JP2023038552A - Il-1ra and col-7 production promoting composition - Google Patents

Abstract

To provide a novel IL-1RA and COL-7 production promoting composition.SOLUTION: The present invention provides an IL-1RA production promoting composition containing at least one selected from yeast extract, inositol and vitamin B2, and L-ascorbic acid 2-glucoside, or a COL-7 production promoting composition containing at least one selected from yeast extract, inositol and vitamin B2, and L-ascorbic acid 2-glucoside.SELECTED DRAWING: Figure 1

Description

The present invention relates to an IL-1 receptor antagonist (IL-1RA) and a composition for promoting type VII collagen (COL-7) production.

IL-1RA is an anti-inflammatory cytokine that increases during inflammation in the body and normalizes it, and is a protein that promotes cell proliferation. The relationship between the behavior of IL-1RA in the skin and skin symptoms has attracted attention.
The present inventors have studied the behavior of IL-1RA in the stratum corneum of human skin, and disclosed a method for indirectly measuring blood flow in the skin using the expression level of IL-1RA in the stratum corneum of the skin as an index. (Patent Document 1). Furthermore, they have found that the state of skin texture and the expression level of IL-1RA in the stratum corneum of the skin have an extremely high correlation, and have disclosed an invention of a new method for evaluating the state of skin texture. (Patent Document 2).
Thus, it has become clear that the expression level of IL-1RA is closely related to understanding the health condition of the skin.

In the course of conducting research on skin aging, the present inventors discovered that the aging phenomenon of the skin is caused by the accumulation and progression of minute damage to the skin and skin cells caused by various factors (such as ultraviolet rays and active oxygen), resulting in wrinkles. It has been found that IL-1RA is involved in this. And in order to speed up the recovery from such minor injuries, we thought that increasing IL-1RA in skin cells would solve the problem. It was found that the amount of production increases, and the expression of type IV collagen (COL-4) and type VII collagen (COL-7), which constitute the basement membrane, increases (Non-Patent Document 1).

Furthermore, the present inventors have searched for substances that produce IL-1RA in skin cells. In the process, it was found that a composition containing yeast extract and vitamin B2 and a composition containing yeast extract and inositol increased IL-1RA in skin cells. (Patent Document 3). In addition, it is disclosed that L-ascorbic acid 2-glucoside also has an effect of promoting IL-1RA production in skin cells (Non-Patent Document 1).

Japanese Patent No. 5731678 Japanese Patent No. 5977268 Japanese Patent No. 6823428

Wu Koyu et al., "Functional analysis of skin aging-related proteins linked to the circadian rhythm," Journal of Cosmetic Dermatology, 2018, 28(2): 230

The present inventors have found that one or more selected from yeast extract, inositol, and vitamin B2 and L-ascorbic acid 2-glucoside synergistically promote the production of IL-1RA and COL-7. .
The present invention was made based on this effect, and an object of the present invention is to provide a novel composition for promoting IL-1RA and COL-7 production.

The inventors of the present invention conducted intensive studies with the aim of searching for a substance having further IL-1RA production-promoting activity. We have found that it promotes the production of 1RA and COL-7, and have completed the present invention.

The main configuration of the present invention is as follows.
1. An IL-1RA production promoting composition containing at least one selected from yeast extract, inositol and vitamin B2, and L-ascorbic acid 2-glucoside.
2. A COL-7 production promoting composition containing at least one selected from yeast extract, inositol and vitamin B2, and L-ascorbic acid 2-glucoside.

The present invention provides novel compositions for promoting IL-1RA and COL-7 production. By using the composition of the present invention as an external preparation for skin, IL-1RA and COL-7 in the skin are increased, promoting the recovery of fine skin damage, suppressing the occurrence of wrinkles, and reducing the appearance of wrinkles. can promote the recovery of

Graph showing the results of testing the effect of promoting IL-1RA production by the combined use of L-ascorbic acid 2-glucoside and yeast extract. Graph showing the results of testing the effect of promoting COL-7 production by the combined use of L-ascorbic acid 2-glucoside and yeast extract. Graph showing the results of testing the IL-1RA production promoting effect of the combined use of L-ascorbic acid 2-glucoside and inositol. Graph showing the results of testing the COL-7 production promoting effect of the combined use of L-ascorbic acid 2-glucoside and inositol. Graph showing the results of testing the IL-1RA production promoting effect of the combined use of L-ascorbic acid 2-glucoside and vitamin B2. Graph showing the results of testing the effect of promoting COL-7 production by the combined use of L-ascorbic acid 2-glucoside and vitamin B2.

The present invention provides an IL-1RA and COL-7 production promoting composition (hereinafter also referred to as a composition) containing at least one selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside. Regarding.
The composition of the present invention can activate the gene expression of IL-1RA and COL-7 expressed in skin cells to increase the amount of IL-1RA and type VII collagen in cells, resulting in skin Cells can be activated to prevent and even restore skin aging.

<Yeast extract>
The yeast extract, which is a component of the composition of the present invention, is a concentrated or dried liquid obtained by autolyzing or acid hydrolyzing yeast cell lysate, or this concentrated or dried liquid is added with water and / Or evaporation residue of a composition obtained by extraction with a solution containing alcohols. That is, the yeast extract of the present invention is obtained by collecting and crushing yeast cells and autolyzing them by the action of digestive enzymes contained in the yeast itself, or by acid or alkali, proteolytic enzymes, or nucleolytic enzymes. The decomposed product may be filtered or not filtered, concentrated or dried, or the evaporation residue of the composition obtained by further extracting this concentrated or dried product with a solution containing water and/or alcohols. . Therefore, yeast extracts are rich in components obtained by yeast culture, such as amino acids and sugars.

A commercially available product can be used as the yeast extract of the present invention. As a commercial product in which the yeast extract is a dried product, Spectrum Chemical Mfg. Corp. can be exemplified by Yeast Extract Powder (YE105). Commercially available products obtained by extracting and dispersing yeast extracts with water and / or alcohols include "Yeast Extract BG" manufactured by Maruzen Pharmaceutical Co., Ltd., "Yeast Liquid 100040" manufactured by Ichimaru Farcos Co., Ltd., and "Chito" manufactured by BIODELL. catalyzer” can be exemplified.
Yeast, which is a raw material for obtaining a yeast extract, is not particularly limited. For example, brewer's yeast represented by Saccharomyces cerevisiae, baker's yeast, Torula yeast (e.g. Candida utilis), etc. any yeast can be used.

Furthermore, the yeast used in the present invention may be not only cultured yeast, but also yeast after brewing beer, sake, or the like. In the case of yeast used for brewing beer (brewer's yeast), since it has bitterness, astringency, etc. derived from hops, it is used for the production of yeast extract after being washed with water and other treatments such as removing bitterness. is desirable.
Moreover, not only fresh yeast but also dry yeast produced by drum drying, spray drying, etc. can be used as raw materials. In order to obtain a yeast extract from yeast as a raw material, the following steps are generally employed.

The starting yeast is autolyzed, or subjected to decomposition treatment by acid decomposition, alkaline decomposition, or protease by a conventional method. After removing insoluble matter by filtration, the mixture is concentrated by heating or under reduced pressure, and in some cases, spray drying. The concentrate or dried product thus obtained is the yeast extract of the present invention, and the yeast extract (1) or You may use what is standardized as a yeast extract (2).
In addition, a composition obtained by extracting yeast extract (1) or yeast extract (2) with a mixed solution of water and / or alcohol is also a yeast extract of the present invention. You may use what is standardized as an extract (3) and a yeast extract (4).
Examples of alcohols used for extraction include C1-C4 alcohols such as ethanol, glycerin, 1,3-butylene glycol, and propylene glycol. Also, these alcohols may be a mixture.

In the dissolving or dispersing operation, the temperature of the solvent to be used, the weight ratio of the solvent to the raw material, and the dissolving operation time can be arbitrarily set according to the raw material and the solvent to be used. The temperature of the solvent can be arbitrarily set in the range of -4°C to 100°C, but is preferably around 10 to 40°C from the viewpoint of the stability of the components contained in the yeast. Also, the weight ratio of the solvent to the raw material can be arbitrarily set, for example, within the range of 4:1 to 1:100 (raw material:solvent), with a weight ratio of 1:1 to 1:20 being particularly preferred. The operation can be performed, for example, by continuously shaking or stirring the mixture of the solvent and the yeast extract for 1 to 10 hours. After the prescribed operation is completed, centrifugation is carried out at 1000 to 15000 G to remove insoluble or non-dispersible precipitates, whereby the yeast extract used in the present invention can be obtained.

After solvent extraction, the yeast extract can be subjected to appropriate purification operations, such as activated charcoal treatment and chromatographic separation. In the case of extraction using 1,3-butylene glycol, the yeast extract of the present invention can be obtained by concentrating the extract by a method such as concentration under reduced pressure.

In the present invention, it is preferable to use standardized yeast extract (3).
Yeast extract (3) is obtained by extracting the dried liquid obtained by autolysis or acid hydrolysis of yeast with water, propylene glycol, 1,3-butylene glycol or a mixture thereof. composition. Commercially available products containing yeast extract (3) include "Yeast Extract Liquid BG" manufactured by Maruzen Pharmaceutical Co., Ltd., which is obtained by spray-drying a digestive juice obtained by autolysis of yeast. was extracted with water, and 1,3-butylene glycol was added. Taking “Yeast Extract Liquid BG” manufactured by Maruzen Pharmaceutical Co., Ltd. as an example, the evaporation residue corresponding to the yeast extract of the present invention is 0.8 to 2.0 mL when 10 mL is dried at 105° C. for 6 hours. 4 w/v %. "Yeast extract BG" manufactured by Maruzen Pharmaceutical Co., Ltd. used in the test examples described later is 1.6% by mass of yeast extract, 49.2% by mass of water, and 49.2% by mass of 1,3-butylene glycol. Become.

<Inositol>
Inositol is a type of cyclitol having a structure (1,2,3,4,5,6-cyclohexanehexaol) in which one hydrogen atom on each carbon of cyclohexane is replaced with a hydroxy group. It is also said to be one of the B vitamins, and in humans, it is known that when inositol is insufficient in the body due to diabetes, etc., adverse effects such as neurological symptoms occur.

<Vitamin B2>
Vitamin B2, which is a component of the composition of the present invention, is also called riboflavin or lactoflavin, and is a physiologically active substance classified as a water-soluble vitamin among vitamins. It is a combination of alcohol ribitol. Vitamin B2 is required in vivo for fat, carbohydrate and protein metabolism and respiration, red blood cell formation, antibody production, and normal development. In addition, vitamin B2 is essential for maintaining normal activity of the thyroid gland and maintaining normal health of the entire body, including skin, nails, and hair. said to cause symptoms.

<L-ascorbic acid 2-glucoside>
L-ascorbic acid 2-glucoside (AA2G) has a structure in which one glucose molecule is bound to the 2-position hydroxyl group of vitamin C (ascorbic acid), and is a highly stable vitamin that is resistant to the effects of heat and light. It is known to be a C derivative, and to be decomposed into vitamin C and glucose by an enzymatic reaction when applied to the skin or taken into the body, and to exert its effect as vitamin C.
As a commercially available product, "AA2G" manufactured by Hayashibara Co., Ltd. can be exemplified.

The composition of the present invention contains one or more selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside, and two or more selected from yeast extract, inositol, and vitamin B2. It can be contained, or all three types can be contained.
In the composition of the present invention, the concentrations of one or more selected from yeast extract, inositol, and vitamin B2 and L-ascorbic acid 2-glucoside are not particularly limited as long as the effects of the present invention are achieved. For example, the yeast extract can be 0.0001% by mass or less than 1% by mass in terms of solid content. Inositol can be 0.001 mass % or 10 mass % or less. Vitamin B2 can be 0.000001% by mass or less than 0.01% by mass. L-ascorbic acid 2-glucoside can be 0.001% by mass or less than 10% by mass.
In addition, the blending ratio of one or more selected from yeast extract, inositol, vitamin B2, and L-ascorbic acid 2-glucoside is, in the case of yeast extract, 2 parts of L-ascorbic acid per 100 parts by mass of yeast extract. -Preferably within the range of 1 part by mass to 1000 parts by mass of glucoside, in the case of inositol, preferably within the range of 1 part by mass to 1000 parts by mass of L-ascorbic acid 2-glucoside per 100 parts by mass of inositol, and in the case of vitamin B2 is preferably in the range of 1,000 parts by mass to 1,000,000 parts by mass of L-ascorbic acid 2-glucoside per 1 part by mass of vitamin B2.

The composition of the present invention can be produced according to commonly used formulation methods, and can be used as pharmaceuticals, quasi-drugs, cosmetics, and the like. For example, the compositions of the present invention include aqueous solutions, oils, lotions, emulsions, gels, creams, ointments, plasters, poultices, aerosols, suppositories, injections, powders, granules, tablets, and pills. , syrups, troches and the like.

The composition of the present invention contains fats and oils such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, and amphoteric surfactants, which are usually blended as pharmaceuticals, quasi-drugs, cosmetics, and the like. Active agents, nonionic surfactants, preservatives, sugars, sequestering agents, polymers such as water-soluble polymers, thickeners, powder ingredients, UV absorbers, UV blockers, such as hyaluronic acid Moisturizers, perfumes, pH adjusters, desiccants, etc. can be contained. Other medicinal and physiologically active ingredients such as vitamins, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavenging agents, anti-inflammatory agents, anticancer agents, whitening agents, and bactericidal agents can also be incorporated.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples.
[Test sample]
AA2G (Hayashibara), yeast extract BG (Maruzen Pharmaceutical), inositol (Tsukino Foods Industry), vitamin B2 (Wako Pure Chemical Industries)
[Test method]
1. Human neonatal keratinocytes (Thermo Fisher Scientific) were seeded in a 12-well plate at a cell density of 200,000 cells/well, and Humedia-KG2 growth additive was added to EpiLife (registered trademark) Medium with 60 μM Calcium (Thermo Fisher Scientific) (Kurabo Co., Ltd.). Cultured in medium for 1 day.
2. After culturing, each test sample was added and cultured for 2 days.
The spiked test samples are shown in Tables 1-3.

3. After culturing, the cells were lysed with M-PER Buffer (Thermo Fisher Scientific), the protein concentration of the lysate was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to a standard method, and a test sample for Western blotting was prepared.

4. 1.5 μg of protein per test sample was electrophoresed on XV PANTERA Gel 15% (DRC) followed by Trans-Blot® Turbo System (Bio-rad) using the Trans-Blot® Turbo PVDF Transfer Pack. , 2.5A for 7 min.
5. This transfer membrane was blocked with Starting Block Blocking Buffer (Thermo Fisher Scientific) or 5% skimmed milk/T-PBS (Wako Pure Chemical Industries).

6. In the IL-1RA production promoting effect test, the primary antibody "IL-1ra antibody (AF-280-NA): (R & D Systems)" was diluted 500 times, and the secondary antibody "anti-Goat IgG (Thermo Fisher Scientific) ” was reacted at 10000-fold dilution.
In the COL-7 production promoting effect test, the primary antibody "COL7 antibody (ab93350): (abcam)" was diluted 1000 times, and the secondary antibody "anti-Rabit IgG (Thermo Fisher Scientific)" was reacted at 10000 times dilution. let me
As a comparison of the expression level, the primary antibody "β-Actin antibody (C4) (Santa Cruz Biotechnology)" was diluted 1000 times, and the secondary antibody "anti-Mouse IgG (Thermo Fisher Scientific)" was reacted at 10000 times dilution. rice field.

7. After that, detection was performed with LAS-4000 mini (Fuji Film Co., Ltd.) using "ECL prime Western Blotting Detection Reagent (GE Healthcare Life Sciences)".
8. The luminescence intensity of the detected band was quantified using Image J (NIH).
9. The intensity of each of the detected bands of IL-1RA and COL7 was corrected by the intensity of β-actin, and the ratio to no treatment or vehicle treatment was calculated.

The results are shown in Figures 1-6.
From the difference between Example 1 and Comparative Example 1, the IL-1RA production promoting effect in Example 1 is 2.54. On the other hand, from the difference between Comparative Examples 2 and 1, the effect of promoting IL-1RA production in Comparative Example 2 is 0.04. Further, from the difference between Comparative Examples 5 and 1, the effect of promoting IL-1RA production in Comparative Example 5 is 1.01. That is, the IL-1RA production promoting effect in Example 1 predicted from Comparative Examples 2 and 5 is 1.05. However, the actual IL1-RA production promoting effect in Example 1 is 2.54, which is significantly higher than the expected 1.05. It was confirmed that the combination synergistically increased IL-1RA production.
Similarly, from each example and comparative example, by combining one selected from yeast extract, inositol, and vitamin B2 with L-ascorbic acid 2-glucoside, IL-1RA and COL-7 production in skin cells is enhanced. A synergistic increase was confirmed.
A similar experiment was conducted with quercetin, which is known to have an IL-1RA and COL-7 production promoting effect, and hydroquinone, which is known to have an IL-1RA production promoting effect, and a synergistic effect was obtained by combining it with L-ascorbic acid 2-glucoside. could not be confirmed.

Claims (2)

An IL-1RA production promoting composition containing at least one selected from yeast extract, inositol and vitamin B2, and L-ascorbic acid 2-glucoside. A COL-7 production promoting composition containing at least one selected from yeast extract, inositol and vitamin B2, and L-ascorbic acid 2-glucoside.

Images