Kallikrein 7 production promoter

Abstract

To provide a novel kallikrein 7 production promoter.SOLUTION: A kallikrein 7 production promoter contains ergothioneine.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an agent for promoting the production of kallikrein 7. [Background technology]

[0002] Kallikrein 7 (hereinafter, also referred to as KLK7) is an S1 serine protease of the kallikrein gene family that exhibits chymotrypsin-like activity. KLK7 is mainly expressed in the skin and is known to be deeply involved in skin physiology. The present applicant has proposed a kallikrein 7 production promoter in Patent Documents 1 and 2.

[0003] Ergothioneine is an amino acid derivative biosynthesized by basidiomycetes such as mushrooms, and is known to have stronger antioxidant power than vitamin C and vitamin E. Ergothioneine is also known to have physiological activity, and for example, Patent Document 3 proposes an ATP production promoter, an epidermal cell activator, and an external skin preparation for improving aging and rough skin, each of which contains ergothioneine as an active ingredient, while Patent Document 4 proposes a vascular wall strengthening agent, each of which contains ergothioneine as an active ingredient. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2016-003198 A [Patent Document 2] JP 2018-131405 A [Patent Document 3] JP 2003-231626 A [Patent Document 4] JP 2022-006753 A Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a novel kallikrein 7 production promoter. [Means for solving the problem]

[0006] The means for solving the problems of the present invention are as follows. 1. A kallikrein 7 production promoter that contains ergothioneine. Effect of the Invention

[0007] The present invention provides a KLK7 production-promoting composition. [Brief description of the drawings]

[0008] [Figure 1] Graph showing that ergothioneine increases the amount of intracellular KLK7 in cultured human epidermal keratinocytes. [Diagram 2] Graph showing that ergothioneine increases the secretion amount of KLK7 in cultured human epidermal keratinocytes. [Diagram 3] Graph showing that the amount of KLK7 in the stratum corneum increases by applying a formulation containing ergothioneine for four weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Ergothioneine has the chemical formula C9H 15 It is an amino acid derivative represented by N3O2S. As ergothioneine, extracts from basidiomycetes such as Pleurotus cornucoides, sake lees, etc. can be used, and these extracts can be used as they are or after purification. In addition, commercially available ergothioneine products can also be used.

[0010] -Kallikrein 7 production promoter Human KLK7 is a secreted protein with a molecular mass of 27,525 Da. By promoting the production of KLK7, it is expected that the amount of KLK7 in the skin will be increased and skin aging will be suppressed. The KLK7 production promoter of the present invention contains ergothioneine. The amount of ergothioneine in the KLK7 production promoter of the present invention is not particularly limited as long as the effects of the present invention are achieved, but the concentration of ergothioneine relative to the total amount is preferably 0.000001% by mass or more and 99.5% by mass or less.

[0011] The KLK7 production promoter of the present invention can be appropriately formulated with oily bases such as vegetable oils and animal oils, analgesics and anti-inflammatory agents, analgesics, germicides, disinfectants, astringents, hormones, vitamins, alcohols, chelating agents, pH adjusters, preservatives, thickeners, colorants, fragrances, etc. that are commonly used in the dosage form, within a range that does not impair the effects of the present invention. The KLK7 production promoter of the present invention can be administered either orally or transdermally, and can be in the form of cosmetics, topical skin preparations, medicines, foods, beverages, etc. The KLK7 production promoter of the present invention is preferably administered transdermally, and among cosmetics, topical skin preparations, medicines, etc., it is particularly preferable to adopt the dosage form of a transdermal composition in the form of an aqueous solution, cream, gel, paste, solid, aerosol, or patch. EXAMPLES

[0012] The effects of the composition of the present invention will be specifically described below with reference to test examples. Experiment 1: "Promotion of KLK7 production in human epidermal keratinocytes" The effect of promoting KLK7 production was confirmed using human epidermal keratinocytes. The cells used were NHEK-Neo (human epidermal keratinocytes, derived from neonatal skin, manufactured by Life Technologies Japan). The culture medium used was EpiLife (registered trademark) Medium with 60 μM Calcium (manufactured by Life Technologies Japan) supplemented with Humedia-KG2 growth additive set (manufactured by Kurabo Industries, Ltd.).

[0013] Human epidermal keratinocytes were cultured in EpiLife with Humedia-KG2 at 2.2 × 10 4 cells / cm 2The cells were seeded in a 6-well plate at a cell density of 1000 x 1000 and cultured overnight in a 37°C 5% CO2 incubator, after which ergothioneine (Cayman Chemical) was added at 0.00003-0.002% by mass. The cells were further cultured for 3 days in a 37°C 5% CO2 incubator, and intracellular and extracellular proteins were collected. The protein amount was adjusted to a constant level using a BCA protein assay kit (Thermo Fisher Scientific), and the expression level of KLK7 was measured by Western blotting. β-actin was quantified as a loading control, and the relative value to β-actin was calculated. For quantification, Human / Mouse Kallikrein7 Antibody (R&D Systems) and β-Actin Antibody (C4) (Santa Cruz) were used as primary antibodies, diluted 1000-fold, and incubated in a 4°C refrigerator for 48 hours. After washing, the secondary antibodies were Goat anti-Mouse IgG (H+L) (Thermo Fisher Scientific) and Rabbit anti-Goat IgG (H+L) (Thermo Fisher Scientific) diluted 10,000 times and incubated at room temperature for 1 hour, followed by ECL. TM Detection was performed using a Western Blotting Detection System (Citiva). The relative amount of KLK7 in the cells is shown in Figure 1, and the relative amount of KLK7 secreted from the cells is shown in Figure 2, compared to the control without ergothioneine.

[0014] ·result It has been confirmed that ergothioneine promotes the production of kallikrein 7.

[0015] Experiment 2: "Ergothioneine increases KLK7 levels in the stratum corneum of the cheek" Twelve subjects were administered a preparation containing 0.00006% by mass of ergothioneine (a product extracted and purified from Pleurotus corneum) shown in Table 1 to the left and right cheeks twice a day, morning and evening. Before and after 4 weeks of application, the stratum corneum of the cheeks was collected by tape stripping using a stratum corneum checker (Asahi Biomed Co., Ltd.). The stratum corneum checker with the collected stratum corneum was placed in a sample tube containing glass beads and 500 μL of Laemmli Buffer (composition: 0.09 M Tris-HCl, pH 6.8, 3% SDS, 10.3% glycerol), and homogenized at 2800 rpm for 180 seconds using a bead-type cell disrupter (Tomy Seiko Co., Ltd.) to extract stratum corneum proteins. The protein content of each sample was measured using a BCA protein assay kit (Thermo Scientific Co., Ltd.), and after the amount of stratum corneum protein was set to a constant level, the expression level of KLK7 was measured by Western blotting. For quantification, Human / Mouse Kallikrein 7 Antibody (R&D Systems) was used as the primary antibody, diluted 3000-fold, and incubated in a refrigerator at 4°C for 18 hours. After washing, Goat anti-Mouse IgG (H+L) (Thermo Fisher Scientific) was used as the secondary antibody, diluted 10000-fold, and incubated at room temperature for 1 hour, followed by ECL. TM Detection was performed using a Western Blotting Detection System (Citiva). The relative values ​​of the amount of KLK7 produced 4 weeks after application to those before application are shown in FIG.

[0016] [Table 1]

[0017] ·result Application of a formulation containing ergothioneine to the cheek increased the amount of KLK7 in the stratum corneum of the cheek compared to before application, which is thought to promote the production of kallikrein 7.

Claims

[Claim 1] A kallikrein 7 production promoter containing ergothioneine.

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