IL-1RA and COL-7 production promoting composition

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

[Technical Field]

[0001] The present invention relates to an IL-1 receptor antagonist (IL-1RA) and a composition for promoting type VII collagen (COL-7) production. [Background technology]

[0002] 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 is attracting attention. The present inventors have studied the behavior of IL-1RA in the stratum corneum of human skin and have disclosed an invention of a method for indirectly measuring skin blood flow using the expression level of IL-1RA in the stratum corneum as an index (Patent Document 1). Furthermore, they have discovered that there is an extremely high correlation between the state of skin texture and the expression level of IL-1RA in the stratum corneum, 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 of the skin.

[0003] Furthermore, in the course of their research into skin aging, the present inventors discovered that skin aging is caused by the accumulation and progression of microdamage to the skin and skin cells due to various factors (e.g., ultraviolet light and reactive oxygen species), which leads to the development of wrinkles, and that IL-1RA is involved in this process. They hypothesized that the recovery of such microdamage could be accelerated by increasing IL-1RA in skin cells, and as a result of extensive research, they found that in epidermal keratinocytes to which IL-1RA had been added, intracellular energy production increased, and the expression of type IV collagen (COL-4) and type VII collagen (COL-7), which constitute the basement membrane, increased (Non-Patent Document 1).

[0004] Furthermore, the present inventors have searched for substances that stimulate the production of IL-1RA in skin cells. In the process, they discovered that a composition containing yeast extract and vitamin B2, and a composition containing yeast extract and inositol, increase IL-1RA in skin cells, and have disclosed an invention of an IL-1RA production-promoting composition comprising these (Patent Document 3). They have also disclosed that L-ascorbic acid 2-glucoside also has the effect of promoting IL-1RA production in skin cells (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5731678 [Patent Document 2] Patent No. 5977268 [Patent Document 3] Patent No. 6823428 [Non-patent literature]

[0006] [Non-Patent Document 1] Woo, Hung-ok et al., "Functional analysis of skin aging-related proteins linked to the circadian rhythm," Journal of the Japanese Society of Aesthetic Dermatology, 2018, 28(2):230 Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have found that one or more selected from yeast extract, inositol, and vitamin B2, together with 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 objective of the present invention is to provide a novel composition for promoting IL-1RA and COL-7 production. [Means for solving the problem]

[0008] The present inventors conducted extensive research to find substances that further promote IL-1RA production, and discovered that combining L-ascorbic acid 2-glucoside with specific other components synergistically promotes the production of IL-1RA and COL-7, thereby completing the present invention.

[0009] The main configuration of the present invention is as follows. 1. A composition for promoting IL-1RA production, comprising one or more members selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside. 2. A composition for promoting COL-7 production, comprising one or more selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside. [Effects of the Invention]

[0010] The present invention provides a novel composition for promoting IL-1RA and COL-7 production. Use of the composition as an external skin preparation increases IL-1RA and COL-7 in the skin, promoting the recovery of minor skin damage and inhibiting the development of wrinkles or promoting the recovery of wrinkles. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing the results of a test on the effect of combined use of L-ascorbic acid 2-glucoside and yeast extract in promoting IL-1RA production. [Figure 2] 1 is a graph showing the results of a test on the effect of combined use of L-ascorbic acid 2-glucoside and yeast extract in promoting COL-7 production. [Figure 3] 1 is a graph showing the results of a test on the effect of combined use of L-ascorbic acid 2-glucoside and inositol on promoting IL-1RA production. [Figure 4] 1 is a graph showing the results of a test on the effect of combined use of L-ascorbic acid 2-glucoside and inositol on promoting COL-7 production. [Figure 5]1 is a graph showing the results of a test on the effect of combined use of L-ascorbic acid 2-glucoside and vitamin B2 on promoting IL-1RA production. [Figure 6] 1 is a graph showing the results of a test on the effect of combined use of L-ascorbic acid 2-glucoside and vitamin B2 on promoting COL-7 production. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to a composition (hereinafter also referred to as a composition) for promoting IL-1RA and COL-7 production, which contains one or more members selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside. The composition of the present invention can activate the gene expression of IL-1RA and COL-7 expressed in skin cells, thereby increasing the amount of IL-1RA and type VII collagen in the cells, thereby activating skin cells and preventing and even reversing skin aging.

[0013] <Yeast extract> The yeast extract, a component of the composition of the present invention, is a concentrated or dried product of the liquid obtained by autolysis or acid hydrolysis of yeast cell lysates, or the evaporation residue of a composition obtained by further extracting this concentrated or dried product with a solution containing water and / or alcohol. In other words, the yeast extract of the present invention may be obtained by collecting and disrupting yeast cells, subjecting them to autolysis by the action of digestive enzymes contained in the yeast itself, or by decomposing them with acid, alkali, or even protease or nuclease, and then concentrating or drying them with or without filtration. It may also be the evaporation residue of a composition obtained by further extracting this concentrated or dried product with a solution containing water and / or alcohol. Therefore, the yeast extract is rich in components obtained by culturing yeast, such as amino acids and sugars.

[0014] Commercially available yeast extracts can be used for the present invention. An example of a commercially available dried yeast extract is Yeast Extract Powder (YE105) from Spectrum Chemical Mfg. Corp. Examples of commercially available products in which yeast extract is extracted and dispersed in water and / or alcohols include "Yeast Extract BG" from Maruzen Pharmaceutical Co., Ltd., "Yeast Liquid 100040" from Ichimaru Pharcos Co., Ltd., and "Cytocatalyzer" from BIODELL. The yeast used as the raw material for obtaining the yeast extract is not particularly limited, and any yeast can be used, including brewer's yeast such as Saccharomyces cerevisiae, baker's yeast, and even Torula yeast (e.g., Candida utilis).

[0015] Furthermore, the yeast used in the present invention may be not only cultured yeast but also yeast used after brewing beer, sake, etc. In the case of yeast used in beer brewing (beer yeast), since it has bitterness, astringency, etc. derived from hops, it is desirable to use it in the production of a yeast extract after washing with water or other treatments to remove bitterness. In addition to fresh yeast, dried yeast produced by drum drying, spray drying, etc. can also be used as a raw material. To produce a yeast extract from yeast, the following steps are generally adopted.

[0016] The starting yeast is subjected to autolysis or hydrolysis with acid hydrolysis, alkali hydrolysis, or protease by conventional methods. After removing insoluble matter by filtration, the resulting mixture is concentrated by heating or vacuum, and optionally spray-dried. The concentrate or dried product thus obtained is the yeast extract of the present invention. Alternatively, yeast extracts standardized as yeast extract (1) or yeast extract (2) in the "Standards for Quasi-drug Ingredients 2006" (hereinafter "Standards") compiled by the Pharmaceutical Affairs Bureau, Ministry of Health, Labor and Welfare (hereinafter "MHLW") may also be used. Furthermore, a composition obtained by further 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, and those standardized as yeast extract (3) or yeast extract (4) in the "Extra-Gen Standards" may also be used. Examples of alcohols used for extraction include C1-C4 alcohols such as ethanol, glycerin, 1,3-butylene glycol, and propylene glycol. These alcohols may also be mixtures.

[0017] During the dissolution or dispersion procedure, the temperature of the solvent used, the weight ratio of the solvent to the raw material, and the dissolution time can each be set arbitrarily depending on the raw material and the solvent used. The solvent temperature can be set arbitrarily between -4°C and 100°C, but from the perspective of the stability of the components contained in the yeast, a temperature of approximately 10 to 40°C is preferred. The weight ratio of the solvent to the raw material can also be set arbitrarily between 4:1 and 1:100, for example, with a weight ratio of 1:1 to 1:20 being particularly preferred. The procedure can be carried out, for example, by continuously shaking or stirring a mixture of the solvent and yeast extract for 1 to 10 hours. After the desired procedure is completed, the yeast extract used in the present invention can be obtained by centrifuging at 1,000 to 15,000 G and removing insoluble or non-dispersible precipitates.

[0018] After solvent extraction, the yeast extract may be further purified as appropriate, for example, by activated carbon treatment or chromatographic separation. In addition, in the case of extraction using 1,3-butylene glycol, the extract may be concentrated by a method such as vacuum concentration to obtain a concentrate, which can be used as the yeast extract of the present invention.

[0019] In the present invention, it is preferable to use a standardized yeast extract (3). Yeast extract (3) is a composition obtained by drying the liquid obtained by autolysis or acid hydrolysis of yeast, followed by extraction with water, propylene glycol, 1,3-butylene glycol, or a mixture thereof. An example of a commercially available product containing yeast extract (3) is "Yeast Extract BG" manufactured by Maruzen Pharmaceutical Co., Ltd., which is prepared by spray-drying the digested liquid obtained by autolysis of yeast, extracting the resulting product with water, and adding 1,3-butylene glycol. Taking "Yeast Extract 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 2.4 w / v% when 10 mL of the product is dried at 105°C for 6 hours. The "Yeast Extract BG" manufactured by Maruzen Pharmaceutical Co., Ltd. used in the test examples described below consists of 1.6% by mass of yeast extract, 49.2% by mass of water, and 49.2% by mass of 1,3-butylene glycol.

[0020] <Inositol> Inositol is a type of cyclitol with a structure (1,2,3,4,5,6-cyclohexanehexaol) in which one hydrogen atom on each carbon atom of cyclohexane is replaced with a hydroxyl group. It is also said to be one of the B vitamins, and in humans, a deficiency of inositol in the body due to diabetes or other conditions is known to have adverse effects such as neurological symptoms.

[0021] <Vitamin B2> Vitamin B2, a component of the composition of the present invention, is also known as riboflavin or lactoflavin. It is a physiologically active water-soluble vitamin, consisting of a heterocyclic isoalloxazine ring bound to the sugar alcohol ribitol. Vitamin B2 is required in the body for the metabolism of fats, carbohydrates, and proteins, respiration, red blood cell formation, antibody production, and normal growth. Vitamin B2 is also essential for maintaining normal thyroid activity and maintaining the normal health of the entire body, including skin, nails, and hair. Vitamin B2 deficiency is said to cause symptoms such as stomatitis, glossitis, dermatitis, and epileptic seizures.

[0022] <L-Ascorbic Acid 2-Glucoside> L-Ascorbic acid 2-glucoside (AA2G) has a structure in which one molecule of glucose is bonded to the 2-position hydroxyl group of vitamin C (ascorbic acid). It is a vitamin C derivative that is not easily affected by heat, light, etc. and has excellent stability. It is known that when applied to the skin or ingested into the body, it is decomposed into vitamin C and glucose by an enzymatic reaction and exhibits the effects of vitamin C. Examples of commercially available products include "AA2G" manufactured by Hayashibara Co., Ltd.

[0023] The composition of the present invention contains one or more selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside. It can also contain two or more selected from yeast extract, inositol, and vitamin B2, or can contain all three. In the composition of the present invention, the concentration of one or more selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside is not particularly limited as long as the effects of the present invention are achieved. For example, the yeast extract can be 0.0001% to 1% by mass in terms of solid content. Inositol can be 0.001% to 10% by mass. Vitamin B2 can be 0.000001% to 0.01% by mass. L-Ascorbic acid 2-glucoside can be 0.001% to 10% by mass. Also, the mixing ratio of one or more selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside is preferably in the range of 1 to 1000 parts by mass of L-ascorbic acid 2-glucoside per 100 parts by mass of yeast extract in the case of yeast extract, preferably in the range of 1 to 1000 parts by mass of L-ascorbic acid 2-glucoside per 100 parts by mass of inositol in the case of inositol, and preferably in the range of 1000 to 1,000,000 parts by mass of L-ascorbic acid 2-glucoside per 1 part by mass of vitamin B2 in the case of vitamin B2.

[0024] The composition of the present invention can be produced according to a commonly used formulation method, and can be made into a medicine, quasi-drug, cosmetic, etc. For example, the composition of the present invention can be made into various dosage forms such as an aqueous solution, oil, lotion, emulsion, gel, cream, ointment, plaster, poultice, aerosol, suppository, injection, powder, granule, tablet, pill, syrup, troche, etc.

[0025] The composition of the present invention may contain oils and fats such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, sugars, sequestering agents, polymers such as water-soluble polymers, thickeners, powder components, UV absorbers, UV blockers, moisturizers such as hyaluronic acid, fragrances, pH adjusters, desiccants, etc. It may also contain other medicinal or physiologically active ingredients such as vitamins, skin activators, blood circulation promoters, resident flora control agents, active oxygen scavengers, anti-inflammatory agents, anticancer agents, whitening agents, and disinfectants. [Example]

[0026] The present invention will be specifically described below with reference to examples. [Test sample] AA2G (Hayashibara), yeast extract BG (Maruzen Pharmaceuticals), inositol (Tsukuno Foods), vitamin B2 (Wako Pure Chemical Industries) [Test method] 1. Human neonatal keratinocytes (Thermo Fisher Scientific) were seeded into 12-well plates at a cell density of 200,000 cells / well and cultured for 1 day in EpiLife® Medium with 60 μM Calcium (Thermo Fisher Scientific) supplemented with Humedia-KG2 growth additive (Kurashiki Boseki Co., Ltd.). 2. After the incubation, each test sample was added and incubated for 2 days. The test samples added are shown in Tables 1 to 3.

[0027] [Table 1]

[0028] [Table 2]

[0029] [Table 3]

[0030] 3. After culturing, the cells were lysed with M-PER Buffer (Thermo Fisher Scientific), the protein concentration of the lysate was measured according to the standard method using a BCA protein assay kit (Thermo Fisher Scientific), and test samples for Western blotting were prepared.

[0031] 4. 1.5 μg of protein per test sample was electrophoresed on XV PANTERA Gel 15% (DRC) and then transferred to a membrane using a Transblot® Turbo PVDF transfer pack in a Transblot® Turbo system (Bio-Rad) at 2.5 A for 7 minutes. 5. This transfer membrane was blocked with Starting Block Blocking Buffer (Thermo Fisher Scientific) or 5% skim milk / T-PBS (Wako Pure Chemical Industries).

[0032] 6. In the test for the effect of promoting IL-1RA production, the primary antibody "IL-1ra antibody (AF-280-NA): (R&D Systems)" was diluted 500 times and reacted with the secondary antibody "anti-Goat IgG (Thermo Fisher Scientific)" at a 10,000-fold dilution. To test the effect of promoting COL-7 production, the primary antibody, "COL7 antibody (ab93350): (abcam)," was diluted 1000 times and reacted with the secondary antibody, "anti-Rabbit IgG (Thermo Fisher Scientific)," diluted 10,000 times. To compare expression levels, the primary antibody, β-Actin antibody (C4) (Santa Cruz Biotechnology), was diluted 1000-fold, and the secondary antibody, anti-Mouse IgG (Thermo Fisher Scientific), was diluted 10000-fold.

[0033] 7. Then, detection was performed using "ECL prime Western Blotting Detection Reagent (GE Healthcare Life Sciences)" with an LAS-4000mini (Fujifilm). 8. The luminescence intensity of the detected bands was quantified using Image J (NIH). 9. The intensity of each detected IL-1RA and COL7 band was corrected by the intensity of β-actin, and the ratio to the untreated or vehicle-treated values ​​was calculated.

[0034] The results are shown in Figures 1 to 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 Example 2 and Comparative Example 1, the IL-1RA production promoting effect in Comparative Example 2 is 0.04. Furthermore, from the difference between Comparative Example 5 and Comparative Example 1, the IL-1RA production promoting effect 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 IL-1RA production promoting effect in Example 1 was 2.54, which is significantly higher than the predicted 1.05. Therefore, it was confirmed from Example 1 that the combination of yeast extract and L-ascorbic acid 2-glucoside synergistically increases IL-1RA production. Similarly, each example and comparative example confirmed that the combination of one selected from yeast extract, inositol, and vitamin B2 with L-ascorbic acid 2-glucoside synergistically increases the production of IL-1RA and COL-7 in skin cells. Furthermore, when similar experiments were conducted using quercetin, which is known to promote IL-1RA and COL-7 production, and hydroquinone, which is known to promote IL-1RA production, no synergistic effect was observed when combined with L-ascorbic acid 2-glucoside.

Claims

1. A composition for promoting IL-1RA production, comprising at least one member selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside.

2. A composition for promoting COL-7 production, comprising one or more members selected from yeast extract, inositol, and vitamin B2, and L-ascorbic acid 2-glucoside.

Images