Anti-skin aging topical composition and senescent cell removal composition

Abstract

To remove senescent cells, and / or to prevent, suppress, or improve skin aging phenomena such as age spots, wrinkles, sagging, dullness, thinning of the skin, weakening of the skin, decreased skin barrier function, and dryness by removing senescent cells. [Solution] A composition containing lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetate, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinyl palmitate, riboflavin, and pantothenyl ethyl ether, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria.

Description

[Technical Field]

[0001] This disclosure relates to topical compositions for anti-skin aging and compositions for removing senescent cells, etc. [Background technology]

[0002] In recent years, cellular senescence, a phenomenon in which cell proliferation permanently stops, has attracted attention. For example, in analyses using genetically modified mice, p16, which is involved in inducing cellular senescence, has been identified. INK4α It has been reported that removing cells that express the gene reduces the incidence of various age-related diseases such as arteriosclerosis, cataracts, sarcopenia, and cancer (Non-Patent Literature 1).

[0003] Cells whose proliferation has permanently ceased are called senescent cells. Senescent cells not only cease proliferation, but are also known to produce and secrete a variety of physiologically active substances, such as proteases including extracellular matrix-degrading enzymes, inflammatory cytokines, chemokines, growth factors, and exosomes. This phenomenon is known as the "senescence-associated secretory phenotype (SASP)" (Non-patent Literature 1). In this disclosure, physiologically active substances secreted by SASP may be referred to as SASP factors.

[0004] SASP factors secreted by senescent cells have been reported to act on neighboring cells, inducing inflammatory responses, promoting or inhibiting cell proliferation, and inducing cellular senescence. In other words, senescent cells can induce inflammatory responses in neighboring normal (non-senescent) cells or transform normal cells into senescent cells through the secretion of SASP factors. Therefore, it is expected that removing senescent cells that secrete SASP factors will suppress inflammatory responses and cellular senescence in surrounding cells. It should be noted that inflammatory responses in the skin are known to increase melanin production and contribute to the formation of age spots.

[0005] As mentioned above, p16 INK4αGenes are known to be involved in inducing cellular senescence. Non-patent document 2 suggests a correlation between the number of p16INK4a-positive cells in skin tissue and skin aging phenomena such as the morphological characteristics of elastic fibers and the degree of wrinkles.

[0006] Furthermore, senescent cells are known to be SA-β-gal (senescence-associated beta galactsidase) positive. Non-patent document 3 reports that when SA-β-gal staining was performed on skin samples taken from subjects of various ages, the frequency of SA-β-gal positive cells was higher in the group of elderly subjects aged 69 and over than in the group of younger subjects aged under 40. More specifically, SA-β-gal positive cells were not found in dermal samples taken from subjects under 40, while SA-β-gal positive cells were found in almost all dermal samples taken from subjects aged 69 and over. In the epidermis, SA-β-gal positive cells were found in all subjects aged 69 and over, while SA-β-gal positive cells were also found in about half of the subjects under 40. However, even in the samples in the group of subjects under 40 in which SA-β-gal positive cells were found, the frequency was lower than in the group of subjects aged 69 and over.

[0007] Furthermore, in the skin of elderly individuals, phenomena such as thinning of the epidermis and dermis, decreased elasticity of the dermis, and decreased connectivity between the epidermis and dermis occur, making the skin of elderly individuals more susceptible to peeling due to mechanical stimuli and more fragile than the skin of young people (Non-Patent Literature 5). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Eiji Hara, "A New Phase in Cellular Senescence: Potential for a More Complete Understanding and Extending Healthy Lifespan," Experimental Medicine, July 2019, Vol. 37, No. 11, pp. 1728-1734. [Non-Patent Document 2] Waaijer ME et al. "P16INK4a Positive Cells in Human Skin Are Indicative of Local Elastic Fiber Morphology, Facial Wrinkling, and Perceived Age." J Gerontol A Biol Sci Med Sci. 2016 Aug;71(8):1022-1028. [Non-Patent Document 3] Dimri GP et al., "A biomarker that identifies senescent human cells in culture and in aging skin in vivo." Proc Natl Acad Sci US A. 1995 Sep 26;92(20):9363-9367. [Non-Patent Document 4] Akiko Takahashi, "Fundamentals of Cellular Senescence and Trends in Senolytics," Experimental Medicine, February 2022, Vol. 40, No. 3, pp. 364-370. [Non-Patent Document 5] Yuko Mizogami, "Effective Preventive Skin Care for Fragile Skin," Journal of the Japanese Society for Pressure Ulcers (2), pp. 169-174, 2005. [Overview of the project] [Problems that the invention aims to solve]

[0009] As mentioned above, p16 INK4αGenes are known to be involved in inducing cell senescence, and since the relationship between the number of p16INK4a-positive cells in skin tissue and skin aging phenomena has been suggested, it is conceivable that cell senescence and skin aging phenomena such as wrinkles and sagging are related. Also, as described above, it has been reported that SASP factors secreted from senescent cells act on neighboring cells and cause induction of inflammatory reactions and induction of cell senescence, etc. Therefore, by removing senescent cells, induction of inflammatory reactions and cell senescence, etc. in surrounding cells are suppressed, and it is expected that the progression of skin aging phenomena such as spots, wrinkles, sagging, dullness, thinning of the skin, fragility of the skin, decline in skin barrier function, and dryness will be suppressed.

[0010] In view of the above circumstances, the inventors of the present invention mainly aim to remove senescent cells and / or prevent, suppress, improve, etc. skin aging phenomena such as spots, wrinkles, sagging, dullness, thinning of the skin, fragility of the skin, decline in skin barrier function, and dryness by removing senescent cells.

Means for Solving the Problems

[0011] The inventors of the present invention have found that at least one selected from the group consisting of lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetic acid, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinol palmitate, riboflavin, and pantothenyl ethyl ether, and chemically possible salts thereof, and Enterococcus bacteria and Lactobacillus bacteria has an action of removing senescent cells. Then, through repeated improvements, the present disclosure has been completed.

[0012] The present disclosure includes, for example, the subject matters described in the following items. Item 1. An external composition for anti-skin aging, containing lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetic acid, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinol palmitate, riboflavin, and pantothenyl ethyl ether, and chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria. Item 2. A composition for removing aging cells, containing lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetic acid, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinol palmitate, riboflavin, and pantothenyl ethyl ether, and chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria. Item 3. An external composition for anti-skin aging and activating skin cells, containing lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, parthenolide, arbutin, tocotrienol, tocopherol, γ-oryzanol, and retinol acetate, and chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria. Item 4. An external composition for removing aging cells and activating skin cells, containing lactic acid, indole lactic acid, phenyl lactic acid, caffeic acid, parthenolide, arbutin, tocotrienol, tocopherol, γ-oryzanol, and retinol acetate, and chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria. Item 5. The composition according to any one of Items 1 to 4, which is used for humans aged 4**0** years or older.

Advantages of the Invention

[0013] According to the technology disclosed herein, senescent cells can be removed, and skin aging phenomena such as age spots, wrinkles, sagging, dullness, thinning of the skin, weakening of the skin, decreased skin barrier function, and dryness can be prevented, suppressed, or improved. [Brief explanation of the drawing]

[0014] [Figure 1] The results of Test Example 1-2 are shown. In the figure, "NHEK" represents NHEK before the procedure in Test Example 1-1 (i.e., untreated), and "SHEK" represents the cells prepared in Test Example 1-1 (i.e., senescent cells). [Figure 2] The results for venetoclax (labeled "ABT-199" in the figure) and navitoclax (labeled "ABT-263" in the figure), which were positive controls, are shown in the results of Test Example 2. The top of the bar graph represents the mean value of cell viability, and the error bars represent ± standard deviation. "DMSO 1 / 1000" indicates that dimethyl sulfoxide (DMSO) was added in an amount equal to 1 / 1000 of the total volume of the evaluation system (the same applies hereafter). [Figure 3] The results for DL-lactic acid (A), DL-indolelactic acid (B), and phenyllactic acid (C) from Test Example 2 are shown below. [Figure 4] The results for catechin (A), caffeic acid (B), umbelliferone (C), and parthenolide (D) from Test Example 2 are shown below. [Figure 5] The results for pyridoxine hydrochloride (A), arbutin (B), hinokitiol (C), and oxaloacetate (D) from Test Example 2 are shown below. [Figure 6] The results for γ-tocotrienol (A), α-tocotrienol (B), α-tocopherol (C), and δ-tocopherol (D) from Test Example 2 are shown below. [Figure 7] The results for γ-oryzanol (A), retinol acetate (B), and retinyl palmitate (C) from Test Example 2 are shown below. [Figure 8]The results for riboflavin (A), pantothenyl ethyl ether (B), Enterococcus faecalis powder (C), and Lactobacillus casei powder (D) from Test Example 2 are shown below. [Modes for carrying out the invention]

[0015] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, topical compositions for anti-skin aging, compositions for senescent cell removal, topical compositions for anti-skin aging and skin cell activation, and compositions for senescent cell removal and skin cell activation, and so on. This disclosure includes everything disclosed herein and recognizable to those skilled in the art. Hereinafter, the topical compositions for anti-skin aging and compositions for senescent cell removal included in this disclosure may be collectively referred to as "the compositions of this disclosure."

[0016] 1. Function / Application The compositions of this disclosure may exhibit a senescent cell scavenging effect. In the technology of this disclosure, the senescent cell scavenging effect is achieved by inhibiting the survival of senescent cells. Whether or not a component has a senescent cell scavenging effect can be confirmed by adding the component to a culture medium and culturing senescent cells. That is, if the survival rate of senescent cells when cultured with the component added is lower than the survival rate of senescent cells when cultured without the component, then the component is determined to have a senescent cell scavenging effect.

[0017] Senescent cells can be prepared, for example, by following the method described in the examples below. Specifically, senescent cells can be prepared by inducing cellular senescence by sequentially adding palbociclib, nutlin-3a, and BI-2536 to the culture medium of normal human neonatal epidermal keratinocytes (NHEK). The fact that a cell is a senescent cell can be confirmed, for example, by being SA-β-gal positive.

[0018] Furthermore, as shown in the examples described later, lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, parthenolide, arbutin, γ-tocotrienol, α-tocopherol, γ-oryzanol, and retinol acetate, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria, can exert not only a senescent cell removal effect but also a skin cell activating effect.

[0019] In the technology of this disclosure, whether or not a certain component has a skin cell activating effect can be confirmed by adding the component to a culture medium and culturing normal skin cells. That is, if the survival rate and / or proliferation rate of normal skin cells when cultured with the component added is higher than the survival rate and / or proliferation rate of normal skin cells when cultured without the component added, then the component is determined to have a skin cell activating effect.

[0020] As described above, senescent cells can trigger inflammatory responses in neighboring normal (non-senescent) cells or transform normal cells into senescent cells through the secretion of SASP factors. Therefore, it is expected that the compositions of this disclosure, which have senescent cell-removing properties, will suppress inflammatory responses and cellular senescence caused by SASP factors. In other words, the compositions of this disclosure may also exert anti-skin aging effects.

[0021] In this disclosure, the term "skin aging" refers to skin changes associated with aging. Specifically, skin changes associated with aging include, in terms of appearance, an increase in the number of age spots, worsening of the severity of age spots (size, darkness, etc.), an increase in the number of wrinkles, worsening of the severity of wrinkles (depth, length, etc.), decreased elasticity (firmness), sagging, dullness, etc., and in terms of changes in skin tissue, a decrease or degeneration of collagen and elastin, thinning of the skin, weakening of the skin, decreased skin barrier function, dryness, etc. In this disclosure, the term "anti-skin aging" encompasses preventing, suppressing, and improving skin aging. Furthermore, such anti-skin aging can be achieved through skin cell activation effects, senescent cell removal effects, etc.

[0022] As described above, Non-Patent Literature 3 reports that when SA-β-gal staining was performed on skin samples taken from subjects of various ages, the frequency of SA-β-gal positive cells was higher in the group of elderly subjects aged 69 and over than in the group of younger subjects under 40 years of age. In other words, senescent cells tend to increase with age. The compositions of this disclosure are preferably applied to people aged 40 years or older, more preferably to people aged 45 years or older, even more preferably to people aged 50 years or older or 55 years or older, and particularly preferably to people aged 60 years or older or 65 years or older. The lower limit of the above range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years of age.

[0023] Furthermore, preferred examples of those to whom the compositions disclosed herein are applicable include people who want to prevent skin aging, people who want to suppress skin aging, people who want to improve skin aging, people who are concerned about age spots, people who want to prevent age spots, people who want to suppress the progression of age spots, people who want to improve age spots, people who are concerned about wrinkles, people who want to prevent wrinkles, people who want to suppress the progression of wrinkles, people who want to improve wrinkles, people who are concerned about sagging, people who want to prevent sagging, people who want to suppress the progression of sagging, people who want to improve sagging, people who are concerned about dullness, people who want to prevent dullness, people who want to suppress the progression of dullness, people who want to improve dullness, people who are concerned about disruptions in skin cell turnover, people who want to improve disruptions in skin cell turnover, and people who are interested in anti-aging.

[0024] 2. Active ingredients The compositions of this disclosure contain lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetate, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinyl palmitate, riboflavin, and pantothenyl ethyl ether, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria. The above components may be collectively referred to as the "active ingredients of this disclosure."

[0025] In the technology of the present disclosure, the content of the active ingredient of the present disclosure is not particularly limited as long as the desired effect is obtained, and may be, for example, 0.0000001 to 5% by mass, 0.000001 to 2% by mass, 0.00001 to 1% by mass, 0.00005 to 0.5% by mass, 0.0001 to 0.2% by mass, 0.0001 to 0.1% by mass, or 0.0001 to 0.05% by mass. The upper or lower limits of the range are, for example, 0.0000001, 0.0000005, 0.000001, 0.000005, 0.00001, 0.00005, 0.0001, 0.00015, 0.0002, 0.00025, 0.0003, 0.00035, 0.0004, 0.00045, 0.0005, 0.00055, 0.0006, 0.00065, 0.0007, 0.00075, 0.0008, 0.00085, 0.0009, 0.00095, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.0 It may also be 0.4, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% by mass.

[0026] Furthermore, the content of the active ingredient in this disclosure may be, for example, 0.01 μg / mL to 100 mg / mL, 0.01 μg / mL to 10 mg / mL, 0.1 μg / mL to 1 mg / mL, 1 to 500 μg / mL, or 10 to 100 μg / mL. The upper or lower limits of the range may be, for example, 0.01, 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, or 900 μg / mL, or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 mg / mL.

[0027] Furthermore, the content of the active ingredient in this disclosure may be, for example, 0.1 μM to 1 M, 1 μM to 100 mM, 10 μM to 10 mM, or 100 μM to 1 mM. The upper or lower limits of the above range may be, for example, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, or 980 μM, or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 30 0, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, or 980 mM, or 1 M.

[0028] Lactic acid The lactic acid used in the technology of this disclosure may be the D-isomer, the L-isomer, or the DL-isomer. There are no particular limitations, but the lactic acid used in the technology of this disclosure is preferably the DL-isomer. There are no particular limitations, but if the composition of this disclosure contains lactic acid, the content may be 0.01 to 1% by mass, preferably 0.02 to 0.5% by mass, more preferably 0.04 to 0.4% by mass, even more preferably 0.05 to 0.3% by mass, and particularly preferably 0.07 to 0.2% by mass.

[0029] Indole lactic acid In this disclosure, indole-3-lactic acid is simply referred to as "indole-lactic acid." The indole-lactic acid used in the technology of this disclosure may be the D-isomer, the L-isomer, or the DL-isomer. Although not particularly limited, the indole-lactic acid used in the technology of this disclosure is preferably the DL-isomer. If the composition of this disclosure contains indole-lactic acid, its content may be 0.001 to 1% by mass, 0.001 to 0.5% by mass, preferably 0.001 to 0.3% by mass, more preferably 0.002 to 0.2% by mass, even more preferably 0.005 to 0.15% by mass, particularly preferably 0.008 to 0.12% by mass, and most preferably 0.01 to 0.1% by mass. Furthermore, if the composition of this disclosure contains indole lactic acid, its content may be 0.001 to 10 mg / mL, 0.01 to 5 mg / mL, preferably 0.01 to 3 mg / mL, more preferably 0.02 to 2 mg / mL, even more preferably 0.05 to 1.5 mg / mL, particularly preferably 0.08 to 1.2 mg / mL, and most preferably 0.1 to 1 mg / mL.

[0030] Phenyllactic acid In this disclosure, 3-phenyllactic acid is simply referred to as "phenyllactic acid." The phenyllactic acid used in the technology of this disclosure may be the D-isomer, the L-isomer, or the DL-isomer. Although not particularly limited, if the composition of this disclosure contains phenyllactic acid, the content may be 0.001 to 1% by mass, 0.001 to 0.5% by mass, preferably 0.005 to 0.3% by mass, more preferably 0.01 to 0.2% by mass, even more preferably 0.015 to 0.15% by mass, particularly preferably 0.02 to 0.12% by mass, and most preferably 0.025 to 0.1% by mass. Furthermore, if the composition of this disclosure contains phenyllactic acid, its content may be 0.001 to 1% by mass, 0.01 to 5 mg / mL, preferably 0.05 to 3 mg / mL, more preferably 0.1 to 2 mg / mL, even more preferably 0.15 to 1.5 mg / mL, particularly preferably 0.2 to 1.2 mg / mL, and most preferably 0.25 to 1 mg / mL.

[0031] Catechin In this disclosure, the term "catechin" refers to catechin in the narrow sense and refers to a compound having the following structure. The structure of (-)-catechin is shown on the left, and the structure of (+)-catechin is shown on the right. In this disclosure, the term "catechin" includes both enantiomers of (-)-catechin and (+)-catechin. There are no particular limitations, but in this disclosure, the use of (-)-catechin is particularly preferred. The molar mass of catechin is 290.27 g / mol. Catechin is generally available on the market, and can be purchased and used from products commercially available under names such as "(-)-catechin, derived from green tea (Fujifilm Wako Pure Chemical Industries, Ltd.)". [ka]

[0032] While not particularly limited, if the composition of the present disclosure contains catechin, its content may be 0.0001 to 1% by mass, 0.0001 to 0.2% by mass, preferably 0.0005 to 0.1% by mass, more preferably 0.001 to 0.07% by mass, even more preferably 0.002 to 0.05% by mass, particularly preferably 0.03 to 0.04% by mass, and most preferably 0.005 to 0.03% by mass. Furthermore, if the composition of the present disclosure contains catechin, its content may be 0.001 to 10 mM, 0.01 to 5 mM, preferably 0.05 to 3 mM, more preferably 0.1 to 2 mM, even more preferably 0.15 to 1.5 mM, particularly preferably 0.17 to 1.2 mM, and most preferably 0.2 to 1 mM.

[0033] Caffeic acid Caffeic acid, also known as 3,4-dihydroxycinnamic acid, is a compound with a molar mass of 180.16 g / mol. The structure of caffeic acid is shown below. [ka]

[0034] While not particularly limited, if the composition of the present disclosure contains caffeic acid, its content may be 0.0001 to 1% by mass, 0.0001 to 0.5% by mass, preferably 0.0001 to 0.1% by mass, more preferably 0.0005 to 0.05% by mass, even more preferably 0.0005 to 0.04% by mass, particularly preferably 0.001 to 0.03% by mass, and most preferably 0.0015 to 0.02% by mass. Furthermore, if the composition of the present disclosure contains caffeic acid, its content may be 0.001 to 10 mM, 0.005 to 5 mM, preferably 0.02 to 3 mM, more preferably 0.05 to 2 mM, even more preferably 0.07 to 1.5 mM, particularly preferably 0.08 to 1.2 mM, and most preferably 0.1 to 1 mM.

[0035] Umbelliferon Umbelliferone, also known as 7-hydroxycoumarin, is a compound with a molar mass of 162.14 g / mol. The structure of umbelliferone is shown below. [ka]

[0036] While not particularly limited, if the composition of the present disclosure contains umbelliferone, its content may be 0.0001 to 1% by mass, 0.0001 to 0.5% by mass, preferably 0.0001 to 0.1% by mass, more preferably 0.0005 to 0.05% by mass, even more preferably 0.0005 to 0.02% by mass, particularly preferably 0.0005 to 0.015% by mass, and most preferably 0.0008 to 0.01% by mass. Furthermore, if the composition of the present disclosure contains umbelliferone, its content may be 1 μM to 10 mM, 1 μM to 5 mM, preferably 5 μM to 2.5 mM, more preferably 10 μM to 1.5 mM, even more preferably 25 μM to 1 mM, particularly preferably 40 μM to 0.8 mM, and most preferably 50 μM to 0.5 mM.

[0037] Parthenolide Parthenolide is a compound with a molar mass of 248.32 g / mol. The structure of parthenolide is shown below. [ka]

[0038] While not particularly limited, if the composition of the present disclosure contains parthenolide, the content may be 0.0000001 to 1% by mass, 0.0000001 to 0.1% by mass, preferably 0.0000005 to 0.01% by mass, more preferably 0.000001 to 0.005% by mass, even more preferably 0.000005 to 0.001% by mass, particularly preferably 0.00001 to 0.0005% by mass, and most preferably 0.00002 to 0.0003% by mass. Furthermore, if the composition of this disclosure contains parthenolide, its content may be 0.001 μM to 1 mM, 0.005 to 500 μM, preferably 0.01 to 200 μM, more preferably 0.05 to 100 μM, even more preferably 0.1 to 50 μM, particularly preferably 0.5 to 20 μM, and most preferably 1 to 10 μM.

[0039] Pyridoxine hydrochloride Pyridoxine hydrochloride is the hydrochloride salt of pyridoxine. The structure of pyridoxine is shown below. [ka]

[0040] While not particularly limited, if the composition of this disclosure contains pyridoxine hydrochloride, its content may be 0.0001 to 5% by mass, preferably 0.0005 to 2% by mass, more preferably 0.001 to 1% by mass, even more preferably 0.002 to 0.5% by mass, particularly preferably 0.005 to 0.2% by mass, and most preferably 0.007 to 0.15% by mass. Furthermore, if the composition of this disclosure contains pyridoxine hydrochloride, its content may be 0.0001 to 100 mg / mL, preferably 0.001 to 50 mg / mL, more preferably 0.005 to 20 mg / mL, more preferably 0.01 to 10 mg / mL, even more preferably 0.02 to 5 mg / mL, particularly preferably 0.05 to 2 mg / mL, and most preferably 0.07 to 1.5 mg / mL.

[0041] Arbutin Arbutin is a glucosylated hydroquinone with a molar mass of 272.25 g / mol. Arbutin exists in α- and β-forms, and the arbutin used in the techniques of this disclosure may be α-form, β-form, or a mixture of α- and β-forms. The structure of α-arbutin is shown below. [ka]

[0042] While not particularly limited, if the composition of the present disclosure contains arbutin, its content may be 0.0001 to 5% by mass, preferably 0.001 to 2% by mass, more preferably 0.005 to 1% by mass, even more preferably 0.01 to 0.5% by mass, particularly preferably 0.02 to 0.4% by mass, and most preferably 0.025 to 0.3% by mass. Furthermore, if the composition of the present disclosure contains arbutin, its content may be 0.01 mM to 1 M, preferably 0.01 to 500 mM, more preferably 0.05 to 100 mM, even more preferably 0.1 to 50 mM, even more preferably 0.5 to 20 mM, particularly preferably 0.8 to 15 mM, and most preferably 1 to 10 mM.

[0043] Hinokitiol Hinokitiol is a compound with a 7-membered ring and a molar mass of 164.2 g / mol. The structure of hinokitiol is shown below. [ka]

[0044] While not particularly limited, if the composition of the present disclosure contains hinokitiol, its content may be 0.000001 to 1% by mass, 0.000001 to 0.1% by mass, preferably 0.000001 to 0.01% by mass, more preferably 0.000001 to 0.001% by mass, even more preferably 0.000005 to 0.0005% by mass, particularly preferably 0.00001 to 0.0002% by mass, and most preferably 0.000015 to 0.0001% by mass. Furthermore, if the composition of this disclosure contains hinokitiol, its content may be 0.001 to 1 mM, 0.001 to 100 μM, preferably 0.005 to 50 μM, more preferably 0.01 to 20 μM, even more preferably 0.05 to 10 μM, particularly preferably 0.08 to 8 μM, and most preferably 1 to 5 μM.

[0045] Oxaloacetate Oxaloacetic acid is a kind of dicarboxylic acid, and its molar mass is 132.07 g / mol. The structure of oxaloacetic acid is shown below. [Chemical formula] <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​(This is a methyl group.)

[0048] The tocotrienol used in the technology of this disclosure may be any of α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol, with α-tocotrienol and / or γ-tocotrienol being preferred. Furthermore, the tocotrienol used in the technology of this disclosure may be the D-isomer, the L-isomer, or the DL-isomer.

[0049] While not particularly limited, if the composition of this disclosure contains tocotrienols, the content may be 0.000001 to 1% by mass, 0.000001 to 0.1% by mass, preferably 0.00001 to 0.01% by mass, more preferably 0.00002 to 0.005% by mass, even more preferably 0.00003 to 0.002% by mass, particularly preferably 0.00004 to 0.0015% by mass, and most preferably 0.00005 to 0.001% by mass. Furthermore, if the composition of this disclosure contains tocotrienol, its content may be 0.01 to 1000 μg / mL, 0.01 to 500 μg / mL, preferably 0.1 to 100 μg / mL, more preferably 0.2 to 50 μg / mL, even more preferably 0.3 to 20 μg / mL, particularly preferably 0.4 to 15 μg / mL, and most preferably 0.5 to 10 μg / mL.

[0050] Tocopherol The structure of tocopherol is shown below. [ka] (Here, α-tocopherol is R 1 , R 2 , and R 3 All of them are methyl groups; β-tocopherol is R 1 is a methyl group, R 2 is a hydrogen atom, R 3 The methyl group is; γ-tocopherol is R 1is a hydrogen atom, R 2 and R 3 The methyl group is; δ-tocopherol is R 1 and R 2 is a hydrogen atom, R 3 (This is a methyl group.)

[0051] The molar mass of α-tocopherol is 430.79 g / mol, and the molar mass of δ-tocopherol is 402.35 g / mol. The tocopherol used in the technology of this disclosure may be any of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, and α-tocopherol and / or δ-tocopherol are preferred. Furthermore, the tocopherol used in the technology of this disclosure may be the D-isomer, the L-isomer, or the DL-isomer.

[0052] While not particularly limited, if the composition of this disclosure contains tocopherol, its content may be 0.00001 to 1% by mass, 0.00001 to 0.1% by mass, preferably 0.00005 to 0.05% by mass, more preferably 0.0001 to 0.02% by mass, even more preferably 0.0002 to 0.01% by mass, particularly preferably 0.0003 to 0.005% by mass, and most preferably 0.0004 to 0.0045% by mass. Furthermore, if the composition of this disclosure contains tocopherol, its content may be 0.5 to 2000 μM, preferably 1 to 1000 μM, more preferably 2 to 500 μM, even more preferably 5 to 200 μM, particularly preferably 7 to 150 μM, and most preferably 10 to 100 μM.

[0053] γ-oryzanol γ-oryzanol is a general term for esters formed by the condensation of ferulic acid and sterols, and is mainly found in rice bran. γ-oryzanol is generally available on the market, and can be purchased and used under names such as "γ-oryzanol (Fujifilm Wako Pure Chemical Industries, Ltd.)". The main γ-oryzanols found in rice bran are cycloartenyl ferulate (oryzanol A), 24-methylenecycloartenyl ferulate (oryzanol C), and campesteryl ferulate, which together account for approximately 80% of the γ-oryzanol contained in rice bran.

[0054] The structure of cycloartenyl ferulate is shown below. [ka] The structure of 24-methylenecycloartenyl ferulate is shown below. [ka] [ka] The structure of campesteryl ferulate is shown below.

[0055] The molar mass of cycloartenyl ferulate is 602.9 g / mol, the molar mass of 24-methylenecycloartenyl ferulate is 616.9 g / mol, and the molar mass of campesteryl ferulate is 576.8 g / mol. The type of γ-oryzanol used in the art of this disclosure is not particularly limited, but it is preferable to include at least one selected from the group consisting of cycloartenyl ferulate, 24-methylenecycloartenyl ferulate, and campesteryl ferulate, and more preferably to include cycloartenyl ferulate.

[0056] While not particularly limited, if the composition of this disclosure contains γ-oryzanol, its content may be 0.00001 to 1% by mass, preferably 0.00001 to 0.5% by mass, more preferably 0.00005 to 0.1% by mass, even more preferably 0.0001 to 0.05% by mass, particularly preferably 0.0005 to 0.01% by mass, and most preferably 0.001 to 0.005% by mass. Furthermore, if the composition of this disclosure contains γ-oryzanol, its content may be 1 μM to 1 mM, preferably 1 to 500 μM, more preferably 5 to 200 μM, even more preferably 10 to 100 μM, particularly preferably 15 to 80 μM, and most preferably 20 to 50 μM.

[0057] Retinol acetate Retinyl acetate is the acetate ester of retinol, with a molar mass of 328.49 g / mol. The structure of retinyl acetate is shown below. [ka]

[0058] While not particularly limited, if the composition of this disclosure contains retinol acetate, its content may be 0.000001 to 1% by mass, 0.000001 to 0.5% by mass, preferably 0.000005 to 0.1% by mass, more preferably 0.00001 to 0.05% by mass, even more preferably 0.00005 to 0.01% by mass, particularly preferably 0.0001 to 0.005% by mass, and most preferably 0.00015 to 0.002% by mass. Furthermore, if the composition of this disclosure contains retinol acetate, its content may be 0.1 μM to 10 mM, preferably 0.5 μM to 1 mM, more preferably 1 to 500 μM, even more preferably 2 to 200 μM, particularly preferably 3 to 100 μM, and most preferably 5 to 50 μM.

[0059] Retinyl palmitate Retinyl palmitate is a palmitic acid ester of retinol, with a molar mass of 524.86 g / mol. The structure of retinyl palmitate is shown below. [ka]

[0060] While not particularly limited, if the composition of the present disclosure contains retinyl palmitate, its content may be 0.00001 to 1% by mass, preferably 0.00001 to 0.5% by mass, more preferably 0.00001 to 0.1% by mass, even more preferably 0.00005 to 0.05% by mass, particularly preferably 0.0001 to 0.01% by mass, and most preferably 0.0005 to 0.006% by mass. Furthermore, if the composition of the present disclosure contains retinyl palmitate, its content may be 0.01 μM to 10 mM, preferably 0.01 μM to 5 mM, preferably 0.1 μM to 2 mM, more preferably 0.5 μM to 1 mM, even more preferably 1 to 500 μM, particularly preferably 5 to 200 μM, and most preferably 10 to 100 μM.

[0061] Riboflavin Riboflavin is a compound also known as vitamin B2, with a molar mass of 376.36 g / mol. The structure of riboflavin is shown below. [ka]

[0062] While not particularly limited, if the composition of the present disclosure contains riboflavin, its content may be 0.00001 to 1% by mass, preferably 0.00005 to 0.5% by mass, more preferably 0.0001 to 0.1% by mass, even more preferably 0.0005 to 0.05% by mass, particularly preferably 0.001 to 0.01% by mass, and most preferably 0.0015 to 0.005% by mass. Furthermore, if the composition of the present disclosure contains riboflavin, its content may be 0.1 μM to 10 mM, preferably 0.1 μM to 5 mM, preferably 1 μM to 1 mM, more preferably 5 to 500 μM, even more preferably 10 to 200 μM, particularly preferably 30 to 150 μM, and most preferably 50 to 100 μM.

[0063] Pantothenyl ethyl ether Pantothenyl ethyl ether is a compound with a molar mass of 233.30 g / mol. The structure of pantothenyl ethyl ether is shown below. [ka]

[0064] While not particularly limited, if the composition of the present disclosure contains pantothenyl ethyl ether, its content may be 0.0001 to 1% by mass, 0.0001 to 0.5% by mass, preferably 0.0005 to 0.1% by mass, more preferably 0.001 to 0.05% by mass, even more preferably 0.005 to 0.01% by mass, particularly preferably 0.0001 to 0.001% by mass, and most preferably 0.0002 to 0.0005% by mass. Furthermore, if the composition of the present disclosure contains pantothenyl ethyl ether, its content may be 0.01 μM to 10 mM, 0.01 μM to 5 mM, preferably 0.1 μM to 1 mM, more preferably 0.5 to 500 μM, even more preferably 1 to 200 μM, particularly preferably 5 to 100 μM, and most preferably 10 to 50 μM.

[0065] Chemically possible salts In the technology of this disclosure, each of the above-mentioned compounds may optionally take the form of a chemically possible salt. Specific examples of salts include, for example, salts with inorganic bases such as aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, potassium salts, sodium salts, and zinc salts. Also, for example, salts with organic bases include salts of primary, secondary, or tertiary amines. Specific examples of amines include, for example, arginine, betaine, caffeine, choline-N-N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine-N-ethylmorpholine-N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine. Examples of salts produced by inorganic acids include hydrochlorides, hydrobroms, sulfates, phosphates, and nitrates. Examples of salts produced by organic acids include acetates, benzoates, tartrates, maleates, fumarates, succinates, citrates, oxalates, p-toluenesulfonates, benzenesulfonates, methanesulfonates, and trifluoroacetates.

[0066] Enterococcus bacteria Enterococcus bacteria are classified as lactic acid bacteria and are Gram-positive cocci. In the technology disclosed herein, it is preferable to use Enterococcus faecalis among the Enterococcus bacteria. In the technology disclosed herein, the Enterococcus bacteria may be live or dead. In the technology disclosed herein, the Enterococcus bacteria may or may not have undergone crushing, heating, drying (freeze-drying, vacuum drying, spray drying, etc.), freezing, lysis, extraction treatment, etc. An example of a commercially available Enterococcus faecalis powder is "La Flora EC-12 (Ichimaru Falcos Co., Ltd.)".

[0067] While not particularly limited, if the composition of the present disclosure contains Enterococcus bacteria, the content may be 0.001 to 20% by mass on a dry weight basis, preferably 0.01 to 10% by mass, more preferably 0.2 to 5% by mass, even more preferably 0.05 to 2% by mass, particularly preferably 0.07 to 1.5% by mass, and most preferably 0.1 to 1% by mass. Furthermore, if the composition of the present disclosure contains Enterococcus bacteria, the content may be 0.01 to 200 mg / mL, preferably 0.1 to 100 mg / mL, more preferably 0.2 to 50 mg / mL, even more preferably 0.5 to 20 mg / mL, particularly preferably 0.7 to 15 mg / mL, and most preferably 1 to 10 mg / mL.

[0068] Lactobacillus bacteria Lactobacillus bacteria are classified as lactic acid bacteria. In the technology disclosed herein, it is preferable to use Lactobacillus casei among Lactobacillus bacteria. In the technology disclosed herein, Lactobacillus bacteria may be live or dead. In the technology disclosed herein, Lactobacillus bacteria may or may not be subjected to crushing, heating, drying (freeze-drying, vacuum drying, spray drying, etc.), freezing, lysis, extraction treatment, etc. An example of a commercially available Lactobacillus casei powder is "La Flora K1 (Ichimaru Falcos Co., Ltd.)".

[0069] While not particularly limited, if the composition of the present disclosure contains Lactobacillus bacteria, the content may be 0.001 to 20% by mass on a dry weight basis, preferably 0.01 to 10% by mass, more preferably 0.2 to 5% by mass, even more preferably 0.05 to 2% by mass, particularly preferably 0.07 to 1.5% by mass, and most preferably 0.1 to 1% by mass. Furthermore, if the composition of the present disclosure contains Lactobacillus bacteria, the content may be 0.01 to 200 mg / mL, preferably 0.1 to 100 mg / mL, more preferably 0.2 to 50 mg / mL, even more preferably 0.5 to 20 mg / mL, particularly preferably 0.7 to 15 mg / mL, and most preferably 1 to 10 mg / mL.

[0070] 3. Preparation of the compositions of this disclosure The compositions of this disclosure may contain the above-described active ingredients of this disclosure individually or in combination of two or more. Furthermore, the compositions of this disclosure may contain known components that can be included in topical compositions, to the extent that they do not impair the effects of this disclosure. Examples of such known components include water, humectants, oils, surfactants, thickeners, film-forming agents, preservatives, pH adjusters, chelating agents, antioxidants, colorants, fragrances, cooling agents, UV absorbers, various powders, and other active ingredients. The compositions of this disclosure may optionally contain one or more of these known components.

[0071] Examples of humectants include sugars such as trehalose, lactulose, and maltitol; sugar alcohols such as sorbitol, mannitol, and maltitol; and polyhydric alcohols such as propanediol, diethylene glycol, polyethylene glycol, 1,3-butylene glycol, glycerin, and diglycerin. Humectants can be used individually or in combination of two or more.

[0072] Examples of oils include vegetable oils, triglycerides, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, and silicone oils. These oils can be used individually or in combination of two or more.

[0073] Examples of surfactants include anionic surfactants such as polyoxyethylene alkyl ether sulfate, alkyl sulfate ester, alkylamide ether sulfate, polyoxyethylene alkylamide ether sulfate, α-olefin sulfonate, alkyl sulfosuccinate, polyoxyethylene alkyl ether acetate, alkyl phosphate, polyoxyethylene alkyl ether phosphate, higher fatty acid salt, N-acyl amino acid salt, N-acyl isethionate, and N-acyl methyl taurate; Cationic surfactants such as quaternary ammonium salts; Amphoteric surfactants such as amidopropyl betaine type, amidoamine oxide type, sulfobetaine type, imidazoline type, and alkylbetaine type: Examples of nonionic surfactants include polyoxyalkylene alkyl ethers, polyoxyalkylene glycols, polyoxyalkylene fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyoxyalkylene glycerin fatty acid esters, polyoxyalkylene fatty acid amides, polyoxyalkylene glycol fatty acid esters, polyoxyalkylene castor oil derivatives, polyoxyalkylene hydrogenated castor oil derivatives, polyglycerin fatty acid esters, sorbitan fatty acid esters, sorbitan fatty acid esters, sucrose fatty acid esters, alkyl glycol fatty acid esters, alkyl polyglycosides, fatty acid alkanolamides, and others.

[0074] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, as well as sodium benzoate, phenoxyethanol, and alkyldiaminoethylglycine hydrochloride.

[0075] Examples of pH adjusting agents include citric acid, phosphoric acid, malic acid, pyrophosphate, lactic acid, tartaric acid, phytic acid, glycerophosphate, acetic acid, nitric acid, or chemically possible salts thereof, as well as sodium hydroxide, potassium hydroxide, and the like.

[0076] Examples of chelating agents include edetic acid, pentetic acid, metaphosphoric acid, gluconic acid, etidronic acid, or chemically possible salts thereof.

[0077] Examples of coloring agents include legally approved pigments such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3.

[0078] Examples of fragrances include menthol, anethole, carvone, eugenol, limonene, wintergreen, cineole, clove oil, rosemary oil, lemon oil, orange oil, ocimene oil, citronellol, and methyl eugenol.

[0079] Examples of cooling agents include menthol, spearmint oil, camphor, thymol, and methyl salicylate.

[0080] The compositions disclosed herein may be pharmaceutical compositions or cosmetic compositions. For example, they may be skincare agents, haircare agents, bath additives, etc. More specifically, they may be lotions, toners, serums, emulsions, creams, ointments, pastes, gels, packs, compresses, sprays, shampoos, conditioners, hair growth agents, scalp lotions, etc.

[0081] The form of the composition of this disclosure is not particularly limited, but may be liquid, cream, gel, paste, semi-solid, solid, powder, etc.

[0082] The compositions of this disclosure can be prepared by known methods or by methods readily conceivable by those skilled in the art from known methods. For example, they can be prepared by mixing the active ingredients and other components of this disclosure. Mixing can be carried out using a known stirrer (e.g., a disper mixer).

[0083] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0084] Furthermore, the various characteristics (properties, numerical values, structure, function, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0085] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0086] Test Example 1. Preparation of senescent cells 1-1. Cellular senescence was induced using normal human neonatal epidermal keratinocytes (NHEK, Lifeline Cell Technology) by known methods. Specifically, cellular senescence was induced using the following methods. Unless otherwise specified, cell culture was performed in a 37°C CO2 5v / v% incubator. (1) Place 8.0 x 10 NHEK in a 96-well plate. 3 The cells were seeded in a cell / well and cultured for 1 day. HuMedia(trademark)-KG2 (Cat. No. KK-2150S, Kurabo Corporation) was used as the culture medium. (2) After removing the culture medium, the medium was replaced with HuMedia-KB2 (Cat. No. KK-2350S, Kurabo Industries Ltd.) containing 1.5 μM Palbociclib (Cat. No. PZ0383, Merck) and 1 v / v% dimethyl sulfoxide (DMSO), and incubated for 24 hours. (3) Remove the culture medium and replace it with HuMedia-KB2 containing 1.5 μM Palbociclib, 9 μM Nutlin-3a (Cat. No. SML0580, Merck), and 1 v / v% DMSO, and incubate for 1 day. (4) Remove the culture medium and replace it with HuMedia-KB2 containing Nutlin-3a 9 μM, BI-2536 (Cat. No. HY-50698, MedChemExpress) 500 nM, and DMSO 1 v / v%, and incubate for 1 day. (5) Remove the culture medium and replace it with HuMedia-KG2 containing BI-2536 500nM and DMSO 1v / v%, and incubate for 3 days. (6) Remove the culture medium, replace it with fresh HuMedia-KG2, and incubate for another 2 days.

[0087] 1-2. The cells prepared in Test Example 1-1, and the NHEK samples before the procedure in Test Example 1-1 (i.e., untreated samples), were each stained with SA-β-gal using the following method. (1) The culture medium in the wells was aspirated and washed twice with PBS. (2) Add the 1× Fixing Solution included with the Cellular Senescence Assay Kit (Cat. No. CBA-230, CELL BIOLABS) and fix at room temperature for 5 minutes. (3) Remove the Fixing Solution and wash three times with PBS. (4) The Cell Staining Working Solution included with the Cellular Senescence Assay Kit was added until the cells were completely submerged. (5) The reaction was carried out overnight in a 37°C incubator, shielded from light. (6) Remove the Cell Staining Working Solution, wash twice with PBS, and fill the wells with PBS. (7) The samples were observed under an optical microscope to confirm whether or not they were stained blue. The resulting micrograph is shown in Figure 1.

[0088] 1-3. As shown in Figure 1, the results of Test Example 1-2 revealed that almost all cells prepared in Test Example 1-1 were SA-β-gal stain-positive, whereas no SA-β-gal stain-positive cells were observed in the NHEK cells before the procedure in Test Example 1-1. This confirms that cellular senescence is induced by the method described in Test Example 1-1, and that normal NHEK cells become senescent cells. Therefore, cells prepared by the method described in Test Example 1-1 were used as senescent cells in subsequent experiments. In this example, senescent cells prepared by the method described in Test Example 1-1 may be referred to as SHEK.

[0089] Test Example 2. Senescent Cell Removal Test 2-1. SHEK was cultured with or without the addition of DL-lactic acid, DL-indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetate, γ-tocotrienol, α-tocotrienol, α-tocopherol, δ-tocopherol, γ-oryzanol, retinyl acetate, retinyl palmitate, riboflavin, pantothenyl ethyl ether, Enterococcus faecalis powder, or Lactobacillus casei powder (hereinafter collectively referred to as "test substances" in this example) to the culture medium. For the catechins used, we used "(-)-catechin, derived from green tea (Fujifilm Wako Pure Chemical Corporation)," for the γ-oryzanol we used "γ-oryzanol (Fujifilm Wako Pure Chemical Corporation)," for the Enterococcus faecalis powder we used "La Flora EC-12 (Ichimaru Pharcos Co., Ltd.)," and for the Lactobacillus casei powder we used "La Flora K1 (Ichimaru Pharcos Co., Ltd.)." In addition, as a positive control, we used venetoclax (labeled "ABT-199" in the figure) or navitoclax (labeled "ABT-263" in the figure), which are Bcl-2 inhibitors that have been reported to have senescent cell-removing effects.

[0090] Water-soluble test substances were dissolved in HuMedia-KB2 medium before being added to the culture medium containing cells. Enterococcus faecalis powder and Lactobacillus casei powder were suspended in HuMedia-KB2 medium before being added to the culture medium containing cells. Oil-soluble test substances were dissolved in dimethyl sulfoxide (DMSO) or ethanol (EtOH) to a concentration 100 times or 1000 times their final concentration before being added to the culture medium containing cells. The concentrations of each test substance added are shown in Figures 2 to 8. The "%" in the figures represents mass percent. HuMedia-KB2 medium (simply labeled "KB2" in the figures) was used as a control for water-soluble test substances. The solvent used to dissolve the oil-soluble test substance (DMSO or ethanol) was used as a control for the oil-soluble test substance. The density of the culture medium was approximately 1 g / mL.

[0091] The specific method for culturing SHEK is shown below. (1) Remove the culture medium from the well containing SHEK obtained in Test Example 1-1 and wash once with PBS. (2) 100 μL of HuMedia-KB2 with the test substance added at the concentrations shown in the figure, or HuMedia-KB2 without the test substance added, was added to each well and incubated for 48 hours.

[0092] 2-2. The viability of SHEK cultured in Test Example 2-1 was measured using the Premix WST-1 Cell Proliferation Assay System (Cat. No. MK400, TaKaRa Bio, hereinafter sometimes referred to as "WST-1 Premix"). WST-1 is a reagent used to quantify cell viability by colorimetric measurement. It is based on the conversion of tetrazolium salt (WST-1) to formazan dye by mitochondrial dehydrogenase in living cells. As the number of living cells increases, the overall activity of mitochondrial dehydrogenase in the sample increases, and this increase in enzyme activity leads to an increase in formazan dye production. Therefore, there is a linear correlation between the amount of formazan dye present and the number of metabolically active cells in the culture medium.

[0093] Specifically, the survival rate was measured using the following method. (1) Remove the culture medium from the wells containing the SHEK cultured in Test Example 2-1 and wash once with PBS. (2) HuMedia-KB2 containing 10 v / v% WST-1 premix was added to each well in 100 μL and incubated for 3 hours. (3) For each well, the absorbance at 450 nm was measured using an xMark® Microplate Spectrophotometer (BIO RAD). (4) Based on the measured absorbance at 450 nm, the cell viability when each test substance was added was calculated using the following formula, with the cell viability when no test substance was added (control) set to 1. As mentioned above, there is a linear correlation between the amount of formazan dye that absorbs at 450 nm and the number of metabolically active cells (i.e., viable cells).

[0094] Cell viability = Absorbance at 450 nm with test substance added / Absorbance at 450 nm without test substance added (control)

[0095] The test was conducted with n=5. The results are shown in Figures 2-8. The top bar of the bar graph represents the mean, and the error bars represent ± standard deviation.

[0096] 2-3. NHEK that had not undergone aging induction was also cultured with or without each test substance added to the culture medium. Specifically, NHEK was cultured using the following method. (1) Place 8.0 x 10 NHEK in a 96-well plate. 3 The cells were seeded in a cell / well and cultured for 2 days. HuMedia-KG2 was used as the culture medium. (2) 100 μL of HuMedia-KB2 with the test substance added at the concentrations shown in the figure, or HuMedia-KB2 without the test substance added, was added to each well and incubated for 48 hours.

[0097] 2-4. For the NHEK cultured in Test Example 2-3, the viability was measured using the WST-1 premix, similar to Test Example 2-2. Specifically, the viability was measured using the following method. (1) Remove the culture medium from the wells containing NHEK cultured in Test Examples 2-3 and wash once with PBS. (2) HuMedia-KB2 containing 10 v / v% WST-1 premix was added to each well in 100 μL and incubated for 3 hours. (3) For each well, the absorbance at 450 nm was measured using an xMark Microplate Spectrophotometer. (4) From the measured absorbance at 450 nm, the cell viability when each test substance was added was calculated based on the following formula, with the cell viability when no test substance was added (control) set to 1. Cell viability = Absorbance at 450 nm with test substance added / Absorbance at 450 nm without test substance added (control)

[0098] The test was conducted with n=5. The results are shown in Figures 2-8. In each figure, the top bar of the bar graph represents the mean, and the error bars represent ± standard deviation.

[0099] 2-5. Discussion As shown in Figure 2, when positive control venetoclax (ABT-199) or navitoclax (ABT-263) was added to the SHEK culture medium, cell viability was lower compared to when no such control was added. This result confirms that this test system can evaluate substances that have senescent cell removal effects.

[0100] As shown in the graphs in Figures 3-8, when each test substance was added to the culture medium of SHEK, the cell viability was lower compared to when the test substance was not added. This result suggests that each test substance has an effect of removing senescent cells. Furthermore, when each test substance was added, the cell viability was lower in SHEK than in NHEK. This result suggests that each test substance can selectively induce cell death in senescent cells.

[0101] Furthermore, when DL-lactic acid, DL-indole lactic acid, phenyl lactic acid, catechin, caffeic acid, parthenolide, arbutin, γ-tocotrienol, α-tocopherol, γ-oryzanol, retinol acetate, Enterococcus faecalis powder, or Lactobacillus casei powder were added to the NHEK culture medium, the cell viability was higher compared to when these substances were not added. Here, NHEK retained its proliferative capacity, and the amount of formazan dye measured in Test Example 2 showed a linear correlation with the number of viable cells in the culture medium. From this, it can be considered that these test substances not only increased the viability of NHEK but also promoted NHEK proliferation. Specifically, DL-lactic acid, DL-indole lactic acid, phenyl lactic acid, catechin, caffeic acid, parthenolide, arbutin, γ-tocotrienol, α-tocopherol, γ-oryzanol, retinol acetate, Enterococcus faecalis powder, and Lactobacillus casei powder were suggested to not only have an effect of removing senescent cells, but also to promote the proliferation of normal skin cells (i.e., a skin cell activating effect).

Claims

1. An anti-skin aging topical composition containing lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetate, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinyl palmitate, riboflavin, and pantothenyl ethyl ether, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria.

2. A composition for removing senescent cells, comprising lactic acid, indole lactic acid, phenyl lactic acid, catechin, caffeic acid, umbelliferone, parthenolide, pyridoxine hydrochloride, arbutin, hinokitiol, oxaloacetate, tocotrienol, tocopherol, γ-oryzanol, retinol acetate, retinyl palmitate, riboflavin, and pantothenyl ethyl ether, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria.

3. An anti-skin aging and skin cell activation topical composition comprising lactic acid, indole-lactic acid, phenyl-lactic acid, catechin, caffeic acid, parthenolide, arbutin, tocotrienol, tocopherol, γ-oryzanol, and retinol acetate, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria.

4. A topical composition for removing senescent cells and revitalizing skin cells, comprising lactic acid, indole-lactic acid, phenyl-lactic acid, caffeic acid, parthenolide, arbutin, tocotrienol, tocopherol, γ-oryzanol, and retinol acetate, as well as chemically possible salts thereof, and at least one selected from the group consisting of Enterococcus bacteria and Lactobacillus bacteria.

5. A composition according to any one of claims 1 to 4, for use in a person aged 40 years or older.

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