JP6823428B2 - IL-1 receptor antagonist (IL-1RA) production promoting composition - Google Patents

Description

The present invention relates to an IL-1RA production promoting composition.

Interleukin-1 (hereinafter abbreviated as "IL-1") is produced from various immunocompetent cells centered on monocytes and macrophages, and has various actions that cause an acute reaction to a disorder suffered by a living body. It is a cytokine with. Due to its various biological actions, it has become known in recent years that it is directly or indirectly involved in the formation of various diseases or pathological conditions. In particular, since IL-1 is associated with its chronicity and tissue destruction in various inflammations, suppressing the action of IL-1 is useful for treating diseases or pathological conditions and further suppressing the progression of aging. , The inhibitor, that is, the IL-1 inhibitor is attracting attention.

Among them, IL-1 receptor antagonists (hereinafter, “IL-1RA”) existing in the living body are attracting attention. Human IL-1RA is known to be present as an IL-1 inhibitor in the urine of patients with monocytic leukemia and patients with fever.

In recent years, attention has been paid to the relationship between the behavior of IL-1RA in the skin and skin symptoms. Non-Patent Document 1 states that there is a difference in the skin wound healing process between IL-1RA-deficient mice and normal mice, and in the deficient mice, angiogenesis in the wound healing process is delayed and skin wound healing is delayed. Has been described. And Non-Patent Document 1 concludes that the presence of IL-1RA is important for the recovery of skin trauma.

The present inventors also studied the behavior of IL-1RA in the stratum corneum of human skin, and indirectly measured the blood flow in the skin using the expression level of IL-1RA in the stratum corneum as an index (Patent Document 1). ) Was invented. Furthermore, they found that the texture of the skin and the expression level of IL-1RA in the stratum corneum had an extremely high correlation, and invented a new method for evaluating the texture of the skin (). 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.

Japanese Patent No. 5731678 Japanese Patent No. 5977268

J Immunol 2006; 176: 5598-5606;

In the process of conducting skin aging research, the present inventors develop wrinkles due to the accumulation and progression of minute damage to the skin and skin cells due to various factors (for example, ultraviolet rays and active oxygen) due to the skin aging phenomenon. It was found that IL-1RA was involved in this. In order to accelerate the recovery of such minute injuries, we have been searching for substances that produce IL-1RA in skin cells, considering that it can be solved by increasing IL-1RA in skin cells. In the process, we focused on the fact that some yeast extracts promote the production of IL-1RA in skin cells and some do not, and further research was conducted to find out the essence of its effect. .. The present invention has been found by such a search.

That is, it is an object of the present invention to provide a composition having a novel IL-1RA production promoting action.

The present invention has the following configuration.
(1) An IL-1RA production promoting composition of skin cells containing a yeast extract and vitamin B2.
(2) The yeast extract is a concentrated or dried product of a solution obtained by autolyzing or acid-hydrolyzing yeast belonging to Saccharomyces or yeast belonging to Candida, or the concentrated or dried product is further watered and dried. / Or the IL-1RA production promoting composition according to (1), which is an evaporation residue of the composition obtained by extracting with a solution containing alcohols.
(3) The IL-1RA production promoting composition according to (1) or (2), which contains vitamin B2 of 0.00018 or more when the total amount of yeast extract and vitamin B2 is 1.
(4) IL-1RA production promoting composition of skin cells containing yeast extract and inositol.
(5) The IL-1RA production promoting composition according to (4), which contains 62 or more inositol with respect to yeast extract 1.
(6) A cosmetic for preventing skin aging containing the IL-1RA production promoting composition according to any one of (1) to (5).

The present invention provides a novel IL-1RA production promoting composition.
By using the composition of the present invention as an external preparation for skin, IL-1RA of the skin is increased, the recovery of minute damages caused on the skin is promoted, and the generation and recovery of wrinkles and the like can be promoted.

It is a graph which shows the result of having tested the difference of the IL-1RA production promotion effect by the difference of the lot of the yeast extract. It is a graph which shows the result of having tested the IL-1RA production promotion effect by the combined use of vitamin B2 and yeast extract. It is a graph which shows the result of having tested the IL-1RA production promotion effect by the combined use of inositol and yeast extract. It is a graph which shows the result of having tested the IL-1RA production promotion effect by the combined use of inositol and yeast extract in human.

The present invention relates to an IL-1RA production promoting composition of skin cells containing a yeast extract and vitamin B2 as active ingredients. The present invention also relates to an IL-1RA production promoting composition of skin cells containing a yeast extract and inositol as active ingredients. The composition of the present invention activates the gene expression of IL-1RA expressed in skin cells to increase the amount of IL-1RA in the cells, and as a result, activates the skin cells and prevents skin aging. , Further recover.

The yeast extract, which is a component of the composition of the present invention, is a concentrated or dried product of a solution obtained by autolysis or acid hydrolysis, or the concentrated or dried product is further added with water and / or alcohols. It is an evaporation residue of the composition obtained by extracting with the containing solution. 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 using acids, alkalis, proteolytic enzymes, and nucleolytic enzymes. The decomposed product may be filtered or not filtered, and may be concentrated or dried, or may be an evaporation residue of a composition obtained by extracting the concentrated or dried product with a solution further containing water and / or alcohols. .. Therefore, the yeast extract is rich in components obtained by culturing yeast, such as amino acids and sugars.

Commercially available products can be used for the yeast extract of the present invention. Yeast Extract Powder (YE105) from Spectrum Chemical Mfg. Corp. can be exemplified as a commercially available product in which the yeast extract is a dried product. Commercially available products obtained by extracting and dispersing yeast extract with water and / or alcohols include "Yeast Extract BG" manufactured by Maruzen Pharmaceuticals Co., Ltd., "East Liquid 100040" manufactured by Ichimaru Falcos Co., Ltd., and "Chito" manufactured by BIODELL. A catalyzer can be exemplified.
The yeast that is the raw material for obtaining the yeast extract is not particularly limited, and for example, brewing yeast typified by Saccharomyces cerevisiae, baker's yeast, as well as tolla yeast (for example, Candida utilis), etc. Any yeast can be used.
Further, the yeast used in the present invention may be not only cultured yeast but also yeast after brewing such as beer and sake. In the case of yeast used for brewing beer (brewer's yeast), since it has hop-derived bitterness, astringency, etc., it is used for producing yeast extract after being washed with water or other de-bitterness treatment. Is desirable.
Further, not only raw yeast but also dried yeast produced by drum drying, spray drying or the like can be used as a raw material. In order to use yeast as a raw material and obtain a yeast extract, the following steps are generally adopted.
The raw material yeast is autolyzed by a conventional method, or decomposed by acid decomposition, alkali decomposition, or protease. Then, after removing the insoluble matter by filtration, heat concentration or vacuum concentration is performed, and in some cases, spray drying treatment is performed. The concentrate or dried product thus obtained is the yeast extract of the present invention, but the yeast extract (1) or the yeast extract (1) or the yeast extract (1) in the "Quasi-drug Raw Material Standards 2006" edited by the Examination Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare A yeast extract (2) standardized may be used.
Further, the composition obtained by further extracting the yeast extract (1) or the yeast extract (2) with a mixed solution of water and / or alcohol is also the yeast extract of the present invention, but this is the yeast in the "outer original rule". Those standardized as the extract (3) and the yeast extract (4) may be used.
Examples of the alcohol used for the extraction include C1-C4 alcohols such as ethanol, glycerin, 1,3-butylene glycol and propylene glycol. Moreover, these alcohols may be a mixture.

In the dissolution or dispersion operation, the temperature of the solvent used, the weight ratio of the solvent to the raw material, and the dissolution operation time can be arbitrarily set depending on the raw material and the solvent 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 stability of the components contained in yeast. Further, the weight ratio of the solvent to the raw material can be arbitrarily set in the range of 4: 1 to 1: 100, for example, the raw material: solvent, and the weight ratio of 1: 1 to 1:20 is particularly preferable. The operation is carried out, for example, by continuously shaking or stirring and mixing a mixture of a solvent and a yeast extract for 1 to 10 hours. Then, after the predetermined operation is completed, centrifugation is performed at 1000 to 15000 G to remove the insoluble or non-dispersible precipitate, whereby the yeast extract used in the present invention can be obtained.

The yeast extract can be further subjected to a purification operation after solvent extraction, and may be subjected to activated carbon treatment, chromatographic separation, or the like. Further, in the case of extraction using 1,3 butylene glycol, the concentrate concentrated from the extract by a method such as concentration under reduced pressure can be used as the yeast extract of the present invention.

In the present invention, it is preferable to use a yeast extract (3) standardized.
Yeast extract (3) was obtained by drying a solution obtained by autolyzing yeast or acid hydrolysis, and further extracting it with water, propylene glycol, 1,3-butylene glycol or a mixture thereof. It is a composition. Examples of the commercially available product containing the yeast extract (3) include the above-mentioned "yeast extract BG" manufactured by Maruzen Pharmaceuticals Co., Ltd., which is obtained by spray-drying the digestive juice obtained by autolysis of yeast. It is extracted with water and added with 1,3-butylene glycol. Taking "yeast extract BG" manufactured by Maruzen Pharmaceuticals Co., Ltd. as an example, the evaporation residue corresponding to the yeast extract of the present invention is 0.8 to 2. When 10 mL is dried at 105 ° C. for 6 hours. It shows 4w / v%. The "yeast extract BG" manufactured by Maruzen Pharmaceuticals Co., Ltd., which was used in the test examples described later, was prepared from 1.6% by mass of yeast extract, 49.2% by mass of water, and 49.2% by mass of 1,3-butylene glycol. Become.

Vitamin B2, which is a component of the composition of the present invention, is also called riboflavin and Lactoflavine, and is a physiologically active substance classified as a water-soluble vitamin among vitamins. It is a sugar in a heterocyclic isoaroxazine ring. It is a combination of the alcohol riboflavin. In vivo, it is required for metabolism and respiration of fats, carbohydrates and proteins, formation of red blood cells, antibody production, and normal development. It is indispensable for maintaining the normal activity of the thyroid gland and maintaining the normal health condition of the whole body including the skin, nails or hair, and it is said that if it is insufficient, symptoms such as aphthous ulcer, glossitis, dermatitis and seizure will occur. There is.
Vitamin B2 is originally contained in yeast and is also contained in a trace amount in the above-mentioned yeast extract, but its content is affected by the dissolution and dispersion operation step of the raw material yeast or a polar solvent, and is constant. It often happens that the content required in the present invention is not reached.
In searching for IL-1RA production-promoting substances in skin cells, the present inventors have noted that some yeast extracts have a high IL-1RA production-promoting effect and some have a low IL-1RA production-promoting effect. As a result of the analysis, it was found that the yeast extract having a high IL-1RA production promoting effect "contains more than 0.00018 of vitamin B2 when the total amount of the yeast extract and vitamin B2 is 1." Therefore, in order to surely obtain the IL-1RA production promoting effect of the present invention, the IL-1RA production promoting composition contains 0.00018 or more of vitamin B2 when the total amount of the yeast extract and vitamin B2 is 1. Add as such. The upper limit of the addition of vitamin B2 is not particularly limited, but in consideration of cost, it is preferable to set the upper limit of vitamin B2 to be 0.0019 when the total amount of the yeast extract and vitamin B2 is 1.

The present invention has also been found that the IL-1RA production promoting effect is enhanced by further adding inositol to the yeast extract.
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 the case of humans, it is known that when inositol is deficient in the body due to diabetes or the like, adverse effects such as neurological symptoms occur. The combined use of inositol and yeast extract synergistically enhances IL-1RA production.

An IL-1RA production promoting composition containing 62 or more inositol with respect to the yeast extract 1 is preferable because the IL-1RA production promoting effect is enhanced. At this time, it is more preferable that vitamin B2 is contained in an amount of 0.00018 or more when the total amount of the yeast extract and vitamin B2 is 1.

The amount of the skin anti-aging cosmetic containing the IL-1RA production promoting composition of the present invention is not limited as long as it can be confirmed that the IL-1RA expression level is enhanced. As a preparation, the IL-1RA production promoting composition is generally blended in an amount of 0.1 to 20% by mass based on the weight of the preparation.
The shape of the skin anti-aging cosmetic containing the IL-1RA production promoting composition of the present invention may be any of liquid, solid, powder, paste and the like.
These formulations can be prepared by conventional formulation means. Examples of the non-toxic carrier for the skin antiaging cosmetics include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester. , Amino acid, albumin, water, physiological saline, fats and oils and the like. In addition, if necessary, conventional additives such as stabilizers, lubricants, wetting agents, emulsifiers, and binders can be appropriately added.

Hereinafter, the present invention will be specifically described with reference to Test Examples and Examples.
1. 1. Test Example 1 (Measurement of vitamin B group content of commercially available yeast extract dispersion)
Containing B vitamins of yeast extract BG (Lot.A Maruzen Pharmaceuticals Co., Ltd.), yeast extract BG (Lot.B Maruzen Pharmaceuticals Co., Ltd.), and chitocatalizer BG30 (BIODELL BIOCHEMICAL SA), which are commercially available cosmetic raw materials. The amount was measured.

(1) Vitamin B1 (thiamine hydrochloride) analysis <HPLC method>
Precisely weigh 0.5 g of each sample (raw material) and add 0.1 mol / L hydrochloric acid solution containing 25% potassium chloride to make exactly 5 mL. The solution obtained by filtering this solution with a membrane filter having a pore size of 0.45 μm is used as a test sample solution.
Separately, weigh accurately 10 mg of thiamine hydrochloride standard and add a 0.1 mol / L hydrochloric acid solution containing 25% potassium chloride to make exactly 50 mL. This solution is appropriately diluted with a 0.1 mol / L hydrochloric acid solution containing 25% potassium chloride so as to be 0.001, 0.005, 0.01, 0.1 ppm, and the solution filtered through a membrane filter having a pore size of 0.45 μm is used as a standard solution.
20 μL of each of the sample solution and the standard solution is tested by liquid chromatography under the following conditions to determine the thiamine hydrochloride content (%) in this product.

<< HPLC analysis conditions >>
Detector: Fluorescent detector Column: L-column ODS 4.6mm ID x 250mm Chemical substance evaluation research organization Column temperature: Constant temperature around 40 ° C Mobile phase: 0.01 mol / L Sodium dihydrogen phosphate solution-0.15 mol / L Sodium perchlorate
Solution (pH 2.2): Methanol = 95: 5 (v / v)
Flow velocity: 1.0 mL / min
Injection volume: 20 μL
Fluorescence excitation wavelength: 375 nm
Fluorescence measurement wavelength: 440 nm
Post column: Reaction coil; Stainless steel tube φ0.25 mm x 50 cm
Reaction test solution: 0.01 mol / L Potassium ferricyanide-containing 15% sodium hydroxide solution Reaction solution flow rate: 0.5 mL / min
Reaction temperature: 40 ° C

(2) Vitamin B2 (riboflavin) analysis <HPLC method>
Precisely weigh 0.5 g of each sample (raw material) and add 0.1 mol / L hydrochloric acid solution to make exactly 5 mL. The solution obtained by filtering this solution with a membrane filter having a pore size of 0.45 μm is used as a test sample solution.
Separately, weigh accurately about 5 mg of riboflavin standard product and add 0.1 mol / L hydrochloric acid solution to make exactly 50 mL.
This solution is appropriately diluted with a 0.1 mol / L hydrochloric acid solution so as to have a concentration of 0.005, 0.01, 0.02, 0.05 ppm, and the solution filtered through a membrane filter having a pore size of 0.45 μm is used as a standard solution. 20 μL of each of the sample solution and the standard solution is tested by liquid chromatography under the following conditions to determine the riboflavin content (%) in this product.

<< HPLC analysis conditions >>
Detector: Fluorescence detector Column: TSK-GEL ODS-80Ts 4.6mm ID x 250mm Tosoh Corporation Column temperature: Constant temperature around 40 ° C Mobile phase: Acetic acid buffer (pH 4.5): Methanol = 65: 35 ( v / v)
Flow velocity: 1.0 mL / min
Injection volume: 20 μL
Fluorescence excitation wavelength: 445 nm
Fluorescence measurement wavelength: 530nm

(3) Vitamin B3 (niacin) analysis <HPLC method>
Weigh accurately 0.5 g of each sample (raw material) and add water to make exactly 5 mL. The solution obtained by filtering this solution with a membrane filter having a pore size of 0.45 μm is used as a sample solution.
Separately, weigh accurately about 5 mg each of nicotinic acid and nicotinamide standard, and add water to make exactly 50 mL. This solution is appropriately diluted with water so as to be 0.05, 0.1, 0.5, 1, 5 ppm, and the solution filtered through a membrane filter having a pore size of 0.45 μm is used as a standard solution.
20 μL of each of the sample solution and the standard solution is tested by liquid chromatography under the following conditions to determine the nicotinic acid and nicotinamide contents (%) in this product, and the two are added together to obtain niacin.

<< HPLC analysis conditions >>
Detector: Ultraviolet spectroscopic detector (detection wavelength: 254 nm)
Column: Capcell pak AQ 4.6mm ID x 250mm Shiseido Co., Ltd. Column temperature: Constant temperature around 45 ° C Mobile phase: 5 mmol / L sodium hexanesulfonate, 20 mmol / LL phosphoric acid (pH 2.3):
Acetonitrile = 93: 7 (v / v)
Flow velocity: 1.0 mL / min

(4) Vitamin B6 (pyridoxine hydrochloride) analysis <HPLC method>
Precisely weigh 0.5 g of each sample (raw material) and add a 50 mmol / L perchloric acid solution to make exactly 5 mL. The solution obtained by filtering this solution with a membrane filter having a pore size of 0.45 μm is used as a test sample solution.
Separately, weigh accurately about 10 mg of pyridoxine hydrochloride standard product and add 50 mmol / L perchloric acid solution to make exactly 50 mL. This solution is appropriately diluted with a 50 mmol / L perchloric acid solution so as to be 0.01, 0.05, 0.1, 1 ppm, and the solution filtered through a membrane filter having a pore size of 0.45 μm is used as a standard solution.
20 μL of each of the sample solution and the standard solution is tested by liquid chromatography under the following conditions to determine the pyridoxine hydrochloride content (%) in this product.

<< HPLC analysis conditions >>
Detector: Ultraviolet spectroscopic detector (detection wavelength: 290 nm)
Column: TSK-GEL ODS-80Ts 4.6mm ID x 250mm Tosoh Co., Ltd. Column temperature: Constant temperature around 45 ° C Mobile phase: 50 mmol / L Perchloric acid Flow velocity: 1.2 mL / min
Injection volume: 20 μL

(5) Results ・ Vitamin B1, B2, B3, B6 concentrations in each raw material (yeast extract dispersion) The measurement results of the B vitamins are shown in Table 1 below. In addition, the content of the yeast extract (evaporation residue) in the yeast extract dispersion and the ratio of vitamin B2 to the yeast extract (total amount including vitamin B2) were calculated and described.

As a result of measuring the vitamin B1, B2, B3, and B6 concentrations of the two lots of yeast extract BG and the chitocatalyzer BG30, the chitocatalyzer had a high content of vitamin B6 and vitamin B2 was below the detection limit. On the other hand, in the yeast extract BG, vitamin B6 was below the detection limit, but the contents of vitamin B2 and B3 were high, and in Lot. B, the content of vitamin B2 was particularly high.

2. 2. Test Example 2 (IL-1RA expression promotion test of yeast extract)
(1) Test method Human Epidermal Keratinocytes, neonatal (HEKn) (manufactured by Thermo Fischer Sientific) was sown on a 24-well plate at 25000 cells / cm ² , and EpiLife® Medium with 60 μM Calcium (Thermo Fisher Scientific) After culturing in a medium containing Humedia-KG2 growth additive (manufactured by Kurashiki Spinning Co., Ltd.) for 2 days, a sample (raw material) was added (n = 3), and 2 days later, cells were subjected to T-PER Buffer (Thermo). Dissolved in Fisher Scientific). The IL-1RA concentration in the lysate was measured by Human IL-1RA / IL-1F3 DuoSet ELISA (manufactured by R & D systems), and the protein concentration was measured by the BCA protein assay kit (manufactured by Thermo Fisher Scientific) according to a standard method.
The amount of IL-1RA produced per unit protein was calculated by the following formula.
[IL-1RA expression level (pg / μg protein)] = [IL-1RA concentration in cell lysate (pg / ml)] / [Protein concentration in cell lysate (μg / ml)]

<Test>
One-way ANOVA was used to analyze the IL-1RA expression level of each sample by the Dunnett method. For synergistic effects, two-way ANOVA was used to analyze multiple comparison tests using the Dunnett method.

(2) Results The IL-1RA production promoting effect of the test sample is shown in FIG.
No promotion of IL-1RA production was confirmed in the chitocatalizer and yeast extract BG (Lot.A), but concentration-dependent and significant IL-1RA production in yeast extract BG (Lot.B). A promoting effect was observed.

3. 3. Test Example 3 (Test to confirm the effect of vitamin B2 addition concentration on IL-1RA expression promotion)
In Test Example 2, a significant difference in IL-1RA expression promoting action was observed between yeast extract BG (Lot.B) and yeast extract BG (Lot.A). This difference was thought to be due to the vitamin B2 content. The effect of adding vitamin B2 to a commercially available yeast extract (yeast extract dispersion) was tested.
(1) Test method The IL-1RA production promoting effect was tested by the method described in Test Example 2.
In the test, vitamin B2 was added to the control group (addition group 1), vitamin B2 (addition group 2, 3), yeast extract BG (Lot.A) (addition group 4, 5), yeast extract BG (Lot.A). There were 5 groups of addition (addition groups 6 and 7) and yeast extract BG (Lot.B) (addition groups 8 and 9). The composition is shown in Table 2.

Human Epidermal Keratinocytes, neonatal (HEKn) (Thermo Fischer Sientific) was sown on a 24-well plate at 25000 cells / cm ² and placed in EpiLife® Medium with 60 μM Calcium (Thermo Fisher Scientific) Human-KG2. After culturing in a medium containing a growth additive (manufactured by Kurashiki Spinning Co., Ltd.) for 2 days, a sample (5 groups) was added (n = 3), and after 2 days, cells were cultured in T-PER Buffer (manufactured by Thermo Fisher Scientific). The IL-1RA concentration in the lysate was measured by Human IL-1RA / IL-1F3 DuoSet ELISA (manufactured by R & D systems), and the protein concentration was measured by the BCA protein assay kit (manufactured by Thermo Fisher Scientific) according to a standard method.
The amount of IL-1RA produced per unit protein was calculated by the following formula.
[IL-1RA expression level (pg / μg protein)] = [IL-1RA concentration in cell lysate (pg / ml)] / [Protein concentration in cell lysate (μg / ml)]

<Test>
One-way ANOVA was used to analyze the IL-1RA expression level of each sample by the Dunnett method. For synergistic effects, two-way ANOVA was used to analyze multiple comparison tests using the Dunnett method.

(2) Results The IL-1RA production promoting effect of the test sample is shown in FIG.
Addition of vitamin B2 to yeast extract BG (Lot.A) clearly promoted IL-1RA production. However, vitamin B2 alone did not show an IL-1RA production promoting effect. In addition, by adding vitamin B2 to yeast extract BG (Lot.A), an IL-1RA expression promoting effect similar to that of yeast extract BG (Lot.B) was observed. From this, a significant synergistic effect between vitamin B2 and yeast extract was confirmed (p = 0.000068) (see FIG. 2). It was found that the IL-1RA production promoting effect was enhanced when vitamin B2 was contained in an amount of 0.00018 or more when the total amount of the yeast extract and vitamin B2 was 1.

4. Test Example 4 (IL-1RA expression promoting effect confirmation test by adding inositol)
A search for substances that affect IL-1RA production revealed a strong IL-1RA production-promoting and enhancing effect against inositol. This confirmation test is shown below.
(1) Test method The IL-1RA production promoting effect was tested by the method described in Test Example 2.
A yeast extract BG (Lot.B) was used, inositol was added thereto, and a test was conducted. The concentration of inositol added was 1% by mass. There were four groups: control, inositol, yeast extract BG, and yeast extract + inositol. The breakdown is as shown in FIG.
Human Epidermal Keratinocytes, neonatal (HEKn) (Thermo Fischer Sientific) was sown in a 24-well plate at 25000 cells / cm ² and placed in EpiLife® Medium with 60 μM Calcium (Thermo Fisher Scientific). After culturing in a medium containing a KG2 growth additive (manufactured by Kurashiki Spinning Co., Ltd.) for 2 days, samples (4 groups) were added (n = 3), and 2 days later, cells were cultured in T-PER Buffer (manufactured by Thermo Fisher Scientific). ) Dissolved. The IL-1RA concentration in the lysate was measured by Human IL-1RA / IL-1F3 DuoSet ELISA (manufactured by R & D systems), and the protein concentration was measured by the BCA protein assay kit (manufactured by Thermo Fisher Scientific) according to a standard method.
The amount of IL-1RA produced per unit protein was calculated by the following formula.
[IL-1RA expression level (pg / μg protein)] = [IL-1RA concentration in cell lysate (pg / ml)] / [Protein concentration in cell lysate (μg / ml)]

<Test>
Two-way ANOVA was used to analyze the IL-1RA expression level of each sample by the Dunnett method.

(2) Results The IL-1RA production promoting effect test results of the test sample are shown in FIG.
As shown in FIG. 3, when 1% by mass of inositol and 1% by mass of yeast extract BG (Lot.B) are used in combination, a synergistic effect equal to or greater than the effect of adding yeast extract BG (Lot.B) or inositol alone is obtained. Indicated. It was revealed that inositol synergistically enhances the IL-1RA production promoting effect of yeast extract BG (Lot.B) by adding 1% by mass or more. Yeast extract BG (Lot.B) is a yeast extract dispersion and contains 1.6% by mass of yeast extract. Therefore, it is calculated that if 62 or more inositol is contained in yeast extract 1, the IL-1RA production promoting effect is obtained. Was found to be enhanced.

Cosmetics were prepared by a conventional method according to the formulation shown below.
Example 1 (Cream) (% by mass)
1. 1. Water residue 2. Glycerin 7
3. 3. Dipropylene glycol 7
4. 1,2-Pentanediol 1
5. Squalene 10
6. Medufoam oil 5
7. Polyglyceryl stearate-10 2
8. Hydrogenated lecithin 0.5
9. Simalgel NS (manufactured by Sepik) 0.5
10. Behenyl alcohol 1
11. Carboxyvinyl polymer 0.1
12. Xanthan gum 0.05
13. Potassium hydroxide 0.03
14. Yeast extract BG (Maruzen Pharmaceuticals (Lot.B)) * 1 0.1
15. Inositol 0.5
* 1 0.0016% by mass of yeast extract is contained in 0.1% by mass of yeast extract BG.

The obtained cosmetic (cream) was used as a test product for an actual use test described later.

5. Confirmation test method of IL-1RA production promoting effect by actual use test
The subjects were healthy Japanese women (9 people) who were 35 years old or older and had wrinkle grades 2 to 3 at the corners of the eyes of the Japan Cosmetic Science Society "Anti-wrinkle product evaluation guideline". After washing the face in the morning and evening, after using a lotion on the entire face, a test product (cream of Example 1) was applied to the half face (no application of the half face), and the face was continuously used for 4 weeks. Before and after use, collect the stratum corneum from the left and right cheeks using a tape stripping method with a single piece of stratum corneum tape (manufactured by Asahi Techno Lab), add T-PER Tissue Protein Extraction Reagent (manufactured by Thermo Fisher Scientific), and add approximately 0.8 g. Glass beads were added and vortexed for 25 minutes to crush the stratum corneum. The IL-1RA concentration in the recovered protein lysate was measured by Human IL-1ra / IL-1F3 DuoSet ELISA (manufactured by R & D systems), and the protein concentration was measured by the BCA protein assay kit (manufactured by Thermo Fisher Scientific) according to a standard method. The amount of IL-1RA per unit protein in the stratum corneum protein was calculated.
[IL-1RA concentration in the stratum corneum (pg / μg protein)] = [IL-1RA concentration in the lysate (pg / ml)] / [Protein concentration in the lysate (μg / ml)]
The amount of variation (ΔIL-1RA) in the amount of IL-1RA in the stratum corneum before and after use without application and the application site was calculated, and the effectiveness of application was evaluated. In the test, the IL-1RA fluctuation values of the coated and non-coated parts were compared and examined by the paired t-test. The results are shown in Table 3 and FIG.

Summary of results The amount of IL-1RA in the stratum corneum was decreased without application, but the decrease was suppressed by the application of the cream of the present invention, and the induction of IL-1RA expression by the application of the cream of the present invention was confirmed. (p = 0.13). The amount of IL-1RA increased by an average of about 16 pg / μg protein in the skin coated with the cream of the present invention as compared with the case without application.

The applicant has confirmed that the IL-1RA expression level in the stratum corneum decreases with age and correlates with the texture and blood flow, which are indicators of skin metabolism, and has registered a patent (Patent No. 5977268, Patent). No. 5731678). The IL-1RA production promoting composition of the present invention and an external preparation containing the same are useful techniques as an invention for preventing skin aging.

Claims (3)

An IL-1RA production-promoting composition for skin cells containing a yeast extract and vitamin B2, which contains 0.00018 parts by mass or more of vitamin B2 with respect to 1 part by mass of the total content of the yeast extract and vitamin B2. -1 RA production promoting composition . An IL-1RA production-promoting composition for skin cells containing a yeast extract and inositol, which contains 62 parts by mass or more of inositol with respect to 1 part by mass of the yeast extract . The yeast extract is a concentrated or dried product of the solution obtained by autolysis or acid hydrolysis of yeast belonging to Saccharomyces or yeast belonging to Candida, or the concentrated or dried product is further water and / or alcohol. The IL-1RA production promoting composition according to claim 1 or 2 , which is an evaporation residue of the composition obtained by extracting with a solution containing the yeast.

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