JP7627449B2 - Ceramide synthase gene expression promoter, skin quality improving agent, functional food and cosmetic for improving skin quality, and method of using dipeptide - Google Patents

Description

The present invention relates to a ceramide synthase gene expression promoter, a skin quality improving agent, a functional food and cosmetic for improving skin quality, and a method for using a dipeptide.

Beautiful and healthy skin is one of the important factors that enhance a person's external attractiveness. For this reason, skin care has traditionally been practiced by both men and women.

In particular, firmness and sufficient moisture are important for maintaining youthful skin. Therefore, ingredients that keep the skin moisturized are important, and one example of such moisturizing ingredients is ceramide.

Ceramide is a type of sphingolipid synthesized within cells, and is the main component of the stratum corneum that forms the surface of the epidermis layer of human skin. Ceramide also prevents excessive evaporation of moisture and acts as a barrier against microorganisms. To keep skin fresh and youthful, it is necessary to maintain an appropriate amount of ceramide in the skin.

However, ceramides tend to be lost with age.

Therefore, in order to promote the biosynthesis of ceramide in epidermal cells of the skin, a ceramide synthesis promoter has been proposed that contains a bacterial culture containing nicotinic acid and/or nicotinamide as an active ingredient.

It is said that such ceramide synthesis promoters can improve rough skin and various skin diseases by activating ceramide synthesis in epidermal cells themselves inside the surface layer of the skin and improving the skin barrier function.

Japanese Patent Application Publication No. 10-259135

However, the above-mentioned conventional ceramide synthesis promoters, which contain bacterial cultures containing nicotinic acid or nicotinamide as active ingredients, have been proposed as external preparations, and their effects when taken orally have not been clarified.

In addition, in promoting ceramide biosynthesis, providing further variations as key components for inducing function would be desirable in terms of expanding the options available to users.

The present invention has been made in consideration of the above circumstances, and provides a ceramide synthase gene expression promoter that employs a specific dipeptide as the main component for inducing function and is capable of increasing the expression level of the ceramide synthase gene.

The present invention also provides skin quality improving agents, functional foods for improving skin quality, and cosmetics that contain a specific dipeptide that also has the function of promoting ceramide synthase gene expression, as well as methods for using the dipeptide.

In order to solve the above-mentioned problems in the conventional art, the ceramide synthase gene expression promoter of the present invention contains a dipeptide represented by Gly-Ile as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene and/or the ceramide synthase 4 (CERS4) gene .

The ceramide synthase gene expression promoter of the present invention is also characterized in that it contains a dipeptide represented by Ile-Gly as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene .

Furthermore, the skin quality improving agent, skin quality improving functional food, and cosmetic (excluding those exhibiting an erythema reaction suppression effect or a whitening effect) of the present invention contain a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for eliciting at least one function selected from improving skin moisture content, suppressing the change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7) .

In addition, in the present invention, a dipeptide represented by Gly-Ile is used as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene and/or the ceramide synthase 4 (CERS4) gene in the production of a ceramide synthase gene expression promoter.
In addition, in the present invention, a dipeptide represented by Ile-Gly is used as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene in the production of a ceramide synthase gene expression promoter.
In addition, in the present invention, a dipeptide represented by Gly-Ile and/or Ile-Gly is used as an active ingredient for the purpose of achieving at least one of the following in the production of functional foods (excluding those that exhibit an erythema reaction suppression effect or a whitening effect): improving skin moisture content, suppressing the change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7).

According to the ceramide synthase gene expression promoter of the present invention, a dipeptide represented by Gly-Ile is contained as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene and/or the ceramide synthase 4 (CERS4) gene . Also, according to the ceramide synthase gene expression promoter of the present invention, a dipeptide represented by Ile-Gly is contained as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene, so that a specific dipeptide is adopted as the main component for inducing the function, and a ceramide synthase gene expression promoter capable of increasing the expression amount of the ceramide synthase gene can be provided.

In addition, if the ceramide synthase gene is the ceramide synthase 3 (CERS3) gene, it is possible to target ceramide synthase 3, which is expressed mainly in skin and keratinocytes, and to provide a more efficient ceramide synthase gene expression promoter.

Furthermore, the skin quality improving agent, functional food for improving skin quality, and cosmetic (excluding those exhibiting an erythema reaction suppression effect or a whitening effect) of the present invention contains a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for eliciting at least one function selected from improving skin moisture content, suppressing the change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7), and therefore can be a skin quality improving agent, functional food for improving skin quality, or cosmetic that can increase the expression level of ceramide synthase gene.

In addition, in the present invention, a dipeptide represented by Gly-Ile is used as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene and/or the ceramide synthase 4 (CERS4) gene in the production of a ceramide synthase gene expression promoter. In addition, in the present invention, a dipeptide represented by Ile-Gly is used as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene in the production of a ceramide synthase gene expression promoter. In addition, in the present invention, a dipeptide represented by Gly-Ile and/or Ile-Gly is used as a participating component for the purpose of at least one selected from improving skin moisture content, suppressing the amount of change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7) in the production of functional foods (excluding those that exhibit an erythema reaction suppressing effect or a whitening effect). Therefore, it is possible to enjoy the effects derived from the increase in the expression level of the ceramide synthase gene by the dipeptide represented by Gly-Ile or Ile-Gly.

FIG. 1 is an explanatory diagram showing the cell viability of HaCaT cells as a result of addition of each dipeptide. FIG. 1 is an explanatory diagram showing changes in expression of ceramide synthase gene in HaCaT cells by addition of each dipeptide. FIG. 1 is an explanatory diagram showing changes in expression of ceramide synthase gene in HaCaT cells by addition of a small amount of dipeptide. FIG. 1 is an explanatory diagram showing test results regarding the moisture content when test foods were ingested. FIG. 1 is an explanatory diagram showing test results regarding transepidermal water loss when test foods were ingested. FIG. 13 is an explanatory diagram showing the test results regarding total skin elasticity R2 when the test food was ingested. FIG. 1 is an explanatory diagram showing test results regarding skin viscoelasticity (R5 (net elasticity)) when test foods were ingested. FIG. 1 is an explanatory diagram showing test results regarding the skin recovery power R7 when the test food was ingested.

The present invention provides a ceramide synthase gene expression promoter that employs a specific dipeptide as the main component for inducing function and is capable of increasing the expression level of the ceramide synthase gene.

The uses of the ceramide synthase gene expression promoter according to this embodiment are not particularly limited, and it can be used as an additive ingredient to impart skin quality improving functions to foods and cosmetics, or as an external agent for improving skin quality, and can also be used, for example, as a reagent for testing and research.

The ceramide synthase gene expression promoter according to this embodiment is characterized in that it contains a dipeptide represented by Gly-Ile (glycine-isoleucine) and/or Ile-Gly (isoleucine-glycine) as the main component for inducing function.

Gly-Ile and Ile-Gly are dipeptides formed by the peptide bond between glycine and isoleucine. There is no particular limitation on the origin of Gly-Ile and Ile-Gly, and they may be derived from any source that is compatible with the purpose, such as dried products or extracts of specific materials or ingredients, digests of specific proteins, or peptide synthesis. In this specification, unless otherwise specified, the left side of the amino acid sequence is written as the N-terminus.

As will be explained later with reference to experimental results, the inventors' intensive research has revealed that Gly-Ile and Ile-Gly promote the expression of ceramide synthase genes, and are expected to have the effect of maintaining or increasing the amount of ceramide in the skin.

Gly-Leu and Leu-Gly are known dipeptides that are believed to have a positive effect on the skin, but the dipeptide proposed in this application is structurally completely different from these substances, and the test results described below make it clear that the dipeptide proposed in this application has a higher effect of promoting ceramide synthase gene expression than Gly-Leu and Leu-Gly.

The present application also provides a skin quality improving agent. The skin quality improving agent according to this embodiment is characterized in that it contains a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for inducing function. In this application, the skin quality improving agent may be taken orally, or may be used as an external preparation for the skin. External preparations for the skin naturally refer to medicines, quasi-drugs, and cosmetics (including medicated cosmetics), but are not limited to these categories and are included in this concept as long as they are an agent that is applied externally to the skin.

This application also provides a cosmetic preparation, which is one aspect of this topical skin preparation. That is, the cosmetic preparation according to this embodiment is characterized in that it contains a dipeptide represented by Gly-Ile and/or Ile-Gly as an active ingredient.

In this application, cosmetics include not only so-called makeup cosmetics, but also basic cosmetics, hair tonics, perfumes, toothpaste, shampoos, rinses, soaps and cleansers used for body washing, bath additives, and other toiletry products, as well as medicated cosmetics that claim to have preventive effects, etc.

Examples of makeup cosmetics include foundation, eyebrow pencil, mascara, eye shadow, eyeliner, lipstick, gloss, blush, face powder, nail polish, etc., while basic cosmetics include, for example, lotion, milky lotion, facial cleanser, cleanser, beauty serum, cream, etc. The above explanations of topical skin preparations and cosmetics are merely examples of equivalent products to aid in the understanding of the present invention, and the interpretation of each term is not limited to these listed products. However, this does not preclude the applicant from limiting the present invention to these products when granting a patent for this application.

The present application also provides a method for using a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for inducing function in a ceramide synthase gene expression promoter. Although several components having the effect of promoting ceramide synthase gene expression have been proposed, no ceramide synthase gene expression promoter has been proposed that contains a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for inducing function.

In addition, proposing new ingredients that can promote ceramide synthase gene expression or induce the effect to users would be useful in increasing the options available to them.

This application also provides a method for using the dipeptide represented by Gly-Ile and/or Ile-Gly as an ingredient involved in improving skin quality in functional foods. Although several ingredients have been proposed that improve skin quality when taken orally, no functional foods have been proposed to date that contain the dipeptide represented by Gly-Ile and/or Ile-Gly as an ingredient involved in improving skin quality.

This application also provides a functional food for improving skin quality, and in particular, a functional food for improving skin quality that can exert an excellent skin moisturizing effect when taken orally.

Functional foods are a concept that encompasses all foods that do not contain pharmaceutical ingredients and are believed to contribute to maintaining and improving health. For example, functional foods include nutritional supplements, health supplements, and nutritionally adjusted foods, as well as general foods such as so-called supplements, and health functional foods such as foods for specified health uses, foods with nutritional functions, and foods with functional claims.

In addition, when it is a supplement, the dosage form is not particularly limited, and any dosage form can be selected, such as tablets, capsules, fine granules, pills, lozenges, liquids, and jellies.

The above explanations of topical agents, cosmetics, and functional foods are merely examples of equivalent products to aid in the understanding of the present invention, and the interpretation of each term is not limited to these listed products. However, this does not preclude the applicant from limiting the present invention to these products when granting rights to this application.

The concentration of Gly-Ile and/or Ile-Gly as a function-inducing component contained in the ceramide synthase gene expression promoter, skin quality improving agent, functional food for improving skin quality, cosmetic product, etc. according to this embodiment is not particularly limited as long as it is a concentration that can induce the above-mentioned function. To give an example, the concentration of Gly-Ile or Ile-Gly contained in a functional food for improving skin quality can be, for example, an amount that allows 20 to 600 μg of Gly-Ile or Ile-Gly to be ingested per day, or an amount that allows 20 to 600 μg of Gly-Ile and Ile-Gly to be ingested per day as the total amount. In addition, if it is a functional food for improving skin quality that is taken at a recommended dose of one tablet per day, the amount of Gly-Ile and/or Ile-Gly contained in one tablet can be adjusted to 20 to 600 μg, and if the recommended dose is two tablets per day, the amount can be adjusted to 10 to 300 μg per tablet. It is also possible to appropriately adjust the amount of Gly-Ile and/or Ile-Gly contained in a unit of intake, such as one tablet, capsule, or packet.

Cosmetics may contain 0.03 to 200 μg/g of Gly-Ile or Ile-Gly, or the total concentration of Gly-Ile and Ile-Gly may be 0.03 to 200 μg/g. The concentrations of Gly-Ile and Ile-Gly in the ceramide synthase gene expression promoter and skin quality improver may be suitable for use in experiments, etc., or as a raw material for the manufacture of foods and cosmetics.

In addition, the ceramide synthase gene expression promoter, skin quality improver, functional food for improving skin quality, cosmetic product, etc. according to this embodiment can contain excipients, additives, auxiliary ingredients, etc. according to the dosage form, food form, etc. in addition to Gly-Ile and/or Ile-Gly as a component for inducing function.

For example, when the dosage form is a solid, excipients can be lactose, crystalline cellulose, starch, etc.

Additives include, for example, stabilizers, surfactants, solubilizers, plasticizers, sweeteners, antioxidants, flavoring agents, colorants, preservatives, inorganic fillers, etc.

The following further describes the ceramide synthase gene expression promoter, skin quality improving agent, functional food and cosmetic for improving skin quality, and method of using the dipeptide according to this embodiment, with reference to the experimental process and results.

1. Ceramide synthase gene expression test
The skin is composed of the epidermis and dermis, and the epidermis is divided into the basal layer, spinous layer, granular layer, and stratum corneum. Between the keratinocytes located in the outermost layer of the skin, a lamellar structure composed of lipids such as ceramide plays a role in retaining moisture in the body and acting as a physical barrier to prevent the invasion of bacteria and viruses.

In this study, the effect of Gly-Ile and Ile-Gly (hereinafter also referred to as the dipeptides of the present application) on improving epidermal cells was evaluated by examining changes in the expression level of the ceramide synthase gene. In addition, Gly-Leu and Leu-Gly (hereinafter also referred to as comparative dipeptides) were used as comparison controls to examine the differences from the dipeptides of the present application.

Human epidermal keratinocytes (HaCaT cells) were precultured in a φ10 cm dish using Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) until confluent.

Thereafter, the cells were washed with phosphate-buffered saline (PBS) and resuspended in medium, after which they were seeded onto a 24-well plate at a density of 1.0×10 ⁵ cells/well and cultured overnight in a CO ₂ incubator (37° C., 5% CO ₂ ).

Twenty-four hours after seeding the HaCaT cells, 1 mL of serum-free DMEM (high glucose) (containing 1% penicillin-streptomycin) containing either Gly-Ile, Ile-Gly, Gly-Leu, or Leu-Gly at the specified final concentration was added to each well, and the cells were cultured for 48 hours in a _CO2 incubator (37°C, 5% _CO2 ).

Next, we examined the changes in gene transcription expression levels using real-time PCR. First, HaCaT cells were harvested 48 hours after the addition of each dipeptide, and total RNA was extracted using the RNeasy Mini kit (Qiagen).

Next, cDNA was synthesized from the extracted total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Real-time PCR was performed using the synthesized cDNA as a template with an AriaMX (Agilent) device. THUNDERBIRD SYBR qPCR Mix (TOYOBO) was used for the real-time PCR reaction.

In addition, for the real-time PCR reaction, 5'-CCGATTACCTGCTGGAGTCAG-3' (sequence number 1) and 5'-GGCGAAGACGATGAAGATGTTG-3' (sequence number 2) were used as primers for the ceramide synthase gene CERS2, 5'-ACATTCCACAAGGCAACCATTG-3' (sequence number 3) and 5'-CTCTTGATTCCGCCGACTCC-3' (sequence number 4) were used as primers for the ceramide synthase gene CERS3, 5'-GGAGGCCTGTAAGATGGTCA-3' (sequence number 5) and 5'-GAGGACCAGTCGGGTGTAGA-3' (sequence number 6) were used as primers for the ceramide synthase gene CERS4, and 5'-GGGTCAGAAGGACTCCTATG-3' (sequence number 7) and 5'-GTAACAATGCCATGTTCAAT-3' (sequence number 8) were used as primers for the internal standard β-actin. The real-time PCR reaction conditions were 40 cycles of a two-step PCR reaction consisting of initial denaturation at 95°C for 60 seconds, denaturation at 95°C for 15 seconds, and annealing/extension at 60°C for 60 seconds.

The dipeptides of the present invention, Ile-Gly and Gly-Ile, and the comparative dipeptides, Leu-Gly and Gly-Leu, were added to HaCaT cells, a human epidermal keratinocyte cell line, and changes in the expression of the ceramide synthase gene were examined. Figure 1 shows the cell viability of HaCaT cells following the addition of each dipeptide.

As shown in Figure 1, the addition of the dipeptide of the present application or the comparative dipeptide tended to increase the cell viability of human keratinocytes HaCaT compared to the control. In particular, when the dipeptide of the present application was added, an increase in cell viability compared to the control was confirmed at all concentrations of 0.1 μg/mL, 10 μg/mL, and 200 μg/mL. It was also confirmed that the cell viability was significantly increased at 10 μg/mL Gly-Leu, 0.1 and 10 μg/mL Ile-Gly, and 200 μg/mL Gly-Ile compared to the control. These results suggest that the dipeptides of the present application, Ile-Gly and Gly-Ile, can be expected to have a keratinocyte activation effect.

Figure 2 shows the change in expression of ceramide synthase genes in HaCaT cells when the dipeptides of the present invention, Ile-Gly and Gly-Ile, and the comparative dipeptides, Leu-Gly and Gly-Leu, were added. Figure 2(a) shows the relative expression levels of the ceramide synthase CERS2 gene to the control, while Figure 2(b) and Figure 2(c) show the relative expression levels of the ceramide synthase CERS3 gene and CERS4 gene, respectively.

It has been reported that the ceramide synthase CERS2 gene is expressed in large amounts in many cell tissues, including the kidney, liver, and intestine. The ceramide synthase CERS3 gene is expressed mainly in the skin and keratinocytes. Furthermore, the ceramide synthase CERS4 gene is known to be expressed not only in the skin, but also in white blood cells, the heart, and the liver.

In this study, each dipeptide was added to HaCaT cells, and then the expression of the ceramide synthase genes CERS2, CERS3, and CERS4 was analyzed using real-time PCR. As a result, as shown in Figure 2 (a), no significant increase in the expression of the CERS2 gene was confirmed at any concentration compared to the control.

In contrast, as shown in Figure 2(b), the expression level of the ceramide synthase CERS3 gene was confirmed to increase at all concentrations by adding the dipeptides of the present application, Ile-Gly and Gly-Ile, compared to the control.

In particular, the addition of Gly-Ile was found to significantly increase expression levels in a concentration-dependent manner, and it is extremely interesting that at a concentration of 200 μg/mL, expression levels increased three-fold compared to the control.

Addition of Ile-Gly also increased the expression level of the CERS3 gene, and although a significant difference was observed when used at a concentration of 0.1 μg/mL, it is notable that the expression level decreased as the concentration increased.

Addition of the comparative dipeptides Leu-Gly and Gly-Leu also confirmed an increase in expression levels of up to approximately 1.6 times compared to the control. However, the increase in expression levels was not as significant as that observed with the dipeptide of the present application.

Although the data is omitted here, the inventors' research experience has demonstrated that even the addition of Ile-Gly or Gly-Ile at a concentration of about 0.03 μg/g significantly increases the expression level of the CERS3 gene compared to the control.

As shown in Figure 2(c), the expression level of the ceramide synthase CERS4 gene was significantly increased by adding Gly-Ile.

These results suggest that the dipeptide of the present application can be expected to have a cell activation effect and to have the effect of enhancing the expression of the ceramide synthase CERS3 and CERS4 genes. It was also suggested that this effect is particularly high in the dipeptide of the present application, Gly-Ile. Thus, the dipeptide of the present application has been shown to have an effect of promoting the expression of ceramide synthase genes, and is expected to have an effect of promoting ceramide synthesis, which plays an important role in the skin's barrier function.

It was also shown that dipeptides represented by Gly-Ile and/or Ile-Gly can be used as a key component for inducing functions in skin quality improving agents, as a component involved in improving skin quality in functional foods, and as an active ingredient for improving skin quality in cosmetics.

2. Ceramide synthase gene expression test (low dose)
Next, the expression of the ceramide synthase gene by the dipeptides of the present invention, Gly-Ile and Ile-Gly, was examined by the same method to see whether it was possible to induce expression at concentrations lower than those mentioned above.

In this study, sample solution S1 contained at least 0.00038 μg/mL of Gly-Ile and at least 0.00097 μg/mL of Ile-Gly, with a total concentration of both dipeptides of 0.00135 μg/mL, and sample solution S2 contained at least 0.00076 μg/mL of Gly-Ile and at least 0.00195 μg/mL of Ile-Gly, with a total concentration of both dipeptides of 0.00271 μg/mL (twice the concentration of sample solution S1). The test solutions were sample solution S2 (solution S2) and sample solution S3 (a solution twice as concentrated as sample solution S2) containing at least 0.00152 μg/mL Gly-Ile and at least 0.0039 μg/mL Ile-Gly, with a total concentration of both dipeptides of 0.00542 μg/mL. 24 hours after seeding of HaCaT cells, 1 mL of final concentration serum-free DMEM (high glucose) (containing 1% penicillin-streptomycin) constituting either of the above test solutions was added to each well, and the cells were cultured for 48 hours in a _CO2 incubator (37°C, 5% _CO2 ).

First, Figure 3(a) shows the cell viability of HaCaT cells when each sample solution S1 to S3 was added. As shown in Figure 3(a), no significant difference was observed between each sample solution S1 to S3 and the control.

Figure 3(b) shows the change in expression of ceramide synthase genes in HaCaT cells after the addition of each of the sample solutions S1 to S3. As shown in Figure 3(b), no significant difference in expression of the CERS2 gene was observed at any concentration compared to the control. On the other hand, the expression of the CERS3 gene and CERS4 gene, which have been reported to be expressed in the skin, was increased by approximately 2- to 4-fold compared to the control. Furthermore, a more significant increase in expression was observed for the CERS3 gene compared to the CERS4 gene.

The above results suggest that the dipeptides Gly-Ile and Ile-Gly of the present application are not cytotoxic and have the effect of enhancing the expression of the ceramide synthase genes CERS3 and CERS4. Therefore, the dipeptides Gly-Ile and Ile-Gly of the present application can be expected to have a ceramide synthesis promoting effect, which plays an important role in the barrier function of the skin, when the total concentration of both dipeptides is 0.00135 μg/mL or more. In addition, it is preferable to use a dipeptide containing at least 0.00038 μg/mL of Gly-Ile and at least 0.00097 μg/mL of Ile-Gly, and a total concentration of both dipeptides of 0.03 μg/mL, for example, to ensure a more consistent effect.

3. Manufacturing of functional foods
Next, functional foods containing the dipeptides represented by Gly-Ile and Ile-Gly as function-inducing components for improving skin quality, for example, as components involved in functional foods, were produced.

The functional food for the test was formulated as a hard capsule. Specifically, 200 mg of powdered dextrin was used as the base material, and Gly-Ile and/or Ile-Gly obtained by peptide synthesis was added and mixed. The resulting mixed powder was filled into a capsule body made of gelatin as the coating material, and a cap was applied to obtain functional food A in the form of a hard capsule.

4. Measurement of Gly-Ile and Ile-Gly in functional foods
Next, the content of the dipeptide of the present invention contained in each tablet of the obtained functional food A was measured.

(4-1. Recovery test of two types of dipeptides)
First, in order to examine whether the test method used this time was appropriate for measuring the Gly-Ile and Ile-Gly contents of functional food A, a test was conducted to confirm the recovery rate of known amounts of Gly-Ile and Ile-Gly that had been added in advance to a sample food prepared to mimic functional food A.

To one capsule not containing mixed powder, 20, 50, 100, and 150 μL of a mixed solution (500 μg/mL) of two types of peptide standards (Gly-Ile, Ile-Gly) was added (final concentration in the measurement solution: 20, 50, 100, 150 ng/mL). Then, 10 mL of ultrapure water was added, and ultrasonic extraction (38 kHz, 200 W) was performed for 40 minutes. During the extraction operation, the extraction solvent was vigorously vibrated 3 to 4 times to uniformly disperse. After extraction, the mixture was centrifuged (3500 rpm, 10 min), and the supernatant was placed in a 25 mL measuring flask. The residue was subjected to ultrasonic extraction and centrifugation twice in the same manner as above with 8 mL and 5 mL of ultrapure water. The three extraction solutions were combined and the volume was adjusted to a constant volume with ultrapure water to prepare the extraction solution (n=3). 20 μL of the internal standard solution (2 μg/mL) was added to 100 μL of the above extraction solution, and 380 μL of acetonitrile was further added, stirred, centrifuged (3500 rpm, 10 min), and the supernatant was filtered through a 0.20 μm PTFE membrane. The filtrate was diluted 4 times with ultrapure water and subjected to LC-MS analysis. The calculation formula for the recovery rate of the added standard is shown in Equation 1, and the analysis conditions are shown in Table 1.

As a result of the LC-MS analysis, the two types of dipeptides were not detected in the hard capsules (capsule body and cap) that did not contain the mixed powder. Therefore, it was confirmed that the capsules did not contain the two types of dipeptides.

Next, to verify the pretreatment method of the sample solution and the accuracy of the quantitative analysis values, we analyzed sample foods in which two types of dipeptides were added to capsules as standards, and calculated the recovery rates. The recovery rates are shown in Table 2.

As can be seen from Table 2, the average recovery rates of the two peptides in the sample foods to which the standard was added were within the range of 97.8% to 104.2% regardless of the amount added. Therefore, the pretreatment method and quantitative analysis method used in this experiment are considered to be valid.

(4-2. Quantitative analysis of two types of dipeptides contained in functional food A)
Next, quantitative analysis was performed on the two types of dipeptides contained in functional food A. 10 mL of ultrapure water was added to one tablet of functional food A, and ultrasonic extraction (38 kHz, 200 W) was performed for 40 minutes. During the extraction procedure, the sample was vigorously vibrated 3 to 4 times to ensure that the extraction solvent was uniformly dispersed.

After the extraction procedure, centrifugation (3500 rpm, 10 min) was performed, and the supernatant was placed in a 25 mL measuring flask. The residue was subjected to ultrasonic extraction and centrifugation twice in the same manner as above using 8 mL and 5 mL of ultrapure water. The three extraction solutions were combined and adjusted to volume with ultrapure water to obtain the extraction solution (n=3).

20 μL of the internal standard solution (2 μg/mL) was added to 100 μL of the above extraction solution, and then 380 μL of acetonitrile was added, stirred, and centrifuged (3500 rpm, 10 min). The supernatant was then filtered through a 0.20 μm PTFE membrane. The filtrate was diluted 4-fold with ultrapure water and subjected to LC-MS analysis. The analytical conditions were as shown in Table 1.

First, the elution times of Gly-Ile and Ile-Gly were confirmed by the analytical results using the standards. As shown in Table 3, the two dipeptide standards showed good linearity ( ^R2 = 0.999) in the calibration curve range from 5 ng/mL to 200 ng/mL.

Furthermore, the dipeptide content of functional food A using the calibration curve is shown in Table 4. It was confirmed that one tablet of functional food A contained 26.25±0.38 μg of Gly-Ile and 22.57±0.48 μg of Ile-Gly.

5. Ingestion Test
Next, the capsule-shaped functional food A obtained in the above-mentioned [3. Production of functional food] was examined for its effect of improving the skin quality of healthy adults.

The study was conducted on 20 healthy women aged 40-50 years old, divided into two groups: 10 in the test product group and 10 in the placebo group. The intake amount was two tablets per day for both the test product group (functional food A) and the placebo group (placebo food). Note that the placebo food did not contain Gly-Ile or Ile-Gly. The intake period was four weeks for both groups.

After the intake period had elapsed, the skin quality was also measured. Specifically, the skin's moisture content, water loss, and viscoelasticity were measured. The measurement site was the left arm.

Measurements were taken using a Cutometer DUAL MPA580 (Courage + Khazaka electronic GmbH). Moisture and viscoelasticity were measured five times each, and the average of the three measurements excluding the lowest and highest values was used as the measured value. R2 (total elasticity), R5 (net elasticity), and R7 (resilience) were used to analyze viscoelasticity.

Standard errors were calculated, and paired and unpaired t-tests were performed to compare pre- and post-measurements (intragroup comparisons) and between groups. The significance level was set at 5%, with a difference of less than 5% considered significant (p<0.05:**) and a difference of less than 10% considered to indicate a significant trend (p<0.1:*).

Of the 20 people enrolled in this study, 19 people were analyzed, excluding one who was unable to participate in the follow-up measurements. Specifically, the placebo group consisted of 10 women with an average age of 44.7 years, and the test product group consisted of 9 women with an average age of 45.0 years.

Figure 4 shows the test results regarding moisture content. Moisture content is the amount of moisture contained in the stratum corneum, and is a value referenced as an index of skin moisture. Figure 4(a) shows the change in moisture content measurements before and after ingestion of the test food or placebo food, and Figure 4(b) shows the change in moisture content in the placebo group and the test food group, respectively.

As can be seen from Figures 4(a) and 4(b), a significant (p<0.05) increase in moisture content was observed in the test product group after ingestion, confirming that skin moisture content improved even in seasonal environments where skin tends to become dry.

Although not shown in the figure, similar moisture measurements were also performed on the subjects' cheeks. In the placebo group, no significant change in cheek moisture was observed before and after intake, but in the test product group, a significant increase was observed (p<0.05).

Figure 5 shows the test results on transepidermal water loss. Transepidermal water loss is the amount of water that evaporates from the surface of the skin, and is a value that serves as an index of barrier function. Figure 5(a) shows the change in transepidermal water loss before and after ingestion of the test food or placebo food, and Figure 5(b) shows the change in transepidermal water loss for the placebo group and the test food group, respectively.

As can be seen from Figure 5(a), the transepidermal water loss in the placebo group increased significantly after ingestion, whereas no significant change was observed in the test product group, indicating that the skin barrier function was maintained by ingestion of the test product.
It was shown that ingestion of the test product significantly suppressed the change in transepidermal water loss compared to ingestion of the placebo product (p<0.05).

Figure 6 shows the test results for total elasticity of the skin (R2). Total elasticity is an index that represents the elasticity of the entire skin. Skin elasticity is maintained by the fibers and water-retaining components in the dermis layer. Figure 6 (a) shows the change in total elasticity before and after the intake of the test food or placebo food, and Figure 6 (b) shows the change in total elasticity for the placebo group and the test food group, respectively.

As can be seen from Figures 6(a) and 6(b), R2, which indicates total elasticity, showed a tendency to decrease in the placebo group, and a significant interaction between the two factors of time and group was observed, and a significant difference was observed between the amount of change in the placebo group and the test product group. From this, it can be said that Gly-Ile and Ile-Gly have the effect of activating the function of the dermis layer and maintaining the firmness and elasticity of the skin in terms of total elasticity of the skin (R2).

Figure 7 shows the test results for skin viscoelasticity (R5 (net elasticity)). Viscoelasticity is a value that serves as an index of skin firmness and elasticity. Figure 7(a) shows the change in viscoelasticity before and after ingestion of the test food or placebo food, and Figure 7(b) shows the amount of change in viscoelasticity for the placebo group and the test food group, respectively.

As can be seen from Figures 7(a) and 7(b), in the test product group, R5, which indicates net elasticity, increased significantly (p<0.05) compared to before ingestion, indicating that ingestion of the test product improves skin elasticity. In addition, a significant interaction between the two factors of time and group was observed, and a significant difference between the groups was also observed in the amount of change.

Figure 8 shows the test results for skin recovery R7 (rebound rate). Recovery is a value that indicates the instantaneous contraction rate of the skin relative to the stretched length when suctioned, and shows a high correlation with skin firmness and sagging. Figure 8(a) shows the change in recovery before and after ingestion of the test food or placebo food, and Figure 8(b) shows the amount of change in recovery for the placebo group and the test product group, respectively.

As can be seen from Figures 8(a) and 8(b), recovery power R7 in the test product group increased significantly (p<0.05) compared to before intake, indicating that intake of the test product improves the skin's recovery power. In addition, a significant interaction between the two factors of time and group was observed in recovery power R7, and the amount of change also showed a significant trend in the difference between the groups.

Taking into account the results of each of these intake tests, it has been shown that functional foods containing the dipeptides represented by Gly-Ile and Ile-Gly as function-inducing ingredients for improving skin quality, for example as participating ingredients in the case of functional foods, can contribute to the retention of skin moisture, the inhibition of skin moisture evaporation, and the maintenance and improvement of skin firmness and viscoelasticity, thereby improving skin quality.

As described above, the ceramide synthase gene expression promoter according to this embodiment contains a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for inducing function, and therefore a specific dipeptide is used as the main component for inducing function, making it possible to provide a ceramide synthase gene expression promoter that can increase the expression level of the ceramide synthase gene.

In addition, the skin quality improving agent, functional food for improving skin quality, and cosmetic product according to this embodiment contain a dipeptide represented by Gly-Ile and/or Ile-Gly as a main component for inducing function, and therefore can be a skin quality improving agent, functional food for improving skin quality, and cosmetic product that can increase the expression level of the ceramide synthase gene and improve skin quality.

Finally, the above-mentioned explanations of each embodiment are merely examples of the present invention, and the present invention is not limited to the above-mentioned embodiments. Therefore, even if the embodiment is different from the above-mentioned embodiments, various modifications can be made depending on the design, etc., as long as they do not deviate from the technical concept of the present invention.

Claims (8)

A ceramide synthase gene expression promoter containing a dipeptide represented by Gly-Ile as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene and/or the ceramide synthase 4 (CERS4) gene. A ceramide synthase gene expression promoter that contains a dipeptide represented by Ile-Gly as the main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene. A skin quality improving agent (excluding those that exhibit an erythema reaction suppressing effect or a whitening effect) that contains a dipeptide represented by Gly-Ile and/or Ile-Gly as a main ingredient for inducing at least one function selected from improving skin moisture content, suppressing the amount of change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7). Functional food for improving skin quality (excluding those that exert an erythema reaction suppressing effect or a whitening effect) containing a dipeptide represented by Gly-Ile and/or Ile-Gly as an ingredient involved in inducing at least one function selected from improving skin moisture content, suppressing the amount of change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7). Cosmetics containing a dipeptide represented by Gly-Ile and/or Ile-Gly as an active ingredient for inducing at least one function selected from improving skin moisture content, suppressing changes in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7) (excluding those that exhibit an erythema reaction suppression effect or a whitening effect). Use of a dipeptide represented by Gly-Ile as a main component for inducing the expression promoting function of the ceramide synthase 3 (CERS3) gene and/or the ceramide synthase 4 (CERS4) gene in the production of a ceramide synthase gene expression promoter. Use of a dipeptide represented by Ile-Gly as a main component for inducing the expression promoting function of ceramide synthase 3 (CERS3) gene in the production of a ceramide synthase gene expression promoter. Use of a dipeptide represented by Gly-Ile and/or Ile-Gly as an ingredient in the production of functional foods (excluding those exhibiting an erythema reaction inhibitory effect or a whitening effect) for the purpose of achieving at least one of the following: improving skin moisture content, suppressing the change in transepidermal water loss, improving total skin elasticity (R2), improving net elasticity (R5), and improving return rate (R7).