in more detail by way of examples. In addition, this invention is not limited by this.

Experiment 1: THP-1 M0, M1 and M2 Differentiation Induction Experiment According to the method described in Non-Patent Document 1, THP-1, a human-derived cell line, was used to induce differentiation into M1 and M2 macrophages. Specifically, by the method shown in FIG. 1a, THP-1 was cultured in RPMI1640 (Nakalai) with the addition of 1 mM Na Pyruvate (Nakalai), 2 mM L-glutamine (Nakalai) and 10% FBS. Thereafter, 100 nM PMA (abcam) was further added to stimulate for 24 hours to differentiate into macrophages. When differentiated into M1, 100 ng/mL LPS (sigma) and 20 ng/mL IFNγ (R & D) were added for 24 hours of stimulation, and when differentiated into M2, 20 ng/ml IL-4 (R & D) and 20 ng/mL were added. IL13 (R&D) was added for 24 hours of stimulation. Microscopic observation confirmed that the morphology changed as shown in FIG. 1b.

In addition, the mRNA of each differentiated or undifferentiated cell is extracted, and realtime PCR is performed by TaqMan Gene expression assay using IL-1beta, TNF-alpha, and IL-10 probes (Applied Biosystems). , the expression level was quantified (Fig. 1c upper panel). Furthermore, using a CD86 antibody (R&D) and a CD206 antibody (BD), PCR was performed in the same manner to quantify the expression level (Fig. 1c, lower figure). Each value was corrected with the expression level of GAPDH mRNA.

As shown in FIG. 1c, macrophages differentiated by the above method produce inflammatory cytokines (IL-1beta, TNF-alpha) characteristic of M1 and anti-inflammatory cytokines (IL-10) characteristic of M2, respectively. It was shown to produce Furthermore, these differentiation-induced macrophages showed increased expression of CD86 and CD206, surface markers of M1 and M2 macrophages, respectively. These results confirmed that differentiation induction was successful. Therefore, M1 and M2 macrophages differentiated by the above method and undifferentiated M0 macrophages were used in the following experiments.

Experiment 2: Search for M1 suppressor/M2 inducer (1)
Various M1/M2 markers were used to search for agents that could prevent and/or improve photoaging and/or dermal pigmentation by adjusting or improving the M1/M2 balance, and cakushinin had a strong M2-inducing effect. was found. The extract used here (without hydrolysis or with hydrolysis) was prepared with reference to Japanese Patent No. 4781842 (Patent Document 9) and International Publication No. 2012/0571243 (Patent Document 10). and was prepared by the following procedure. It should be noted that the procedures for preparing the extracts of Chinese cinnamon that can be used in the present invention are not limited to those described below.

<Preparation of Hakushinin extract>
Chinese cinnamon seeds (unpulverized, if necessary the outer shell was removed) were immersed in 5 volumes (v/w) of acetone, ethyl acetate (ethyl acetate) or hexane, and extracted at room temperature for 10 days. Residues were removed in advance with a nylon mesh (100 mesh) and filtered with filter paper. After removing the solvent from the filtrate on a rotary evaporator, 0.5-15N NaOH was added as NaOH in the range of 100-300 g/l extract while stirring with a stirrer blade (temperature 50°C). The treatment was carried out for 5 hours while stirring at 200 rpm. Thereafter, 5N H ₂ SO ₄ was gradually added, the pH was lowered to around 1 while stirring, and the separated oily substance was collected. An equal volume of water was added to the recovered oily substance and washed with water to remove impurities, salts and excess acid. The oily substance was dried under reduced pressure to obtain an alkali-treated product (Hakushinin extract with hydrolysis). As a control, the extract before alkali treatment was used (Hakushinin extract without hydrolysis).

The THP-1 cells were used in the undifferentiated state of M0 and cultured overnight at 37°C. After that, 3 ppm of unhydrolyzed (without alkali treatment) hakushinin (control) dissolved in a solvent (DMSO), or 0.3 ppm, 1 ppm, or 3 ppm of hakushinin hydrolyzate was added, and the mixture was heated at 37°C. cultured overnight. Cells were harvested and RNA was extracted, the expression levels of CD86, CCR7, TNF-alpha, CD206, CD163, IL-10 and GAPDH were quantified by real-time PCR, and the expression level of each gene was divided by the expression level of GAPDH. bottom. As a result, the hakushinin hydrolyzate concentration-dependently reduced the expression levels of the CD86 gene and TNF-α gene, and increased the expression levels of the CD206 gene and IL-10 gene. From this, it was found that the Hakushinin hydrolyzate has the effect of suppressing M1 induction and promoting M2 induction (Fig. 2).

Experiment 3: Effect of increasing melanin phagocytosis by hakushinin Immature (M0) THP-1 cells were added with 3 ppm of unhydrolyzed (no alkali treatment) hakushinin (control) dissolved in a solvent (DMSO), or Hakushinin was added. Decomposition products were added to 1.0 ppm and 3.0 ppm. After 48 hours of culture at 37° C., a melanin solution (Sigma) was added and observed under a microscope (Bio Station CT (Nikon)).

The results are shown in Figures 3a-b. Thirty minutes after the addition of melanin, the macrophages to which hairpin had been added began to take up melanin (black arrows in Fig. 3b). After 3 hours, many cells still had no melanin uptake in the control, but all the cells in the sample with the addition of hakushinin hydrolyzate had taken up melanin.

The ratio of M1 increases due to photoaging, and the higher the ratio of M2, the higher the amount of skin collagen and the higher the pigment phagocytosis (Patent Document 11). It is suggested that the pigmentation inhibitory effect is high in the dermis.

Experiment 4: Intracellular activity of macrophages For M0 macrophages, M1 macrophages and M2 macrophages differentiated from THP-1 in the same manner as in Experiment 1, an extracellular flux analyzer XFe24 (for 24 wells) manufactured by Agilent Technologies (formerly Seahorse Bioscience) ) (hereinafter referred to as “flux analyzer”), Nat Immunol, 2014. 15(9): Intracellular activity was measured with reference to pp846-855 (Non-Patent Document 7). The flux analyzer is a device that enables noninvasive and highly sensitive temporal measurement of the state of glycolysis, which is the main energy metabolism pathway of cells, and aerobic respiration by mitochondria.

Oligomycin (ATP synthase inhibitor) (“Oligo”), FCCP (uncoupler), and rotenone (mitochondrial complex I inhibitor) plus antimycin A (mitochondrial complex III inhibitor) using a flux analyzer (“Rot+Ant”) were added at given times and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured. As the ECAR and OCR values, the basal OCR value and basal ECAR value 18.3 minutes after measurement were used. Each count value was corrected by the cell number obtained by counting the cell nuclei stained by adding Hoechst.

As a result, it was shown that mitochondrial respiratory activity was high in the order of M2 macrophages, M0 macrophages, and M1 macrophages (Fig. 4).

　Experiment 5: Influence of Hakushinin extract on intracellular activity of macrophages

M0, M1, and M2 differentiated from THP-1 were prepared in the same manner as in Experiment 1, and Hakushinin extract (Hakushinin hydrolyzate) (0.3 ppm, 1 ppm) prepared in the same manner as in Experiment 2 was added to M0. or 3 ppm) was added. After 48 hours of incubation, oxygen consumption rate (OCR) was measured with a flux analyzer. Each measured value was corrected by the cell number obtained by counting the cell nuclei stained by adding Hoechst (Fig. 5).

As a result, by adding the Hakushinin extract, the value of Basal OCR (Fig. 5) at 18.3 minutes after the start of measurement (measured value of the third point) and the value of mitochondrial respiration (18.3 minutes after the start of measurement It was revealed that the value obtained by subtracting non-mitochondrial inspiration (oxygen consumption unrelated to mitochondria) 95.1 minutes after the start of measurement from the value of Basal OCR (Fig. 6) increased significantly.

　Experiment 6: Influence of each component contained in Hakushinin extract on the intracellular activity of macrophages (1)

Hakushinin extract is known to contain unsaturated fatty acids such as linolenic acid and juniperonic acid, which are ω3 fatty acids, and linoleic acid, which is ω6 fatty acid. It has been reported that unsaturated fatty acids (EPA, DHA) act on M1 inflammation-related genes to suppress inflammation (Non-Patent Document 8 (Cell Metab. 2017 Feb 7; 25(2): 412-427). ). Therefore, we investigated the effect of unsaturated fatty acids contained in the extract of Hakushinin on the intracellular activity of macrophages.

Prepare M0 and M2 differentiated from THP-1 in the same manner as in Experiment 1, add linoleic acid, linolenic acid or juniperonic acid (3 ppm) to M0, and after 48 hours, in the same manner as in Experiment 15 Oxygen consumption rate (OCR) was measured with a flux analyzer. Each count value was corrected by the cell number obtained by counting the cell nuclei stained by adding Hoechst.

As a result, the values of Basal OCR and mitochondrial respiration to M0 macrophages by the addition of linoleic acid, linolenic acid, and juniperonic acid contained in the cinnamon extract were not significantly different from the control to which only solvent (DMSO) was added. (Fig. 7).

　Experiment 7: Influence of each component contained in Hakushinin extract on the intracellular activity of macrophages (2)

　M0 Basal OCR and mitochondrial respiration values were investigated when the incubation time was changed to 24 hours after the addition of the cinnamon extract or unsaturated fatty acids. The experimental method was the same as the method described in Experiment 6, except that the incubation time after the addition of the cinnamon extract or the unsaturated fatty acid was changed to 24 hours.

As a result, no increase in Basal OCR and mitochondrial respiration values was observed 24 hours after the addition of the cinnamon extract and each unsaturated fatty acid (Fig. 8).

　Experiment 8: Influence of each component contained in Hakushinin extract on the intracellular activity of macrophages during M1 differentiation

M0 differentiated from THP-1 by the same method as in Experiment 1 was further induced to differentiate into M1. Changes in activity were analyzed using a flux analyzer (Fig. 9).

When M0 differentiated from THP-1 was further induced to differentiate into M1, the oxygen consumption rate (OCR value) and extracellular acidification were measured using a flux analyzer 24 hours after the addition of cinnamon extract or unsaturated fatty acid. Velocity (ECAR value) was measured.

As a result, a significant increase in Basal OCR and mitochondrial respiration was observed only when the hakushinin extract was added (Fig. 10 (A) and (B)). Only linolenic acid, which has been reported to have a strong anti-inflammatory action, was observed to have a strong inhibitory effect on the ECAR value, and as a result, an increase in the OCR/ECAR value was observed.

Mitochondrial respiration of macrophages during M1 differentiation was significantly increased by the extract of Hakushinin, but not by the unsaturated fatty acids contained in the extract of Hakushinin alone. It was suggested that there is a possibility that it is a specific effect due to the mixture of various components contained.

Experiment 9: Search for M1 suppressor/M2 inducer (2)
As shown in Experiment 4 above, repair-type M2 macrophages have higher mitochondrial respiration than undifferentiated M0 macrophages and inflammatory M1 macrophages, and obtain energy through mitochondrial respiration. Inflammatory M1 macrophages depend on glycolysis for energy. Therefore, when searching for substances capable of inducing M2 or M2-like macrophages using intracellular activities (mitochondrial respiration and glycolysis) as indices, it was found that nicotinamide can significantly increase the mitochondrial activity of macrophages. (Fig. 11).

Specifically, in the same manner as Experiment 1, M0 was prepared by differentiation induction from THP-1, and nicotinamide (0 mM (control), 0.6 mM or 3.0 mM) was added to M0. After 24 hours of incubation, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with a flux analyzer. Each count value was corrected by the cell number obtained by counting the cell nuclei stained by adding Hoechst.

As a result, the addition of nicotinamide significantly increased the Basal OCR value (Fig. 11 (B)) and the OCR/ECAR value (Fig. 11 (D)) 18.3 minutes after the start of measurement, Respiration value (value obtained by subtracting non-mitochondriary respiration (oxygen consumption not related to mitochondria) 95.1 minutes after the start of measurement from the Basal OCR value 18.3 minutes after the start of measurement) (Fig. 11 (C )) is on the increase.

From the above, it was revealed that nicotinamide is a substance that can increase the oxygen consumption rate (OCR) that is characteristic of M0 macrophages and M2 or M2-like macrophages.

Experiment 10: Effect of nicotinamide on intracellular activity of macrophages during M1 differentiation

The change in intracellular activity when nicotinamide was added in the step of further inducing the differentiation of M0 from THP-1 by the same method as in Experiment 1 into M1 was analyzed using a flux analyzer (FIG. 12, Figure 13).

When further inducing the differentiation of M0 from THP-1 into M1, the oxygen consumption rate (OCR value) and the extracellular acidification rate (ECAR value) were measured using a flux analyzer 24 hours after the addition of nicotinamide. was measured.

As a result, when nicotinamide was added to M1 during induction of differentiation, a suppressive effect on ECAR values was observed (Fig. 13 (D)), and as a result, an increase in OCR/ECAR values was observed (Fig. 13 (C )).

Experiment 11: Effect of nicotinamide on intracellular activity of macrophages during M2 differentiation

The change in intracellular activity when nicotinamide was added in the step of further inducing the differentiation of M0 from THP-1 by the same method as in Experiment 1 into M2 was analyzed using a flux analyzer (Fig. 14, Figure 15).

When further inducing the differentiation of M0 from THP-1 into M2, the oxygen consumption rate (OCR value) and the extracellular acidification rate (ECAR value) were measured using a flux analyzer 24 hours after the addition of nicotinamide. was measured.

As a result, when nicotinamide was added to M2 during induction of differentiation, a suppressive effect on ECAR values was observed (Fig. 15 (D)), and as a result, an increase in OCR/ECAR values was observed (Fig. 15 (C )).

　Experiment 12: Effects of nicotinic acid amide and cinnamon extract on the intracellular activity of M0 macrophages

In the same manner as in Experiment 1, M0 was prepared by differentiation induction from THP-1, and Hakushinin extract (prepared in the same manner as in Experiment 2. 10 ppm), nicotinic acid amide (0.1 mM) or Hakushinin extract was added to M0. A mixture (10 ppm) and nicotinamide (0.1 mM) was added. After 24 hours of incubation, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with a flux analyzer. Each count value was corrected by the cell number obtained by counting the cell nuclei stained by adding Hoechst.

As a result, cinnamon extract (10 ppm) and nicotinamide (0.1 mM), which did not exhibit their effects when added for 24 hours, exhibited a synergistic effect by mixing them and adding them, increasing the OCR value of M0 macrophages. was found to be significantly increased (Fig. 16(B)).

　Experiment 13: Effects of nicotinamide and cinnamon extract on intracellular activity of macrophages during M1 differentiation

In the step of further inducing the differentiation of M0 from THP-1 by the same method as in Experiment 1 into M1, the case where a cinnamon extract, nicotinic acid amide, or a mixture (mix) of a cinnamon extract and nicotinic acid amide was added. was analyzed using a flux analyzer (Fig. 17).

When further inducing the differentiation of M0 from THP-1 into M1, a succinicin extract (prepared in the same manner as in Experiment 2. 10 ppm), nicotinic acid amide (0.1 mM), or a succinicin extract (10 ppm). Oxygen consumption rate (OCR value) and extracellular acidification rate (ECAR value) were measured using a flux analyzer 24 hours after addition of nicotinamide (0.1 mM) mixture (mix).

As a result, when nicotinamide was added to M1 during induction of differentiation, cinnamon extract (10 ppm) and nicotinamide (0.1 mM), which did not exert its effect when added for 24 hours, were mixed and added. By this, it was found that the synergistic effect was exhibited, the OCR value of macrophages was increased when M1 was induced to differentiate, the ECAR value was decreased, and the OCR/ECAR value was increased (Fig. 17 (B) , (C)).

Experiment 14: Effect of nicotinamide on intracellular activity of macrophages during M2 differentiation

In the step of further inducing the differentiation of M0 from THP-1 by the same method as in Experiment 1 into M2, the case where a succinicin extract, nicotinic acid amide, or a mixture (mix) of a succinicin extract and nicotinic acid amide was added. was analyzed using a flux analyzer (Fig. 18).

When further inducing the differentiation of M0 from THP-1 into M2, a succinicin extract (prepared in the same manner as in Experiment 2. 10 ppm), nicotinic acid amide (0.1 mM), or a succinicin extract (10 ppm) was used. Oxygen consumption rate (OCR value) and extracellular acidification rate (ECAR value) were measured using a flux analyzer 24 hours after addition of nicotinamide (0.1 mM) mixture (mix).

As a result, the addition of nicotinamide during differentiation induction to M2 did not exert its effect when added for 24 hours. Thus, it was revealed that a synergistic effect was exhibited, and the OCR value of macrophages was increased during induction of differentiation to M2 (Fig. 18(B)).

Based on the above results, nicotinamide and succinicin extract induce or activate M2 or M2-like macrophages, and as a result, modulate or improve the balance of M1/M2, thereby preventing photoaging and/or dermal pigmentation. It was suggested that it can be prevented and/or improved.

Claims

An agent for inducing or activating M2 or M2-like macrophages, comprising nicotinamide or a pharmaceutically acceptable salt thereof as an active ingredient.
The inducing or activating agent according to claim 1, which prevents and/or improves pigmentation in the dermis via the M2 or M2-like macrophages.
The inducing or activating agent according to claim 1 or 2, which suppresses glycolysis of macrophages.
The inducing or activating agent according to any one of claims 1 to 3, which promotes aerobic respiration of macrophages.
The inducing or activating agent according to any one of claims 1 to 4, further comprising a cakushinin extract.
A composition comprising nicotinic acid amide or a pharmaceutically acceptable salt thereof and an extract of Chinese cinnamon.
A method of inducing or activating M2 or M2-like macrophages in a subject in need thereof, comprising:
A method comprising applying nicotinamide or a pharmaceutically acceptable salt thereof to said subject to induce or activate M2 or M2-like macrophages.
The method according to claim 7, wherein pigmentation in the dermis of said subject is prevented and/or improved via said M2 or M2-like macrophages.
The method according to claim 7 or 8, which suppresses glycolysis of macrophages.
The method according to any one of claims 7 to 9, which promotes aerobic respiration of macrophages.
The method according to any one of claims 7 to 10, further comprising a cactidennis extract.
A method of preventing and/or improving dermal pigmentation in a subject in need thereof, comprising:
A method comprising applying to said subject a composition comprising nicotinamide or a pharmaceutically acceptable salt thereof and a cinnamon extract.
Use of nicotinamide or a pharmaceutically acceptable salt thereof for the manufacture of a composition for inducing or activating M2 or M2-like macrophages.
The use according to claim 13, which prevents and/or improves pigmentation in the dermis of a subject via said M2 or M2-like macrophages.
The use according to claim 13 or 14, which suppresses glycolysis of macrophages.
The use according to any one of claims 13 to 15, which promotes aerobic respiration of macrophages.
The use according to any one of claims 13 to 16, further comprising a cakushinin extract.
　Use of nicotinamide or a pharmaceutically acceptable salt thereof and cinnamon extract for the manufacture of a composition for preventing and/or improving pigmentation in the dermis.