EHF gene expression promoting agent, skin barrier function improving agent, and epidermal turnover improving agent

Abstract

The object of the present invention is to provide novel various improving agents (agents for promoting EHF gene expression, agents for improving skin barrier function, and agents for improving epidermal turnover). [Solution] The present invention provides an EHF gene expression promoting agent, etc., comprising bakuchiol and stearyl glycyrrhetinate.

Description

[Technical Field]

[0001] The present invention relates to an agent for promoting EHF gene expression, an agent for improving skin barrier function, and an agent for improving epidermal turnover. [Background Art]

[0002] Skin improving agents and the like are blended with various components according to the effect to be obtained, from the viewpoint of improving or maintaining skin condition. For example, Patent Document 1 proposes an agent for suppressing cell atrophy caused by UVA irradiation in fibroblasts, which contains bakuchiol as an active ingredient. [Prior Art Literature] [Patent Literature]

[0003] [Patent Document 1] Japanese Unexamined Patent Publication No. 2022-073838 [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] On the other hand, the present inventor has found that combining a plurality of components can exert excellent improving effects that are difficult to achieve when each component is used alone.

[0005] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide various novel improving agents: an agent for promoting EHF gene expression, an agent for improving skin barrier function, and an agent for improving epidermal turnover. [Means for Solving the Problem]

[0006] As a result of studies conducted by the present inventor, it has been found that the combination of bakuchiol and stearyl glycyrrhetinate significantly promotes EHF gene expression, and the present invention has been completed. More specifically, the present invention provides the following.

[0007] (1) An EHF gene expression promoter comprising bakuchiol and stearyl glycyrrhetinate.

[0008] (2) An agent for improving skin barrier function, comprising bakuchiol and stearyl glycyrrhetinate.

[0009] (3) An agent for improving epidermal turnover, comprising bakuchiol and stearyl glycyrrhetinate. [Effects of the Invention]

[0010] According to the present invention, novel improving agents (agents for promoting EHF gene expression, agents for improving skin barrier function, and agents for improving epidermal turnover) are provided. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the results of the effect on EHF gene expression in the examples. [Modes for carrying out the invention]

[0012] The following describes embodiments of the present invention, but the present invention is not limited thereto.

[0013] (1) EHF gene expression promoter In one embodiment of the present invention, the EHF gene expression promoting agent of the present invention comprises bakuchiol and stearyl glycyrrhetinate.

[0014] The inventors explored combinations of various components that can be incorporated into skin conditioning agents to find combinations that exhibit beneficial interactions. They focused on EHF gene expression as one of the indicators for this exploration. Promoting EHF gene expression can lead to improvements in skin barrier function, epidermal turnover, and other effects.

[0015] As a result, the inventors have found the unexpected finding that while bakuchiol alone does not exhibit an EHF gene expression promoting effect, it exhibits a remarkable EHF gene expression promoting effect when combined with stearyl glycyrrhetinate, and thus completed the present invention.

[0016] In the present invention, the "EHF gene expression promoting effect" includes that the use of the agent for promoting EHF gene expression of the present invention increases the expression level of EHF gene compared to when the agent is not used. Whether EHF gene expression has increased can be identified by an in vitro system (for example, the method described in the Examples). In addition, when the agent for promoting EHF gene expression is applied to any site of a living body (such as skin), whether EHF gene expression has increased can be indirectly determined, for example, by identifying whether the skin barrier function or epidermal turnover has been improved.

[0017] The term "EHF" is an abbreviation for "ETS (erythroblast transformation specific) homologous factor".

[0018] Hereinafter, the constitution of the agent for promoting EHF gene expression of the present invention will be described in detail.

[0019] (1-1) Bakuchiol Bakuchiol (CAS Registry Number: 10309-37-2, chemical formula: C 18 H 24 O) is a meroterpene, which is also called "4-[(S,E)-3-ethenyl-3,7-dimethyl-1,6-octadienyl]phenol" or the like. The inventors of the present invention have found the unexpected finding that while bakuchiol alone does not have an EHF gene expression promoting effect, it exerts a remarkable EHF gene expression promoting effect when combined with stearyl glycyrrhetinate.

[0020] The content of bakuchiol in the agent for promoting EHF gene expression of the present invention is not particularly limited, and can be appropriately adjusted according to the effect to be obtained and the like. In a preferred embodiment of the present invention, the lower limit of the bakuchiol content relative to the agent for promoting EHF gene expression is preferably 0.01% by mass or more, more preferably 0.50% by mass or more, and still more preferably 1.00% by mass or more. In a preferred embodiment of the present invention, the upper limit of the bakuchiol content relative to the agent for promoting EHF gene expression is preferably 10.00% by mass or less, more preferably 5.00% by mass or less.

[0021] The content of bakuchiol contained in the agent for promoting EHF gene expression is specified by a conventional method such as high performance liquid chromatography (HPLC).

[0022] (1-2) Stearyl Glycyrrhetinate Stearyl glycyrrhetinate (CAS Registration No.: 13832-70-7, chemical formula: C 48 H 82 O4) is an ingredient that has anti-inflammatory effects and the like, and is formulated into various cosmetics. The present inventors have confirmed that stearyl glycyrrhetinate alone has a certain EHF gene expression promoting effect. However, the inventors have found that the combination of bakuchiol and stearyl glycyrrhetinate exhibits a more remarkable EHF gene expression promoting effect.

[0023] The content of stearyl glycyrrhetinate in the agent for promoting EHF gene expression of the present invention is not particularly limited, and can be appropriately adjusted according to the effect to be obtained and the like. In a preferred embodiment of the present invention, the lower limit of the stearyl glycyrrhetinate content relative to the agent for promoting EHF gene expression is preferably 0.05% by mass or more, more preferably 0.10% by mass or more. In a preferred embodiment of the present invention, the upper limit of the stearyl glycyrrhetinate content relative to the agent for promoting EHF gene expression is preferably 0.50% by mass or less, more preferably 0.40% by mass or less, and still more preferably 0.30% by mass or less.

[0024] The amount of stearyl glycyrrhetinate contained in EHF gene expression promoters is determined by conventional methods such as high-performance liquid chromatography (HPLC).

[0025] (1-3) Ratio of bakuchiol to stearyl glycyrrhetinate The ratio of bakuchiol and stearyl glycyrrhetinate incorporated into the EHF gene expression promoter is not particularly limited and can be set as appropriate depending on the desired effect.

[0026] From the viewpoint of more easily achieving the effects of the present invention, it is preferable that the concentration of stearyl glycyrrhetinate in the EHF gene expression promoting agent is higher than the concentration of bakuchiol. In a preferred embodiment of the present invention, the concentration ratio of stearyl glycyrrhetinate to bakuchiol (stearyl glycyrrhetinate / bakuchiol) in the EHF gene expression promoting agent is preferably 0.01 to 50, more preferably 0.1 to 50.

[0027] (1-4) Other ingredients The EHF gene expression promoting agent may contain, or may not contain, components other than bakuchiol and stearyl glycyrrhetinate, as long as they do not inhibit the effects of the present invention.

[0028] Other components besides bakuchiol and stearyl glycyrrhetinate can be appropriately selected depending on the desired effect and the form of the preparation.

[0029] In a preferred embodiment of the present invention, components other than bakuchiol and stearyl glycyrrhetinate include any components that can be incorporated into a topical skin preparation. Such components include bases, solvents, fragrances, antioxidants, excipients, colorants, surfactants, preservatives, pH adjusters, and the like. In the present invention, "topical skin preparation" includes any preparation that is applied to the surface of the skin of a living organism (e.g., by coating, spraying), and examples include the following. • Non-emulsified formulations (liquid formulations, etc.), emulsified formulations (oil-in-water emulsions, water-in-oil emulsions) Solid preparations, liquid preparations, ointments, creams, sprays

[0030] (1-5) Form of the EHF gene expression promoting agent of the present invention The form of the EHF gene expression promoting agent of the present invention is not particularly limited. The EHF gene expression promoting agent of the present invention may be an agent comprising bakuchiol and stearyl glycyrrhetinate, or an agent containing bakuchiol, stearyl glycyrrhetinate, and other components.

[0031] In a preferred embodiment of the present invention, the EHF gene expression promoting agent of the present invention may be used as part of a component of a topical skin preparation, or the EHF gene expression promoting agent of the present invention may be prepared as a topical skin preparation itself. In the former embodiment, the EHF gene expression promoting agent of the present invention may also be referred to as an "additive for topical skin preparations," etc.

[0032] The amount and frequency of use of the EHF gene expression promoting agent of the present invention are not particularly limited and can be appropriately determined depending on the form of the EHF gene expression promoting agent of the present invention and the effect to be obtained.

[0033] (1-6) Method for producing the EHF gene expression promoting agent of the present invention The method for producing the EHF gene expression promoting agent of the present invention is not particularly limited, and any method can be used depending on the form of the agent, for example.

[0034] (2) Agents for improving skin barrier function In one embodiment of the present invention, the skin barrier function improving agent of the present invention comprises bakuchiol and stearyl glycyrrhetinate.

[0035] As described above, the inventors of this invention discovered the surprising finding that bakuchiol alone did not promote EHF gene expression, but when combined with stearyl glycyrrhetinate, it exhibited a remarkable effect in promoting EHF gene expression. Promoting EHF gene expression leads to improved skin barrier function.

[0036] The improvement in skin barrier function can be determined, for example, based on transepidermal water loss (TEWL). If a downward trend in transepidermal water loss is observed, it can be concluded that the skin barrier function has improved.

[0037] Each component of the skin barrier function improving agent of the present invention can adopt the same configuration as the EHF gene expression promoting agent described above.

[0038] (3) Agents for improving epidermal turnover In one embodiment of the present invention, the epidermal turnover improving agent of the present invention comprises bakuchiol and stearyl glycyrrhetinate.

[0039] As described above, the inventors of this invention discovered the surprising finding that bakuchiol alone did not promote EHF gene expression, but when combined with stearyl glycyrrhetinate, it exhibited a remarkable effect in promoting EHF gene expression. Promoting EHF gene expression leads to improved epidermal turnover.

[0040] The effect of improving epidermal turnover can be evaluated based on the epidermal turnover time (the time from when cell division begins in the basal layer until the stratum corneum peels off) using any conventionally known method. If the epidermal turnover time is shortened, it can be concluded that epidermal turnover has improved.

[0041] Each component of the epidermal turnover improving agent of the present invention can adopt the same configuration as the EHF gene expression promoting agent described above. [Examples]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0043] <Experiment 1: Gene expression analysis using keratinocytes> The following methods were used to investigate the effects of bakuchiol and stearyl glycyrrhetinate on gene expression in keratinocytes.

[0044] (1) Preparation of the sample The following samples were prepared and subjected to the culture test described later. • Bactiol (manufactured by Sytheon Ltd.) • Stearyl glycyrrhetinate (manufactured by Maruzen Pharmaceutical Co., Ltd.)

[0045] (2) Culture test Place keratinocytes (Kurabo Corporation) in a 24-well plate in a culture medium, 5.0 × 10⁶ 4 Seeds were seeded to a concentration of cells / mL and incubated at 37°C under a 5% CO2 atmosphere for 48 hours. "EpiLife-KG2" medium (manufactured by Kurabo Industries Ltd.) was used as the culture medium.

[0046] Next, each sample was added to the culture to reach the predetermined final concentration, and the culture was further incubated at 37°C under a 5% CO2 atmosphere for 18 hours. The conditions for the sample added to the culture medium are as follows: • Cont (control): Culture medium only • Bac (Bactiol): Final concentration 0.0001% by mass (1 ppm) GRS (Stearyl Glycyrrhetinate): Final concentration 0.0025% by mass (25 ppm) • GRSBac (Stearyl Glycyrrhetinate and Bakuchiol): GRS final concentration 0.0025% by mass, Bac final concentration 0.0001% by mass, concentration ratio (Stearyl Glycyrrhetinate (=25) / Bakuchiol (=1)) = 25

[0047] After culturing, cells were harvested from each culture using "RLT buffer" (Qiagen), and RNA was purified from the harvested cells using "RNeasy Mini Kit" (Qiagen). The purified RNA was sequenced using "Novaseq" (Illumina). Based on the sequencing data, the expression levels of the transcript units were calculated using the "DRAGEN Bio-IT Platform" (Illumina). The obtained gene list was used to perform differential gene expression extraction and pathway analysis using "iDEP.96" (South Dakota State University).

[0048] The Tukey-Kramer method was used for the statistical analysis of the obtained results. In each method, we determined whether or not there was a significant improvement compared to the control group.

[0049] (3) Results Figure 1 shows the results of the EHF gene expression analysis (the vertical axis represents TPM (transcripts per million)). EHF is involved in the differentiation of epidermal cells, and it is expected that the higher the expression level of the EHF gene, the greater the effect on improving epidermal turnover and skin barrier function. A 5% statistically significant difference was observed between "control" and "GRSBac," as well as between "Bac" and "GRSBac." In "GRS," EHF gene expression was higher than in the control, but this was not statistically significant. This indicates that the combination of bakuchiol (Bac) and stearyl glycyrrhetinate (GRS) has a synergistic effect in promoting EHF gene expression.

Claims

1. An EHF gene expression promoter containing bakuchiol and stearyl glycyrrhetinate.

2. A skin barrier function improving agent containing bakuchiol and stearyl glycyrrhetinate.

3. An agent for improving epidermal turnover, containing bakuchiol and stearyl glycyrrhetinate.

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