MMP9 gene expression inhibitor, wrinkle improvement agent, and firmness improvement agent

Abstract

The object of the present invention is to provide novel various improving agents (an agent for suppressing MMP9 gene expression, an agent for improving wrinkles, and an agent for improving skin firmness). [Solution] The present invention provides an agent for suppressing MMP9 gene expression, etc., comprising bakuchiol and tranexamic acid.

Description

[[Technical Field]]

[0001] The present invention relates to an agent for inhibiting MMP9 gene expression, an agent for improving wrinkles, and an agent for improving skin firmness. [[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 Document]] [[Patent Document]]

[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 an excellent improving effect that is difficult to achieve with each component 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 inhibiting MMP9 gene expression, an agent for improving wrinkles, and an agent for improving skin firmness. [[Means for Solving the Problem]]

[0006] As a result of studies conducted by the present inventor, it was found that the combination of bakuchiol and tranexamic acid significantly inhibits MMP9 gene expression, and the present invention has been completed. More specifically, the present invention provides the following.

[0007] (1) An agent for inhibiting MMP9 gene expression, comprising bakuchiol and tranexamic acid.

[0008] (2) A wrinkle-improving agent containing bakuchiol and tranexamic acid.

[0009] (3) A skin firming agent containing bakuchiol and tranexamic acid. [Effects of the Invention]

[0010] According to the present invention, novel improving agents (an agent for suppressing MMP9 gene expression, an agent for improving wrinkles, and an agent for improving skin firmness) are provided. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the results of the effect on MMP9 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) Agents that suppress MMP9 gene expression In one embodiment of the present invention, the MMP9 gene expression inhibitor of the present invention comprises bakuchiol and tranexamic acid.

[0014] The inventors explored combinations of various components that can be incorporated into skin conditioning agents to find those that exhibit beneficial interactions. They focused on MMP9 gene expression as one of the indicators for this exploration. Suppression of MMP9 gene expression can inhibit collagen degradation and, furthermore, may lead to wrinkle reduction, improved skin firmness, and other benefits.

[0015] As a result, we discovered the unexpected finding that bakuchiol alone did not suppress MMP9 gene expression, but when combined with tranexamic acid, it exhibited a remarkable suppressive effect on MMP9 gene expression, thus completing the present invention.

[0016] In the present invention, the "MMP9 gene expression inhibitory effect" includes a reduction in the MMP9 gene expression level when the agent for inhibiting MMP9 gene expression of the present invention is used, as compared with when the agent is not used. Whether MMP9 gene expression has decreased can be specified by an in vitro system (for example, the method shown in the Examples). In addition, when the agent for inhibiting MMP9 gene expression is applied to any site of a living body (such as skin), whether MMP9 gene expression has decreased can be indirectly determined, for example, by specifying whether skin wrinkles and firmness have been improved.

[0017] The term "MMP9" is an abbreviation for "matrix metalloproteinase-9".

[0018] Hereinafter, the configuration of the agent for inhibiting MMP9 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, also called "4-[(S,E)-3-ethenyl-3,7-dimethyl-1,6-octadienyl]phenol" or the like. The present inventors have found the surprising finding that although bakuchiol alone does not have an MMP9 gene expression inhibitory effect, the combination of bakuchiol and tranexamic acid exerts a remarkable MMP9 gene expression inhibitory effect.

[0020] The content of bakuchiol in the agent for inhibiting MMP9 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 inhibiting MMP9 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 is preferably 10.00% by mass or less, more preferably 2.00% by mass or less, relative to the agent for inhibiting MMP9 gene expression.

[0021] The content of bakuchiol contained in the agent for inhibiting MMP9 gene expression is determined by a conventional method such as high performance liquid chromatography (HPLC).

[0022] (1-2) Tranexamic acid Tranexamic acid (CAS Registry Number: 1197-18-8, chemical formula: C8H 15 NO2) is an ingredient having anti-inflammatory effects and the like, and is blended in various cosmetics. The present inventors confirmed that tranexamic acid alone exhibits a certain degree of MMP9 gene expression inhibitory effect. However, the inventors have found that the combination of bakuchiol and tranexamic acid exerts a more remarkable MMP9 gene expression inhibitory effect.

[0023] The content of tranexamic acid in the agent for inhibiting MMP9 gene expression of the present invention is not particularly limited, and can be appropriately adjusted according to the intended effect and the like. In a preferred embodiment of the present invention, the lower limit of the tranexamic acid content is preferably 0.05% by mass or more, more preferably 1.00% by mass or more, relative to the agent for inhibiting MMP9 gene expression. In a preferred embodiment of the present invention, the upper limit of the tranexamic acid content is preferably 3.00% by mass or less, more preferably 2.00% by mass or less, relative to the agent for inhibiting MMP9 gene expression.

[0024] The content of tranexamic acid contained in the agent for inhibiting MMP9 gene expression is determined by a conventional method such as high performance liquid chromatography (HPLC).

[0025] (1-3) Ratio of bakuchiol and tranexamic acid The ratio of bakuchiol and tranexamic acid to be formulated in the agent for inhibiting MMP9 gene expression is not particularly limited, and can be appropriately set according to the intended effect and the like.

[0026] From the viewpoint of more easily achieving the effects of the present invention, it is preferable that the concentration of tranexamic acid in the MMP9 gene expression inhibitor is higher than the concentration of bakuchiol. In a preferred embodiment of the present invention, the concentration ratio of tranexamic acid to bakuchiol (tranexamic acid / bakuchiol) in the MMP9 gene expression inhibitor is preferably 0.1 to 1000, more preferably 0.2 to 1000, and even more preferably 1 to 1000.

[0027] (1-4) Other ingredients The MMP9 gene expression inhibitor may or may not contain components other than bakuchiol and tranexamic acid, as long as they do not inhibit the effects of the present invention.

[0028] Other ingredients besides bakuchiol and tranexamic acid 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 tranexamic acid 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 MMP9 gene expression inhibitor of the present invention The form of the MMP9 gene expression inhibitor of the present invention is not particularly limited. The MMP9 gene expression inhibitor of the present invention may be an agent comprising bakuchiol and tranexamic acid, or an agent containing bakuchiol, tranexamic acid, and other components.

[0031] In a preferred embodiment of the present invention, the MMP9 gene expression inhibitor of the present invention may be used as part of a component of a topical skin preparation, or the MMP9 gene expression inhibitor of the present invention may be prepared as a topical skin preparation itself. In the former embodiment, the MMP9 gene expression inhibitor 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 MMP9 gene expression inhibitor of the present invention are not particularly limited and can be appropriately set according to the form of the MMP9 gene expression inhibitor of the present invention and the effect to be obtained.

[0033] (1-6) Method for producing the MMP9 gene expression inhibitor of the present invention The method for producing the MMP9 gene expression inhibitor 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) Wrinkle-improving agents In one embodiment of the present invention, the wrinkle-improving agent of the present invention comprises bakuchiol and tranexamic acid.

[0035] As described above, the inventors of this invention discovered the surprising finding that bakuchiol alone did not exhibit an inhibitory effect on MMP9 gene expression, but when combined with tranexamic acid, it exhibited a remarkable inhibitory effect on MMP9 gene expression. Suppressing MMP9 gene expression leads to wrinkle improvement.

[0036] The wrinkle-reducing effect can be determined, for example, by an anti-wrinkle evaluation test in accordance with the "Guidelines of the Japanese Society of Cosmetic Scientists." For example, if a decrease of one or more of the following is observed on any given skin surface: wrinkle area ratio, maximum average wrinkle depth, maximum maximum wrinkle depth, and total average wrinkle depth, then it can be determined that the wrinkles have improved.

[0037] Each component of the wrinkle-improving agent of the present invention can adopt the same configuration as the MMP9 gene expression inhibitor described above.

[0038] (3) Agents for improving skin firmness In one embodiment of the present invention, the skin firming agent of the present invention comprises bakuchiol and tranexamic acid.

[0039] As described above, the inventors of this invention discovered the surprising finding that bakuchiol alone did not exhibit an inhibitory effect on MMP9 gene expression, but when combined with tranexamic acid, it exhibited a remarkable inhibitory effect on MMP9 gene expression. Suppression of MMP9 gene expression leads to improved skin firmness.

[0040] The effect of improving skin firmness can be evaluated by measuring skin elasticity (skin viscoelasticity, etc.) using any conventionally known method. For example, an increase in skin viscoelasticity can be interpreted as an improvement in skin firmness.

[0041] Each component of the firming agent of the present invention can adopt the same configuration as the MMP9 gene expression inhibitor 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 tranexamic acid 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.) • Tranexamic acid (manufactured by Changzhou Yinsheng 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) • TA (Tranexamic Acid): Final concentration 0.05% by mass (500 ppm) • BacTA (Bactiol and Tranexamic Acid): Final concentration of Bac 0.0001% by mass, final concentration of TA 0.05% by mass, concentration ratio (Tranexamic Acid (=500) / Bactiol (=1)) = 500

[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 expression analysis of the MMP9 gene (the vertical axis represents TPM (transcripts per million)). MMP9 is involved in skin inflammation, and it is expected that the lower the expression level of the MMP9 gene, the more collagen breakdown is suppressed, leading to improved wrinkles and firmness. A 5% significance level was observed between "control" and "BacTA," "TA" and "Bac," and "Bac" and "BacTA." In "TA," the expression level of the MMP9 gene was lower than that of the control, but this was not statistically significant. This indicates that the combination of bakuchiol (Bac) and tranexamic acid (TA) has a synergistic effect on suppressing MMP9 gene expression.

Claims

1. An agent for suppressing MMP9 gene expression, containing bakuchiol and tranexamic acid.

2. A wrinkle-improving agent containing bakuchiol and tranexamic acid.

3. A skin firming agent containing bakuchiol and tranexamic acid.

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