Dermal extracellular matrix production promoter
Patent Information
- Application Number
- JP2022153659
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2022-09-27
- Publication Date
- 2025-06-30
AI Technical Summary
Existing methods fail to effectively promote dermal extracellular matrix production, leading to dermal wrinkles and skin aging, which are not adequately addressed by current cosmetic and therapeutic approaches.
A dermal extracellular matrix production promoter containing carbon dioxide gas is applied to the skin for at least 3 minutes, creating a low pH environment that enhances the production of dermal extracellular matrix components like elastin and collagen by regulating gene expression of relevant enzymes.
Promotes the production of dermal extracellular matrix, thereby reducing wrinkles and improving skin elasticity and firmness by increasing the expression of genes involved in elastin and collagen fiber formation while decreasing the expression of enzymes involved in their degradation.
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Abstract
Description
[Technical field]
[0001] The present invention relates to a dermal extracellular matrix production-promoting agent that increases the amount of extracellular matrix in the dermis. [Background technology]
[0002] Wrinkles seen on the skin are broadly divided into epidermal wrinkles and dermal wrinkles. Epidermal wrinkles are temporary and result from the drying of the epidermis, whereas dermal wrinkles are formed due to quantitative and qualitative changes in the structure that constitutes the dermis deep in the skin caused by aging and photoaging. They are easily noticeable and often give the impression of aging or fatigue. Therefore, preventing and improving dermal wrinkles is an important issue in considering the improvement of the quality of life (QOL) of middle-aged and elderly people.
[0003] Extracellular matrix (ECM) is a dermal structure involved in wrinkle formation, and it is known that the extracellular matrix decreases or degenerates due to aging or ultraviolet light. Major extracellular matrices include collagen, elastin, hyaluronic acid, etc., and it is believed that the decomposition or degeneration of these induces the formation of dermal wrinkles. Therefore, it is believed that inhibiting the decomposition or degeneration of this dermal extracellular matrix and maintaining or increasing the amount of the extracellular matrix is useful for preventing or improving wrinkles.
[0004] On the other hand, the physiological effect of carbon dioxide gas is generally known to be the promotion of blood circulation, and cosmetics and bath additives that utilize the blood circulation promoting effect of carbon dioxide gas are being considered (Patent Document 1). In recent years, a therapy called carbon dioxide mesotherapy, in which carbon dioxide gas is directly injected into the skin (intradermally or subcutaneously), is known to be useful for inducing the exfoliation of the dermis layer and the reconstruction of collagen, as well as improving skin firmness, luster, and wrinkles by promoting blood flow. Carbon dioxide packs that have carbon dioxide gas dissolved in them are also known to have the effect of regenerating the skin and improving blood flow. It has also been reported that carbon dioxide has a wound healing effect (Non-Patent Document 1). [Prior art documents] [Patent documents]
[0005] [Patent Document 1] JP 2011-93877 A [Non-patent literature]
[0006] [Non-Patent Document 1] PLoS ONE. 2015; 10(2): e0117106. Summary of the Invention [Problem to be solved by the invention]
[0007] The present invention relates to providing a technique for promoting the production of dermal extracellular matrix and preventing or ameliorating dermal skin aging. [Means for solving the problem]
[0008] The present inventors have found that a specific low pH environment is important for promoting the production of dermal extracellular matrix, and that a specific method of applying carbon dioxide gas is useful for realizing said pH environment.
[0009] That is, the present invention relates to the following. A dermal extracellular matrix production promoter containing carbon dioxide gas as an active ingredient, said dermal extracellular matrix production promoter being used by applying an aerosol preparation containing 0.1 to 5 parts by mass of carbon dioxide gas per 100 parts by mass of aerosol concentrate to the skin and leaving it for 3 minutes or more. Effect of the Invention
[0010] According to the present invention, it is possible to effectively promote the production of dermal extracellular matrix and prevent or improve wrinkles. [Brief description of the drawings]
[0011] [Figure 1] Carbon dioxide induces a decrease in pH in the dermis layer of a human skin model. [Diagram 2] Effect of carbon dioxide application time on pH changes in the dermis layer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] As shown in the Examples below, when an aerosol preparation containing a predetermined amount of carbon dioxide gas is added to a three-dimensional skin model and left for 3 minutes or more, the carbon dioxide gas permeates into the dermis layer, and the pH is transiently induced to be less than 7. In addition, when low pH stimulation is performed multiple times by exposing human dermal fibroblasts to a low pH environment for a predetermined period of time, increased expression of ELN, FBN1, and MFAP-4, which are involved in the formation of elastin fibers, decreased expression of NEP, which is involved in the degradation of elastin fibers, increased expression of COL1A1 and COL3A1, which are involved in the formation of collagen fibers, decreased expression of MMP1, which is involved in the degradation of collagen fibers, and further increased expression of HAS2, which is involved in the synthesis of hyaluronic acid, and decreased expression of HYBID, which is involved in the degradation, are observed. Therefore, application of an aerosol preparation containing a prescribed amount of carbon dioxide gas to the skin and leaving it on for 3 minutes or more can promote the production of dermal extracellular matrix. In addition, an aerosol preparation containing a predetermined amount of carbon dioxide gas, which is applied to the skin and left for at least 3 minutes, can serve as a dermal extracellular matrix production promoter and can be used as a medicine, quasi-drug, or cosmetic for promoting the production of dermal extracellular matrix.
[0013] Here, the use of carbon dioxide gas on humans may be therapeutic or non-therapeutic. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, treatment, or diagnosis on humans, and more specifically, does not include methods of surgery, treatment, or diagnosis performed on humans by doctors, medical professionals, or persons under the instructions of doctors. In the present invention, non-therapeutic use includes the use of carbon dioxide gas to promote the production of dermal extracellular matrix for cosmetic or aesthetic purposes, such as use by estheticians, hairdressers, barbers, trimmers, etc.
[0014] In the present invention, the term "dermal extracellular matrix" refers to an extracellular matrix present in the dermis, and examples thereof include collagen, elastin, hyaluronic acid, proteoglycan, fibronectin, laminin, etc., with collagen, elastin, and hyaluronic acid being preferred. In the present invention, "promotion of the production of dermal extracellular matrix" means promoting the production ability or suppressing the decomposition ability of dermal extracellular matrix, thereby increasing the amount of extracellular matrix in the dermis. In one embodiment, the promotion of the production of dermal extracellular matrix can be promotion of the expression of dermal extracellular matrix, and the promotion of the expression includes promotion of the expression at the gene level and the protein level of the extracellular matrix or the enzyme involved in its synthesis. In another embodiment, the promotion of the degradation of dermal extracellular matrix can be suppression, and the suppression of the degradation includes suppression of the expression at the gene level and the protein level of the enzyme involved in the degradation of the extracellular matrix. Here, the promotion / suppression of expression at the gene level includes promotion / suppression of mRNA expression and promotion / suppression of transcription to mRNA, and the promotion / suppression of expression at the protein level includes promotion / suppression in translation. Each expression level can be measured based on any parameter. Examples of genes encoding enzymes involved in the extracellular matrix or its synthesis include ELN, FBN1, and MFAP-4, which are involved in the formation of elastin fibers, COL1A1 and COL3A1, which are involved in the formation of collagen fibers, and HAS2, which is involved in the synthesis of hyaluronic acid. On the other hand, examples of genes encoding enzymes involved in the degradation of the extracellular matrix include NEP, which is involved in the degradation of elastin fibers, MMP1, which is involved in the degradation of collagen fibers, and HYBID (KIAA1199), which is involved in the degradation of hyaluronic acid. The production of dermal extracellular matrix is promoted and the amount of extracellular matrix in the dermis is increased, thereby improving or preventing wrinkles and improving skin elasticity. Therefore, the use of carbon dioxide gas in the present invention makes it possible to prevent or improve dermal skin aging, such as the formation of dermal wrinkles, the occurrence of dermal sagging, and reduced skin elasticity, which are caused or associated with a decrease in the extracellular matrix.
[0015] In the present invention, carbon dioxide gas is applied to the skin using an aerosol preparation containing carbon dioxide gas (also called a "carbonate aerosol"). Carbonate aerosol is an aerosol formulation that contains a propellant containing carbon dioxide gas, and is composed of an aerosol concentrate and a propellant containing carbon dioxide gas. As the propellant, in addition to carbon dioxide gas, other propellants can be used in combination. Examples of propellants other than carbon dioxide gas include liquefied petroleum gas (ethane, propane, ethylene, isobutane, normal butane, propylene, etc., mixed gases thereof (e.g., mixed gases of isobutane and propane, propane and butane, etc.)), ether-based propellants (dimethyl ether, etc.), fluorocarbons (fluorocarbons, chlorofluorocarbons, bromochlorofluorocarbons, etc.), compressed gases (nitrogen, air, mixed gases thereof, etc.), and fluorocarbon gases (monochlorodifluoroethane, tetrafluoroethane, etc.), among which nitrogen, dimethyl ether, and liquefied petroleum gas are preferred. When using other propellants together with carbon dioxide gas, any one of them can be used alone or in combination of two or more.
[0016] When a propellant other than carbon dioxide gas is used in combination as a propellant, in order not to impair the effect of carbon dioxide gas, the ratio of carbon dioxide gas in the total propellant is preferably 20% or more, more preferably 40% or more, even more preferably 60% or more, and even more preferably 80% or more, based on the volume of the gaseous state (1013.25 hPa, 25°C).
[0017] The aerosol concentrate may contain various ingredients that are commonly used in skin care products such as cosmetics, quasi-drugs, and pharmaceuticals. Specifically, from the viewpoint of discharging the skin care product in a foam form, improving the spread of the foam on the skin, providing an excellent feeling of use, and sustaining the foam to enhance the effect of carbon dioxide, examples of the ingredients that may be included include water, polyhydric alcohols, oils, water-soluble thickeners, surfactants, preservatives, powders, ethanol, antioxidants, colorants, fragrances, moisturizers, blood circulation promoters, cooling agents, antiperspirants, disinfectants, whitening agents, anti-inflammatory agents, and skin activators. Note that each of these ingredients is not limited to its intended use, and may be used for other purposes or in combination with other purposes depending on the purpose.
[0018] Among these, the polyhydric alcohol may be any one used in ordinary cosmetics, and examples thereof include dihydric polyhydric alcohols such as ethylene glycol, propylene glycol, propanediol, 1,3-butylene glycol, 1,3-propanediol, dipropylene glycol, polyethylene glycol, and polypropylene glycol; and trihydric or higher polyhydric alcohols such as glycerin and sorbitol. Among these, from the viewpoint of improving the penetration feeling and uniform application of the ejected agent into the skin, glycerin and dihydric polyols are preferred, glycerin, propylene glycol, propanediol, 1,3-butylene glycol, 1,3-propanediol, and dipropylene glycol are more preferred, glycerin, 1,3-propanediol, and dipropylene glycol are even more preferred, and it is even more preferred to include glycerin and dipropylene glycol.
[0019] The oil agent is not limited as long as it is one that is commonly used in cosmetics, and examples thereof include hydrocarbon oils, silicone oils, ester oils, ether oils, and fluorine oils. More specifically, for example, linear or branched hydrocarbon oils such as light isoparaffin, liquid paraffin, liquid isoparaffin, squalane, and squalene; silicone oils such as dimethylpolysiloxane, cyclomethicone, dimethicone, trisiloxane methyltrimethicone, ethyltrisiloxane, dimethylcyclopolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, and higher alcohol-modified organopolysiloxane; monoester oils such as isononyl isononanoate, isotridecyl isononanoate, and alkyl benzoate (C12-15) which is an ester of benzoic acid and an aliphatic alcohol having 12 to 15 carbon atoms; diester oils such as neopentyl glycol dicaprate; triester oils such as tri(capryl / capric acid)glyceryl, and triglyceryl 2-ethylhexanoate; alkyl-1,3-dimethylbutanoate; Examples of suitable oils include ether oils such as ethyl ether, dicaprylyl ether, and dicaprylyl ether; and fluoro oils such as fluoropolyether and perfluoroalkyl ether silicone. From the viewpoint of improving foam extension, one or more oils selected from hydrocarbon oils, ester oils, and silicone oils are preferred, with hydrocarbon oils, monoester oils, triester oils, and silicone oils being more preferred, and monoester oils and silicone oils being more preferred. From the viewpoint of improving the permeability of carbon dioxide gas into the skin, the viscosity of the oil is 1 mPa·s or more, preferably 2 mPa·s or more, more preferably 3 mPa·s or more, even more preferably 4 mPa·s or more, and 100 mPa·s or less, preferably 50 mPa·s or less, more preferably 30 mPa·s or less, and even more preferably 20 mPa·s or less. The oil has a viscosity of 1 to 100 mPa·s, preferably 2 to 50 mPa·s, more preferably 3 to 30 mPa·s, and even more preferably 4 to 20 mPa·s. Here, the viscosity was measured at 25°C using a BM viscometer (manufactured by Toki Sangyo Co., Ltd.) (rotor No. 1, 60r pm, 1 min).
[0020] Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. In the present invention, nonionic surfactants are preferably used from the viewpoints of the amount of carbon dioxide dissolved in the original solution, solubility promotion, and foaming property during application. The nonionic surfactant used in the present invention is a nonionic surfactant with an HLB of 3 to 20. The HLB of the nonionic surfactant is preferably 7 or more, more preferably 9 or more, and even more preferably 11 or more from the viewpoints of improving the penetration feeling of the foam into the skin and the storage stability of the composition, and is preferably 18 or less, more preferably 16 or less, and even more preferably 15 or less from the viewpoint of improving the film feeling of the skin after application. The HLB of the nonionic surfactant is preferably 7 to 18, more preferably 9 to 16, and even more preferably 11 to 15. Here, HLB (Hydrophilic-Lipophilic Balance) indicates the molecular weight of the hydrophilic group in the total molecular weight of the surfactant, and is calculated by Griffin's formula. The HLB value of the mixed surfactant is calculated by averaging the HLB values of each nonionic surfactant based on their blending ratio.
[0021] The water-soluble thickener may be any one used in ordinary cosmetics, and examples thereof include carrageenan, dextrin, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, carboxyvinyl polymer, acrylic acid / alkyl methacrylate copolymer, xanthan gum, carboxymethyl chitin, chitosan, etc. These have the effect of increasing the viscosity of the original solution, suppressing the sudden generation of bubbles, and improving stability. Furthermore, from the viewpoint of improving the permeability of carbon dioxide gas into the skin and the film feeling on the skin after application, carboxyvinyl polymer and acrylic acid / alkyl methacrylate copolymer are preferred, and acrylic acid / alkyl methacrylate copolymer is more preferred. Here, the acrylic acid / alkyl methacrylate copolymer refers to C 10-30It is a copolymer of alkylacrylic acid and acrylic acid, methacrylic acid or a lower alkyl ester thereof, and is crosslinked with an allyl ether of sucrose or an allyl ether of pentaerythritol. Commercially available products such as Pemulen TR-1, Pemulen TR-2, Carbopol ETD2020, Carbopol 1342, and Carbopol 1382 (all from Lubrizol Advanced Materials) can be used.
[0022] Water acts as a solvent, and is preferably 55% by mass or more, more preferably 65% by mass or more, even more preferably 75% by mass or more, and is preferably 95% by mass or less, more preferably 93% by mass or less, and even more preferably 90% by mass or less in the original solution. The content of water in the original solution is preferably 55 to 95% by mass, more preferably 65 to 93% by mass, and even more preferably 75 to 90% by mass.
[0023] From the viewpoints of improving stability, foam ejection, retention of the aerosol formulation on the skin, and penetration of carbon dioxide into the skin, the viscosity of the aerosol concentrate at 25°C is preferably 500 mPa·s or more, more preferably 1000 mPa·s or more, even more preferably 1500 mPa·s or more, preferably 20000 mPa·s or less, more preferably 10000 mPa·s or less, and even more preferably 7000 mPa·s or less. The viscosity of the concentrate at 25°C is preferably 500 to 20000 mPa·s, more preferably 1000 to 10000 mPa·s, and even more preferably 1500 to 7000 mPa·s. Here, the viscosity was measured at 25°C using a BM viscometer (manufactured by Toki Sangyo Co., Ltd.) with rotor No. 3, 12 rpm, and 1 minute. When the viscosity exceeded 10,000 mPa s, the value was measured with rotor No. 3, 6 rpm, and 1 minute.
[0024] Carbonic acid aerosols can be produced by preparing the above-mentioned aerosol concentrate and filling it into a pressure-resistant container together with a propellant containing carbon dioxide gas. The form of the spray is preferably a foam type that is discharged in the form of bubbles.
[0025] The ratio of carbon dioxide gas to 100 parts by mass of the aerosol concentrate is 0.1 parts by mass or more, preferably 0.5 parts by mass or more, more preferably 1 part by mass or more, and 5 parts by mass or less, preferably 4 parts by mass or less, more preferably 3 parts by mass or less, from the viewpoint of improving the solubility of carbon dioxide gas in the aerosol concentrate, the foam viscosity, and the sprayability. Also, it is 0.1 to 5 parts by mass, preferably 0.5 to 4 parts by mass, and more preferably 1 to 3 parts by mass.
[0026] The dermal extracellular matrix production promoter of the present invention is preferably applied to humans who wish to prevent or improve symptoms caused by skin aging due to aging and photoaging due to chronic ultraviolet radiation, such as wrinkles, sagging, loss of elasticity, etc. The dermal extracellular matrix production promoter is applied in an appropriate amount to the skin, particularly the skin of the whole body except the scalp, preferably the face, body, limbs, etc., more preferably the face, neck, décolleté, back of the hand, outer forearm, outer upper arm, etc., where wrinkles and sagging tend to occur. The application may be carried out by taking it in the hands and spreading it on the target area, or by directly discharging it on the target area. In the case of a foam type, it is preferable to take it in the hands and apply it without crushing the foam. When taking the foam with both hands, the application amount may be, for example, about 0.5 to 5.0 g. The amount of aerosol preparation to be applied to the skin is, for example, 1 cm of skin. 2 The amount is 0.01 to 1 g, preferably 0.1 to 0.5 g.
[0027] After application, the carbon dioxide aerosol is left on the skin for 3 minutes or more, preferably 5 minutes or more, more preferably 10 minutes or more, without being blended with the skin. By contacting the carbon dioxide gas with the skin for a certain period of time, it is possible to induce a low pH environment (pH less than 7) in the dermis. The time for which the carbon dioxide aerosol preparation is left on the skin may be long, but is usually 20 minutes or less, preferably 15 minutes or less, from the viewpoint of maintaining the foam state. Therefore, in one embodiment, the time for which the carbonated aerosol is left on the skin is 3 to 20 minutes, preferably 5 to 15 minutes, and more preferably 10 to 15 minutes.
[0028] It is preferable to repeatedly apply the dermal extracellular matrix production-promoting agent at regular intervals in terms of the effect of promoting extracellular matrix production. The interval is not particularly limited, but may be, for example, 1 to 48 hours, preferably 1 to 24 hours, and more preferably 1 to 15 hours. There is no limit to the number of applications, but it is preferable to apply the agent at least twice and continuously for a certain period of time. For example, a preferred application pattern includes continuous application twice a day with an interval of 1 to 15 hours (for example, application in the morning and evening). EXAMPLES
[0029] Example 1 Carbon dioxide induces a decrease in pH in the dermis (1) Test specimen The following three types of test products were used. 1) Carbon dioxide gas-containing formulation: Aerosol formulation containing carbon dioxide gas (2.4%) (original viscosity: 4970 mPa·s), pH 6.1 (discharged in foam form) 2) Placebo formulation: An aerosol formulation that does not contain carbon dioxide gas (viscosity of original solution: 4970 mPa s), pH 6.9 3) Hydrochloric acid preparation: A preparation adjusted with HCl so that the pH of 2) is equivalent to that of the carbon dioxide-containing preparation (original viscosity: 3860 mPa s), pH 6.1
[0030] (2) Effect of carbon dioxide-containing formulations on the pH of the dermis layer of a human skin model Using a reconstructed human full-thickness skin model (T-Skin, NIKODERM RESEARCH) (hereafter referred to as a three-dimensional skin model) and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF, DOJINDO) to visualize and quantify pH changes, we performed an evaluation following a previous report (Fukagawa S, Takahashi A, Sayama K, Mori S, Murase T (2020) Carbon dioxide ameliorates reduced desquamation in dry scaly skin via protease activation. Int J Cosmet Sci. 42(6), 564-572.). BCECF is a soluble fluorescent dye whose fluorescence intensity increases depending on the pH and does not permeate the cell membrane, making it possible to capture pH changes between cells. The three-dimensional skin model was pre-cultured in the attached assay medium at 37°C and 5 Vol% CO2 for 24 hours, after which the medium was replaced with Hanks' Balanced Salt Solution (HBSS, GIBCO) containing BCECF (final concentration 10 μM) and allowed to acclimate for 15 minutes before use in the experiment. The test sample was added to the stratum corneum side of the three-dimensional skin model conditioned with BCECF-containing HBSS in an amount of about 0.7 g, and the fluorescence intensity I440 (Ex / Em=440 / 515) and I490 (Ex / Em=490 / 515) were measured for 100 minutes using a fluorescent plate reader (Infinite (registered trademark) M200PRO, TECAN) before and every 2 minutes after the addition. The measurement was performed by measuring the fluorescence intensity from below, irradiating the excitation light from the lower dermis side of the plate, and multi-point measurement (9 points / well) was performed to reduce the variation in the well, and the average value was used as the measured value. During the measurement, the added test sample was left without being touched at all. Separately, a calibration curve was created from the fluorescence intensity ratio (I490 / I440) of a three-dimensional skin model conditioned for 15 minutes with a pH standard solution (pH 5.5, 6.5 or 7.5) containing BCECF (final concentration 10 μM), and the pH was calculated. The results are shown in Figure 1. As is clear from Figure 1, the addition of the carbon dioxide gas-containing formulation induced a decrease in pH, and it was confirmed that the pH remained below 6.5 for approximately 30 minutes from 2 minutes after addition. On the other hand, no significant change in pH was observed when the placebo formulation or hydrochloric acid formulation was added. This means that when the carbon dioxide gas-containing formulation was added to the stratum corneum side of the three-dimensional skin model, the carbon dioxide gas penetrated into the dermis layer, inducing a transient decrease in pH. In addition, it was visually confirmed that when the carbon dioxide gas-containing formulation was added to human skin (inner forearm) and left to stand, it remained in a state of retaining carbon dioxide bubbles for 100 minutes.
[0031] Example 2: Effect of lowering the pH of the extracellular environment on the promotion of extracellular matrix production by dermal fibroblasts (1)Cell culture Normal human dermal fibroblasts (fibroblasts) derived from foreskin were purchased from Kurabo Industries, Ltd. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) containing 10% (v / v) Fetal Bovine Serum (FBS) (Sigma-Aldrich) at 37°C and 5 Vol% CO2.
[0032] (2) Effect of short-term (repeated) low pH stimulation on gene expression of extracellular matrix-related factors in fibroblasts 1.25 × 10 fibroblasts (1) 5 The cells were seeded in a 24-well plate at a density of 100 cells / well using DMEM medium supplemented with 2% (v / v) FBS. The next day, the cells were washed with PBS, and then replaced with a low pH stimulation medium in which the pH of the FBS-free DMEM medium was adjusted to pH 6.5 with HCl, and left to stand in an incubator for 10 or 30 minutes. As a control, a pH-unadjusted FBS-free DMEM medium (pH 7.6-7.8) was used and left to stand in the same manner (no low pH stimulation). After standing, the cells were washed with PBS, and all wells were replaced with FBS-free DMEM medium (pH unadjusted) and cultured overnight. The above process was considered as one low pH stimulation, and this process was repeated two or three times, so that the fibroblasts were given a low pH stimulation for 10 or 30 minutes two or three times. After two or three low pH stimulation steps, the cells were washed with PBS, and total RNA was extracted using an RNeasy Mini Kit (QIAGEN) according to a standard method. Using the extracted total RNA as a template, cDNA was synthesized by reverse transcription using High Capacity RNA to cDNA Kit (Thermo Fisher Scientific). ProFlex PCR System (Thermo Fisher Scientific) was used for the reaction. Subsequently, gene expression analysis was performed by quantitative PCR using the synthesized cDNA and TaqMan (registered trademark) probe. The probe and primer specific to the ELN, FBN1, MFAP-4, NEP, COL1A1, COL3A1, MMP1, HAS2, and HYBID (KIAA1199) genes were TaqMan Gene Expression Assays (P / N 4331182) manufactured by Thermo Fisher Scientific. The expression level of each gene was corrected by the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reaction conditions were performed according to the standard method using StepOnePlus System (Thermo Fisher Scientific). The results are shown in Table 1. The results are shown as relative values, with the expression level without low pH stimulation set at 1. As is clear from Table 1, under conditions of two or more low pH stimulations, the expression of ELN, FBN1, and MFAP-4, which are involved in the formation of elastin fibers, was increased, and the expression of NEP, which is involved in the degradation of elastin fibers, was decreased. In addition, the expression of COL1A1 and COL3A1, which are involved in the formation of collagen fibers, was increased, and the expression of MMP1, which is involved in the degradation of collagen fibers, was decreased. Furthermore, the expression of HAS2, which is involved in the synthesis of hyaluronic acid, was increased, and the expression of HYBID, which is involved in the degradation, was decreased. Regarding the stimulation time, the direction and degree of the expression change were roughly consistent for both 10 and 30 minutes. These mean that repeated lowering of the dermis pH can increase the production of the main dermis components, elastin fibers, collagen fibers, and hyaluronic acid, in fibroblasts in a complex manner.
[0033] [Table 1]
[0034] Example 3: Induction of pH decrease in the dermis depending on carbon dioxide application time (1) Test specimen The following two types of test products were used. 1) Carbon dioxide gas-containing formulation: Carbon dioxide gas (2.4%)-containing aerosol formulation (original viscosity: 4780 mPa·s), pH 6.3 (discharged in foam form) 2) Placebo formulation, pH 6.3: 1) An aerosol formulation that does not contain carbon dioxide, and whose pH was adjusted to be equivalent to that of the carbon dioxide-containing formulation using HCl (viscosity of the original solution: 2940 mPa s)
[0035] (2) Effect of different application times of a carbon dioxide-containing formulation on pH changes in the dermis layer in a human skin model Evaluation was performed using the method described in Example 1. The three-dimensional skin model was pre-cultured in the attached assay medium at 37°C and 5 Vol% CO2 for 24 hours, and then the medium was replaced with Hank's Balanced Salt Solution (HBSS, GIBCO) containing BCECF (final concentration 10 μM), and the model was allowed to acclimate for 15 minutes before use in the experiment. The test sample was added to the stratum corneum side of the three-dimensional skin model conditioned with BCECF-containing HBSS to a volume of about 0.7 g, and after 30 seconds, 1 minute, or 3 minutes, the test sample was removed from the skin using a medicine spoon. Using a fluorescent plate reader (Infinite (registered trademark) M200PRO, TECAN), fluorescence measurements (Ex / Em = 440 / 515, 490 / 515) were performed for 30 minutes before and every 2 minutes from 4 minutes after addition. The measurements were performed by bottom fluorescence measurements in which excitation light was irradiated from the lower dermis side of the plate, and multi-point measurements (9 points / well) were performed to reduce variation within the well, and the average value was used as the measured value. Separately, a calibration curve was created from the fluorescence intensity ratio (I490 / I440) of a three-dimensional skin model conditioned for 15 minutes with a pH standard solution (pH 5.5, 6.5, or 7.5) containing BCECF (final concentration 10 μM), and the pH was calculated. The results are shown in Figure 2.
[0036] As is clear from Figure 2, when the carbon dioxide gas-containing formulation was added, a strong decrease in pH was induced depending on the application time, whereas no significant change in pH was observed when the placebo formulation was added. Furthermore, by applying the carbon dioxide gas-containing formulation for 3 minutes, the pH was decreased to less than 7, and the lower pH was maintained than when the placebo formulation was applied for 3 minutes.
[0037] From the above, it can be said that after applying a carbon dioxide gas-containing preparation to the skin, in order to induce a sufficient decrease in pH in the dermis and the resulting promotion of extracellular matrix production, it is important to retain the foam after ejection for at least 3 minutes.
Claims
1. A promoter for promoting the production of dermal extracellular matrix, which contains carbon dioxide gas as an active ingredient. The promoter for promoting the production of dermal extracellular matrix is used by applying an aerosol preparation containing 0.1 to 5 parts by mass of carbon dioxide gas to 100 parts by mass of the aerosol stock solution to the skin and leaving it for 3 minutes or more.
2. The application is an application of an aerosol preparation at 0.01 to 1 g per 1 cm of skin ２ of the promoter for promoting production of dermal extracellular matrix according to claim 1.
3. The promoter for promoting the production of dermal extracellular matrix according to claim 1 or 2, wherein the application is repeated at intervals of 1 to 48 hours.
4. The promoter for promoting the production of dermal extracellular matrix according to claim 1, wherein the dermal extracellular matrix is one or more selected from collagen, elastin, and hyaluronic acid.
5. The promoter for promoting the production of dermal extracellular matrix according to claim 1, for preventing or improving dermal skin aging.