Il-8 inhibitor, Anti-aging agent for skin, and method for controlling skin aging using same

Abstract

To provide an IL-8 inhibitor, an anti-aging agent for the skin, and a method for controlling skin aging by using the same.SOLUTION: Provided are an IL-8 inhibitor, an IL-8 translation inhibitor, and an anti-aging agent for skin, comprising at least one selected from a creeping thyme extract, a fennel extract, a rose extract, a marjoram extract, and a cabbage rose extract as an active ingredient. The anti-aging agent for skin of the present application achieves anti-aging of the skin through IL-8 inhibition.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an IL-8 inhibitor, a skin anti-aging agent, and a method for inhibiting skin aging using the same. [Background technology]

[0002] Various strategies are being investigated to inhibit skin aging. It has been reported that a phenomenon known as senescence-associated secretory phenotype (SASP) is involved in aging, and cytokines such as IL-8 and IL-6 are known as SASP factors (Non-Patent Documents 1-3). Therefore, it is expected that skin aging can be inhibited by substances that inhibit IL-8.

[0003] For example, Patent Document 1 discloses a method for screening anti-wrinkle agents and / or anti-inflammatory agents, which includes a step of applying pressure to epidermal cells from the lateral direction, and uses the activity of inflammatory factors such as IL-8 released by the epidermal cells as an indicator.

[0004] As a substance that suppresses IL-8 in skin cells, Patent Document 2 discloses a gel-forming polysaccharide obtainable from microalgae cells from the order Prasinococcales. Patent Document 3 discloses a composition containing Bacteroides thetaiotaomicron or a biotype thereof, which is used to reduce the expression of pro-inflammatory genes such as IL-8, and is used for the treatment or prevention of inflammatory disorders such as atopic dermatitis and / or autoimmune and / or allergic disorders. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-171441 [Patent Document 2] Patent No. 6270822 [Patent Document 3] Japanese Patent Application Laid-Open No. 2018-90591 [Non-patent literature]

[0006] [Non-Patent Document 1] Juan C. Acosta, Ana Loghlen, Ana Banito, Selina Raguz & Jesus Gil (2008), "Control of senescence by CXCR2 and its ligands", Cell Cycle, 7:19, 2956-2959, DOI: 10.4161 / cc.7.19.6780 [Non-patent document 2] Francis Rodier and Judith Campisi, "Four faces of cellular senescence", J. Cell Biol. Vol. 192 No. 4, 547-556, doi / 10.1083 / jcb.201009094 [Non-patent document 3] Eiji Hara, "New Aspects of Cellular Senescence," Experimental Medicine Vol. 37, No. 11 (July issue) 2019, pp. 1728-1734 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an IL-8 suppressant, a skin anti-aging agent, and a method for suppressing skin aging using the same. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have searched for substances that inhibit IL-8, particularly the translation of IL-8, and have come up with the idea that skin aging can be suppressed by applying an IL-8 inhibitor, thereby achieving the present invention.

[0009] This application encompasses the following inventions: (1) An IL-8 inhibitor containing at least one of a musk extract, a fennel extract, a rose extract, a marjoram extract, and a rose extract as an active ingredient. (2) An IL-8 translation inhibitor containing at least one of a musk extract, a fennel extract, a rose extract, a marjoram extract, and a rosa communis extract as an active ingredient. (3) A skin anti-aging agent comprising the agent according to (1) or (2). (4) The skin anti-aging agent according to (3), wherein skin anti-aging is achieved by promoting the proliferation of skin stem cells. (5) The skin anti-aging agent according to (3) or (4), wherein skin anti-aging is achieved by inhibiting the proliferation of skin aging cells. (6) The skin anti-aging agent according to any one of (3) to (5), wherein the skin anti-aging is achieved by inhibiting aging of skin stem cells with IL-8. (7) A cosmetic method for suppressing skin aging by applying the agent according to any one of (1) to (6). [Effects of the Invention]

[0010] According to the present invention, an agent and method are provided that are effective in inhibiting skin aging by applying an IL-8 inhibitor. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of Experiment 1, showing the amount of IL-8 (pg / 100 μg protein) in skin cells obtained from subjects of each age. [Figure 2] Figure 2 shows the results of Experiment 2. Photographs (top) show skin cell colonies formed without IL-8 addition (cont: left), with IL-8 addition (IL-8: center), and with IL-8 and SB225002 addition (IL-8 + SB225002: right), along with a graph (bottom) showing the number of colonies counted. [Figure 3]Figure 3 shows the results of Experiment 3. The expression level of Lrig1 (LRIG1 / B2M) in skin keratinocytes without IL-8 addition (control: left) and with IL-8 addition (IL-8: right) is shown as a percentage of the level without addition, which is 100%. [Figure 4] Figure 4 shows the results of Experiment 4. Photographs of SA-β-Gal-stained epidermal keratinocytes (top panel) are shown without IL-8 (cont: left) and with IL-8 (IL-8: right), and a graph (bottom panel) shows the percentage (%) of SA-β-Gal-positive cells. [Figure 5] FIG. 5 shows the results of Experiment 5, and is a graph showing the number of epidermal keratinocytes (%) and the area of ​​lymphatic vessels (%). [Figure 6] Figure 6 shows the results of Experiment 7, showing the IL-8 / B2M value (upper left), IL-8 protein amount (pg / mL) (upper right), and IL-8 translation rate (%) (lower left) of the Musk extract. [Figure 7] FIG. 7 shows the results of Experiment 7, showing the IL-8 / B2M value (upper left), IL-8 protein amount (pg / mL) (upper right), and IL-8 translation rate (%) (lower left) of the fennel extract. [Figure 8] FIG. 8 shows the results of Experiment 7, showing the IL-8 / B2M value (upper left), IL-8 protein amount (pg / mL) (upper right), and IL-8 translation rate (%) (lower left) of the rose extract. [Figure 9] Figure 9 shows the results of Experiment 7, showing the IL-8 / B2M value (upper left), IL-8 protein amount (pg / mL) (upper right), and IL-8 translation rate (%) (lower left) of marjoram extract. [Figure 10] FIG. 10 shows the results of Experiment 7, showing the IL-8 / B2M value (upper left), IL-8 protein amount (pg / mL) (upper right), and IL-8 translation rate (%) (lower left) of the rose extract. DETAILED DESCRIPTION OF THE INVENTION

[0012] As described in Patent Document 1 and Non-Patent Documents 1 to 3, it is expected that skin aging can be suppressed by substances that suppress IL-8. The present inventors have confirmed that the amount of IL-8 actually increases with aging, that IL-8 reduces the expression of skin stem cell genes and increases senescent skin cells, and that IL-8 reduces the proliferation ability of skin cells, but that inhibition of IL-8 restores this proliferation ability.

[0013] Therefore, the present inventors have conceived that skin aging can be inhibited by inhibiting IL-8, particularly by inhibiting IL-8 translation, and have searched for novel substances with IL-8 inhibitory activity. As a result of extensive research, the present inventors have discovered that thyme extract, fennel extract, rose extract, marjoram extract, and European rose extract have high IL-8 inhibitory activity.

[0014] Based on these findings, the present invention provides an IL-8 suppressor, an IL-8 translation inhibitor, and a skin anti-aging agent (hereinafter, these may be collectively referred to as "the agent of the present invention"), as well as a method for suppressing skin aging by applying the same. The method of the present invention may be a method for cosmetic purposes and may not be treatment by a doctor or medical professional.

[0015] IL-8 suppression refers to translational suppression of the IL-8 gene. Translational suppression of the IL-8 gene can mean, for example, a statistically significant decrease (e.g., Student's t-test) in the concentration or amount of IL-8 protein produced by expression of the IL-8 gene or the translation rate (ratio of IL-8 protein concentration or amount relative to control / ratio of IL-8 gene expression level relative to control × 100 (%)) when the agent of the present invention is administered compared to a state in which the agent of the present invention is not administered (control), or a decrease of, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0016] In one embodiment, suppression of IL-8 may include suppression of IL-8 gene expression. Suppression of IL-8 gene expression may mean, for example, that when the agent of the present invention is administered, compared to a state in which the agent of the present invention is not administered (control), IL-8 gene expression is reduced with a statistically significant difference (e.g., Student's t-test) at a significance level of 5%, or that the expression is reduced by, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0017] The expression level of the IL-8 gene may be measured, for example, using quantitative PCR as in the Examples, or using a commercially available kit, but is not limited to these methods, and any known technique can be used.

[0018] The concentration or amount of IL-8 protein may be measured, for example, by staining with an antibody or by using a commercially available kit, but is not limited to these, and any known technique can be used.

[0019] As used herein, skin anti-aging refers to the inhibition of aging of skin cells such as keratinocytes and fibroblasts by suppressing IL-8. In one embodiment, skin anti-aging is achieved by promoting the proliferation of skin stem cells and / or suppressing the increase of skin aging cells. The inventors of the present application have also discovered that IL-8 promotes the aging of skin stem cells, such as epidermal stem cells (data not shown). Therefore, in one embodiment, skin anti-aging is achieved by suppressing the aging of skin stem cells by IL-8.

[0020] Promotion of skin stem cell proliferation can be determined by counting the number of stem cells, measuring the expression levels of stem cell markers such as Lrig1 and integrin β1, and evaluating the proliferation potential of skin cells, for example, counting the number of colonies formed by skin cells. Suppression of the increase in skin senescent cells can be determined by counting the number of senescent cells and measuring the expression levels of senescent cell markers such as SA-β-gal and X-gal. Skin stem cell senescence can be determined by combining the above-mentioned evaluation criteria for stem cells and senescent cells, such as double staining for the above-mentioned markers. It has become clear that senescent cells lose their proliferation ability and release factors that inhibit the differentiation of other epidermal cells. Promotion of skin stem cell proliferation, inhibition of the increase in senescent cells, and / or inhibition of skin stem cell senescence are expected to be effective in skin anti-aging by promoting cell turnover, accelerating recovery from undesirable skin conditions such as pigmentation, and reducing the above-mentioned differentiation-inhibiting factors.

[0021] The present inventors have found that the number of skin stem cells and lymphatic vessels decreases with aging, and that there is a correlation between the number of skin stem cells and lymphatic vessel area. Although the theory is unclear, it is possible that IL-8 that is not fully collected by lymphatic vessels promotes skin cell aging, that IL-8 has a negative effect on lymphatic vessel cells, resulting in a decrease in lymphatic vessel cells and, as a result, various waste products that are not fully collected by lymphatic vessels promote skin cell aging, or both. Therefore, skin aging may be caused by a decrease in the efficiency of waste product excretion due to a decrease in lymphatic vessel function caused by an increase in IL-8. Suppressing IL-8 may improve lymphatic vessel function, thereby promoting waste product excretion and thereby inhibiting skin aging. The present invention also provides an IL-8 inhibitor for improving cutaneous lymphatic vessel dysfunction and a skin anti-aging agent that achieves skin anti-aging by improving cutaneous lymphatic vessel dysfunction. Skin lymphatic vessel function can be measured, for example, by measuring the expression level using immunostaining with markers such as LYVE-1 and MCSP, or by measuring the lymphatic vessel area using image analysis.

[0022] The agent of the present invention may consist of or contain as an active ingredient musk extract, fennel extract, rose extract, marjoram extract, and / or rosa communis extract.

[0023] The agent of the present invention may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.

[0024] Creeping thyme (scientific name: Thymus Serpyllum, also known as wild thyme, wild thyme, and wild thyme) is a perennial plant of the Lamiaceae family. Extracts of Creeping thyme have been reported to promote the production of profilaggrin and filaggrin, improve barrier function, and so on. The extract of the above-ground parts of Creeping thyme is preferred, but since the roots, flowers, fruits, peels, seeds, etc. of Creeping thyme also contain active ingredients, extracts of one or more of these can also be used.

[0025] Fennel (scientific name: Foeniculum vulgare, also known as fennel, English name: Fennel) is a perennial plant of the Apiaceae family. It has been reported to have effects such as stomachic, expectorant, carminative, and analgesic properties. The fennel extract is preferably an extract of fennel fruit, but since active ingredients are also contained in the roots, leaves, stems, flowers, seeds, etc. of Thymus vulgare, extracts of one or more of these can also be used.

[0026] In this specification, rose extract refers to an extract of Rosa canina. Rosa canina (scientific name: Rosa canina, also known as Dog Rose, Brier Bush, Rose Hip, English names: Dog Rose, Brier Bush, Rose Hip) is a deciduous shrub in the Rosaceae family. Rosa canina is rich in vitamin C and has been reported to have effects such as improving blemishes and producing collagen. The rose extract is preferably an extract of Rosa canina fruit, but since active ingredients are also contained in the roots, leaves, stems, flowers, peel, seeds, etc. of Rosa canina, extracts of any one or more of these can also be used.

[0027] Marjoram (scientific name: Origanum majorana, Majorana hortensis, also known as origanum, or marjoram, English name: Sweet Marjoram) is a perennial plant of the Lamiaceae family. Marjoram contains flavanones, flavones, tannins, etc., and has been reported to promote the production of epidermal hyaluronic acid, promote the production of type I collagen, and inhibit the production of phospholipase A2. The marjoram extract is preferably an extract of marjoram leaves, but since active ingredients are also contained in marjoram roots, stems, flowers, fruits, peels, seeds, etc., extracts of any one or more of these can also be used.

[0028] European rose (scientific name: Rosa centifolia, also known as rose, centifolia rose, English names: Cabbage Rose, Provance Rose) is a variety of the Rosaceae family. European rose has been reported to have antibacterial, deodorizing, antiviral, and other effects. The European rose extract is preferably an extract of European rose flowers, but since active ingredients are also contained in the roots, stems, leaves, fruit, peel, seeds, etc. of European rose, extracts of one or more of these can also be used.

[0029] The above-mentioned various extracts may be commercially available as cosmetic raw materials or health food ingredients, or may be obtained by conventional methods. The extraction method is not particularly limited, but examples include extraction methods using a solvent. Extraction can be performed by immersing the raw material in an extraction solvent at room temperature or under heating, or by heating under reflux, followed by filtration and concentration. While the raw material can be used as is, grinding it into granules or powder and then subjecting it to extraction allows for extraction of active ingredients under mild conditions with high extraction efficiency in a short time. The extraction temperature is not particularly limited and can be set appropriately depending on the particle size of the ground material, the type of solvent, etc. It is usually set within the range from room temperature to the boiling point of the solvent. The extraction time is also not particularly limited and can be set appropriately depending on the particle size of the ground material, the type of solvent, the extraction temperature, etc. Furthermore, during extraction, stirring may be performed, the mixture may be left standing without stirring, or ultrasound may be applied.

[0030] Any solvent commonly used for extraction can be used as the extraction solvent. For example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as various alcohols including lower alcohols such as ethanol, propylene glycol, 1,3-butylene glycol, and glycerin, or their hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc. can be used alone or in combination, for example, as a mixed solvent of water and 1,3-butylene glycol.

[0031] By such an extraction procedure, the active ingredient is extracted and dissolved in the solvent. The solvent containing the extract may be used as is, or may be subjected to conventional purification treatments such as sterilization, washing, filtration, bleaching, and deodorization before use. If necessary, the extract may be concentrated by lyophilization or diluted with an appropriate solvent before use. Furthermore, the extract may be used after volatilizing the solvent to obtain a solid (dried product), or after redissolving the dried product in an appropriate solvent.

[0032] Furthermore, the squeezed liquid obtained by squeezing the raw material also contains the same active ingredients as the extract, so the squeezed liquid can also be used instead of the extract.

[0033] The present application also provides a composition comprising the agent of the present invention. The composition of the present invention may be a cosmetic composition or a food composition. The composition of the present invention may be, for example, a composition that suppresses skin aging by promoting the proliferation of skin stem cells and / or suppressing the increase of skin aging cells and / or suppressing IL-8-induced skin stem cell aging and / or improving lymphatic dysfunction through the inhibitory effect of IL-8.

[0034] The subject to which the agent or composition of the present invention is applied may be a subject in need of skin aging inhibition from an objective or subjective perspective, such as inflammation, sagging skin, delayed turnover, a decrease in stem cells, an increase in senescent cells, or a decrease in lymphatic vessels, or a subject who desires to preventively inhibit skin aging.

[0035] The agent or composition of the present invention can be administered by any route, such as topical or oral administration, but is preferably incorporated into an external skin preparation that can be applied directly to the skin. The external administration form can be selected from various forms, such as liquid, emulsion, cream, solid, sheet, spray, gel, foam, and powder. It may also be a cosmetic composition, such as an emulsion, cream, serum, lotion, pack, or facial cleanser. Oral administration can be selected from various forms, such as tablets, supplements, beverages, and powders. The agent or composition of the present invention can be appropriately formulated with optional ingredients used in cosmetic and pharmaceutical compositions, as needed, as long as the ingredients do not impair their efficacy. Examples of optional ingredients include excipients, carriers, diluents, oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, UV absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, and neutralizers. Furthermore, other medicinal ingredients, such as those that promote the activation of skin stem cells, may also be included.

[0036] The administration frequency can be selected arbitrarily, such as once every 4 weeks, once every 2 weeks, once a week, once every 3 days, once every 2 days, once a day, twice a day, three times a day, four times a day, five times a day, or administration as needed, but is not limited to these.

[0037] However, the forms that the agent or composition of the present invention can take are not limited to the dosage forms and shapes described above.

[0038] The amount of active ingredient of the musk extract, fennel extract, rose extract, marjoram extract, or rosa spreada extract in the agent or composition of the present invention can be appropriately determined depending on the type, purpose, form, method of use, etc. For example, the amount of the musk extract, fennel extract, rose extract, marjoram extract, or rosa spreada extract can be 0.0001 to 100 wt%, 0.0001 to 90 wt%, 0.001 to 50 wt%, 0.001 to 5.0 wt%, 0.001 to 1.0 wt%, 0.01 to 1.0 wt%, 0.001 to 0.1 wt%, or 0.01 to 0.1 wt%, per total weight of the agent or composition of the present invention, but is not limited thereto as long as the effects of the present invention are exhibited.

[0039] The present application also provides a musk extract, a fennel extract, a rose extract, a marjoram extract, and a rosa communis extract for use in inhibiting skin aging by promoting the proliferation of skin stem cells, inhibiting the increase of skin aging cells, inhibiting IL-8-mediated aging of skin stem cells, and improving decreased lymphatic function through the inhibition of IL-8. [Example]

[0040] The present invention will now be described in more detail with reference to examples, although the present invention is not limited thereto.

[0041] <Experiment 1: Changes in IL-8 levels in skin cells with aging> Preparation of epidermal keratinocytes from various age groups: Epidermal keratinocytes derived from skin of 0, 21, and 62 year olds purchased from KAC Co., Ltd. were used. 2 The cells were seeded into a flask at 500,000 cells / flask / 20 mL and cultured in normal human epidermal keratinocyte growth medium (HuMedia-KB2, KURABOU). The medium was changed every two days until the cells became subconfluent. After passage using trypsin, the cells were seeded into a 6-well plate at 250,000 cells / well.

[0042] Measurement of IL-8 levels by ELISA assay: Two days after seeding, IL-8 levels were measured by ELISA assay in epidermal keratinocytes. Epidermal keratinocytes from each age group cultured in 6-well plates were washed with 1 mL of PBS, solubilized with RIPA Buffer (Nacalai), and collected using a scraper. Protein levels were measured using a BCA assay, and the protein concentration of each sample was adjusted to 500 μg / mL. The adjusted protein concentrations were quantitatively analyzed using the Quantikine HS human IL-8 ELISA kit (R&D systems, HS800).

[0043] result: The results are shown in Figure 1. The older the subjects, the higher the amount of IL-8 in skin cells. This result suggests that the amount of IL-8 in skin cells increases with age.

[0044] <Experiment 2: Effect of IL-8 on skin cell proliferation> Preparation of epidermal keratinocytes: Human epidermal keratinocytes-fetal purchased from ScienCell were cultured in iMatrix-511-coated flasks using normal human epidermal keratinocyte growth medium (HuMedia-KB2, KURABOU). IL-8 (15 ng / ml) was added to the cells after one passage, and they were subjected to three passages under IL-8 exposure. Controls included no IL-8 and cells containing IL-8 (15 ng / ml) and an IL-8 inhibitor (SB225002: 40 nM). After passage, the cells were used for the colony formation evaluation described below.

[0045] Creating a Feeder layer: Mouse 3T3 cells were cultured in a 175cm flask at 2.5×10 5Cells / flask were seeded in 10% FBS-DMEM and the medium was changed every two days. After the cells reached subconfluence, the medium was changed with 10 μg / mL mitomycin C medium (200 μL of 1 mg / mL mitomycin C / PBS + 20 mL of 10% FBS-DMEM) and incubated at 37°C for 3 hours. The medium was then changed with 20 mL of 10% FBS-DMEM and cultured overnight before recovery with trypsin. The recovered cells were frozen (3.0 × 10 6 cells / mL / tube).

[0046] Colony formation performance evaluation: Mitomycin C-treated 3T3 cells (3.0 × 10 6 cells / mL / tube) into a 6-well plate. 5 The cells were seeded at 1.0 × 10 cells / well and cultured overnight in 10% FBS-DMEM. 3 Cells were seeded at 1000 cells / well and cultured in Humedia-KG2 with medium changes every two days. After 14 days of culture, the cells were fixed in mild form (10% formalin) at room temperature for 10 minutes. After washing, 1 ml / well of crystal violet solution (0.2 g crystal violet, 20 mL methanol + 80 mL milliQ) was added and allowed to stand at room temperature for 30 minutes. After washing with PBS, the cells were air-dried and photographed. Colony counts and colony sizes were calculated using image analysis with a Winroof scanner.

[0047] result: The results are shown in Figure 2. When IL-8 was added, the number of colonies was significantly reduced compared to when no IL-8 was added, but the decrease in colony number was suppressed when an IL-8 inhibitor was added. These results suggest that IL-8 reduces the proliferation ability of skin cells, but that this is restored when IL-8 is inhibited.

[0048] <Experiment 3: Effect of IL-8 on epidermal stem cell gene expression> Preparation of epidermal keratinocytes: As in Experiment 2, human epidermal keratinocytes-fetal (ScienCell) were cultured in iMatrix-511-coated flasks using normal human epidermal keratinocyte growth medium (HuMedia-KB2, KURABOU). IL-8 (15 ng / ml) was added to the first passaged cells, and the cells were subjected to three passages under IL-8 exposure. A control group was used without IL-8. After passage, mRNA was extracted using the RNase Easy Mini Kit, and cDNA was prepared using the Superscript VILO cDNA synthesis kit and used as a sample for qPCR.

[0049] qPCR: Using the qPCR samples prepared by the above method, quantitative analysis was performed using StepOne (Applied Bioscience) with Platinum SYBER Green qPCR SuperMix-UDG (Invitrogen) and the following primers to analyze the expression of the stem cell gene Lrig1. B2M forward: 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1) B2M reverse: 5'-CAATCCAAATGCGGCATCT-3' (SEQ ID NO: 2) LRIG1 forward: 5'-CTTGACCTGGGTTCTGGGTA-3' (SEQ ID NO: 3) LRIG1 reverse: 5'-GGCCAAAGGAACATTTGAAG-3' (SEQ ID NO: 4)

[0050] result: The results are shown in Figure 3. Addition of IL-8 significantly reduced the expression level of Lrig1 in epidermal keratinocytes. This result suggests that IL-8 has the effect of reducing the proliferation ability of skin stem cells.

[0051] <Experiment 4: IL-8-induced aging of skin cells> Skin keratinocyte culture: A portion of the epidermal keratinocytes cultured as described in Experiment 3 was used in the SA-β-Gal assay to detect senescent cells in this experiment.

[0052] SA-β-Gal assay: Senescence was detected using a Senescence Detection Kit (ab65351, Abcam). The cells were washed with 1 ml of PBS, and then added with Fixative Solution and incubated for 15 minutes at room temperature. The Fixative Solution was removed, and 500 μl of Staining Solution Mix was added and incubated overnight at 37°C. Nuclei were stained with Hoechst, and the total number of cells and the number of SA-β-Gal-positive cells were counted. The ratio of SA-β-Gal-positive cells to the total number of cells (= (number of SA-β-Gal-positive cells / total number of cells) × 100) was calculated.

[0053] result: The results are shown in Figure 4. Addition of IL-8 increased the number and percentage of SA-β-Gal-positive cells. This result suggests that IL-8 has the effect of promoting the aging of skin cells.

[0054] <Experiment 5: Effects of aging on epidermal keratinocyte stem cells and dermal lymphatic vessels> Histological immunostaining of human cheek skin block: Skin samples from the cheeks of four healthy individuals in their 20s and 30s (young group) and seven healthy individuals in their 70s and 80s (old group) were prepared in AMeX paraffin blocks and then cut into 3 μm-thick sections. The sections were deparaffinized in xylene, immersed in EtOH, and then immersed in PBS. After incubation with primary antibodies overnight at 4°C, the primary antibodies used were anti-LYVE-1 (Rabbit IgG, ReliaTech) and anti-MCSP (Mouse IgG2a, Millipore). The antibodies were diluted 1:100 in 12% BSA / PBS. After washing three times with PBS, the sections were incubated with fluorescently labeled secondary antibodies (Alexa488 and Alexa594) corresponding to the animal species of each primary antibody at room temperature for 1.5 hours in the dark. The secondary antibodies were diluted 2:200 in 12% BSA / PBS. After washing three times with PBS, the cells were mounted in a mounting medium containing DAPI and then subjected to fluorescent observation. The number of epidermal stem cells was counted and their percentage (%) of the total cell number was calculated. The area of ​​the stained lymphatic vessels was measured and their percentage (%) of the total cell area was calculated. The correlation between the number of epidermal stem cells and lymphatic vessel area was calculated using Pearson's product-moment correlation coefficient.

[0055] result: Both the number of epidermal stem cells and lymphatic vessel area in the elderly group were lower than those in the young group (data not shown). Furthermore, a correlation was found between the number of epidermal stem cells and the area of ​​lymphatic vessels (Figure 5). These results suggest that epidermal stem cells decrease with age, leading to a decline in lymphatic vessel function. The results also suggest that there is some relationship between the increase in IL-8 with age, the aging of skin cells, and the decrease in lymphatic vessels. While the theory is unclear, it is possible that IL-8 that is not fully collected by lymphatic vessels promotes skin cell aging, or that IL-8 has a negative effect on lymphatic vessel cells, impairing lymphatic vessel function, resulting in waste products that are not fully collected by lymphatic vessels promoting skin cell aging.

[0056] The results of Experiments 1-5 suggest that IL-8 increases with age, and that increased IL-8 accelerates the aging of skin cells. Therefore, it is expected that suppressing IL-8 can achieve anti-aging of skin cells. Therefore, the following experiments were conducted to search for substances that suppress IL-8.

[0057] <Experiment 6: Search for IL-8 inhibitors> Sample preparation: The following samples were used to evaluate the IL-8 suppression activity. [Table 1]

[0058] A total of 168 candidate samples were prepared, including natural ingredients such as animal and plant extracts and synthetic ingredients.

[0059] Cell culture: Normal human fetal epidermal keratinocytes (ScienCell, Cat No. 2120) were cultured in epidermal keratinocyte culture medium (Kurabo, KK-2150S). After passage, 2.5 × 10 5 Cells were seeded into a 6-well plate at 2 mL per well, and drugs were added 24 hours later. The drugs were diluted 1000-fold with DMSO to a final concentration of 0.01%, and the control was DMSO diluted 1000-fold. mRNA was collected 24 hours after drug addition. Protein was collected 48 hours after drug addition for protein analysis using ELISA.

[0060] 1. Primary screening of substances that inhibit IL-8 expression based on gene expression analysis: RNA was extracted from the cells 24 hours after drug addition using the Quiagen Rneasy mini Kit (Quiagen), and cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). IL-8 and B2M gene expression levels were measured using the Syber Green method with Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used were as follows: B2M forward: 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1) B2M reverse: 5'-CAATCCAAATGCGGCATCT-3' (SEQ ID NO: 2) IL-8 forward: 5'-GGGTACCCAGTTAAATTTTCATTTC-3' (SEQ ID NO: 5) IL-8 reverse: 5'-CAAGTTTCAACCAGCAAATTACT-3' (SEQ ID NO: 6)

[0061] The primary screening was performed with N=1 and a drug concentration of 0.01%. In the primary screening, 82 substances were selected from 168 substances that did not show IL-8 / B2M values ​​significantly higher than the control IL-8 / B2M values ​​as candidates for substances with IL-8 expression inhibitory activity.

[0062] 2. Secondary screening of substances with IL-8 translation inhibitory activity based on protein quantity analysis: The 82 products selected in the primary screening were subjected to secondary screening based on the amount of IL-8 protein at a drug concentration of 0.01% (N=3). After 48 hours of drug addition, cells (6-well plate) were washed once with 2 mL of PBS and then lysed in RIPA Buffer (Nacalai, Cat. No. 08714-04). Insoluble proteins were precipitated using a centrifuge, and the supernatant was collected and used as the protein solution. Protein amount was corrected using the BCA method, and IL-8 concentration was measured using an IL-8 ELISA kit (Abcam, Cat. No. ab46032).

[0063] Based on the results of the secondary screening, 16 substances were selected from 82 as candidates for substances with IL-8 translation inhibitory activity based on the protein expression inhibitory effect.

[0064] 3. Tertiary screening based on analysis of protein levels at different drug concentrations: For the 16 products selected in the secondary screening, gene expression levels (IL-8 / B2M) and IL-8 protein concentrations (pg / mL) were measured using the same methods as in the primary and secondary screening, except that drug concentrations were 0.001%, 0.01%, and 0.1%, with each concentration measured in triplicate. For gene expression levels (IL-8 / B2M), the expression level (% of control) of each drug-treated group relative to the control was calculated, with the control set at 100%. Similarly, protein concentrations were calculated as a percentage of the control (% of control). The translation rate was calculated according to the following formula, and tertiary screening was performed based on the IL-8 translation rate.

number

[0065] From the results of the tertiary screening, five substances were selected as having a concentration-dependent inhibitory effect on IL-8 translation: musk extract, fennel extract, rose extract, marjoram extract, and rose extract.

[0066] Experiment 7: Effects of selected drugs on IL-8 translation in skin cells The reproducibility of the IL-8 suppression effect on epidermal keratinocytes was confirmed for the five products selected in Experiment 6. Epidermal keratinocytes from each age group cultured in 6-well plates were washed with 1 mL of PBS, solubilized in RIPA Buffer (Nacalai), and harvested using a scraper. Protein content was measured using a BCA assay, and the protein concentration of each sample was adjusted to 500 μg / mL. The protein-adjusted samples were quantitatively analyzed using a Quantikine HS human IL-8 ELISA kit (R&D systems, HS800), and the amount of IL-8 (pg / mL) and IL-8 translation rate (%) were calculated using the same method as in Experiment 6.

[0067] result: The results are shown in Figures 6-10. The IL-8 protein concentration and translation rate were reduced in the extracts of musk, fennel, rose, marjoram, and rosehip, demonstrating a reproducible inhibitory effect on IL-8 translation.

[0068] These results indicate that musk extract, fennel extract, rose extract, marjoram extract, and rose extract are effective in suppressing IL-8, particularly in inhibiting IL-8 translation. IL-8 levels increase with age, but suppressing IL-8 is effective in promoting the proliferation of skin stem cells, inhibiting the increase in skin senescent cells, and inhibiting skin stem cell aging, thereby inhibiting skin aging. Furthermore, a relationship between the increase in IL-8 and skin cell aging and lymphatic vessel loss with age is suggested.

Claims

1. An IL-8 inhibitor containing at least one of a musk extract, a fennel extract, a rose extract, a marjoram extract, and a European rose extract as an active ingredient.

2. An IL-8 translation inhibitor comprising at least one of a musk extract, a fennel extract, a rose extract, a marjoram extract, and a rosa communis extract as an active ingredient.

3. A skin anti-aging agent comprising the agent according to claim 1 or 2.

4. The skin anti-aging agent according to claim 3, wherein skin anti-aging is achieved by promoting the proliferation of skin stem cells.

5. The skin anti-aging agent according to claim 3 or 4, wherein skin anti-aging is achieved by inhibiting the proliferation of skin senescent cells.

6. The skin anti-aging agent according to any one of claims 3 to 5, wherein skin anti-aging is achieved by inhibiting aging of skin stem cells with IL-8.

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