JP7807070B2 - Agents for promoting elastin production - Google Patents

Description

The present invention relates to an agent for promoting elastin production, for use in, for example, humans, etc.

As the first line of defense against direct influence from the external environment, the skin plays an important role in maintaining the internal environment of the body. Therefore, while skin function never completely ceases, its functions gradually decline with age, exposure to ultraviolet rays, and exposure to chemicals, resulting in the appearance of wrinkles, age spots, dullness, sagging skin, and other signs of aging. For example, the presence or absence of skin "firmness" is considered one indicator of health and the degree of aging. "Firmness" can be divided into two categories: firmness derived from the stratum corneum/epidermis, and firmness derived from the dermis. Dermal firmness, in particular, refers to a state in which the skin has an elasticity that pushes back when pressed with a finger, and quickly returns to its original state when the finger is removed; in physical terms, this is also known as viscoelasticity. The detailed mechanism behind the decline in skin viscoelasticity is not always clear, but thinning of the skin is thought to be one factor contributing to this decline. (Patent Document 1)

Elastin is a fiber that provides elasticity to skin tissue. As humans age, production of elastin decreases, and its degradation and denaturation are accelerated by ultraviolet rays. A decrease in normal elastin reduces skin elasticity, which can lead to wrinkles and sagging. Therefore, if elastin production can be promoted, it is believed that wrinkles and sagging will be less likely to occur, and that symptoms of skin aging, such as loss of firmness and loss of elasticity, can be prevented and/or improved (Patent Document 1).

Patent 7144878

The problem that the present invention aims to solve is to find naturally occurring substances (natural products) that have the effect of promoting elastin production, and to provide agents for promoting elastin production.

Therefore, the inventors of the present invention conducted extensive research to find substances that have the activity of promoting elastin production, and as a result discovered that elastin production can be achieved by adding proteoglycan to certain cells, etc. (for example, without adding elastin), and thus completed the present invention.
[Item 1] An agent for promoting elastin production, which contains proteoglycan.
[Item 2] The agent according to [Item 1] for promoting extracellular matrix production.
[Item 3] The agent according to [Item 2] for promoting collagen production and/or hyaluronic acid production.
[Item 4] The agent according to [Item 2] or [Item 3] for improving skin elasticity.
[Item 5] An oral composition containing the agent according to any one of [Item 1] to [Item 4].

The present invention provides a new oral composition for promoting elastin production.

The following describes how to implement the present invention.

(proteoglycan)
Proteoglycans (PGs) are glycoproteins composed of a core protein covalently bound to sugar chains called glycosaminoglycans (GAGs), such as chondroitin sulfate and dermatan sulfate. PGs are widely distributed throughout the body, including skin and cartilage, as one of the major components of the extracellular matrix. GAG chains have a long, unbranched, linear structure. Due to the numerous sulfate and carboxyl groups, they are negatively charged, and the GAG chains assume an extended shape due to their electrical repulsion. Furthermore, PGs can retain large amounts of water due to the water affinity of sugars. The numerous GAG chains contained in PG flexibly retain water like a sponge, while also providing cartilage-specific functions such as elasticity and impact resistance.

The core protein of PG has the property of binding to various molecules in the matrix. In the case of cartilage PG, it has binding domains at the N-terminus that bind to hyaluronic acid and link protein, and can bind to these substances and sometimes associate with the same molecules. The C-terminus contains lectin-like domains and EGF-like domains, which bind to various other molecules. This property allows PG to build structures that are suited to each tissue.

Salmon nasal cartilage-derived PG is PG obtained by extraction from salmon nasal cartilage. Here, the salmon is, for example, a fish belonging to the genus Oncorhynchus, and preferably, salmon with the scientific name "Oncorhynchus keta" is selected from the viewpoint of efficiently regulating immune responses. The proteoglycan contained in the agent or composition of this embodiment is prepared, for example, by a method described in a publication (Japanese Patent No. 6317053). Furthermore, the lower limit of the proteoglycan content in a composition (1 g) containing the composition of this embodiment is preferably 0.1 μg/g or more, more preferably 100 μg/g or more, and even more preferably 1 mg/g or more, from the viewpoint of, for example, effectively exerting elastin production.

An example of a commercially available PG product is Proteoglycan F (manufactured by Ichimaru Falcos).

(Promotes elastin production)
Increasing the amount of elastin in the body (promoting elastin production) can increase the strength and elasticity of the dermis, ligaments, tendons, blood vessel walls, bones, cartilage, etc. Agents for elastin production are thought to be used to prevent and/or improve various disorders associated with elastin reduction (for example, for treating or preventing vascular diseases such as arteriosclerosis caused by a lack of normal elastin), and to prevent and/or improve cosmetic problems (for example, for preventing and/or improving wrinkles or sagging skin caused by elastin reduction due to aging, ultraviolet light, active oxygen, stress, etc., or for preventing and/or improving reduced skin elasticity or firmness).

(extracellular matrix)
The extracellular matrix (ECM) is a complex of sugars and proteins present in the basement membrane that lines tissues and in the intercellular spaces. Examples of structures formed by the ECM include the basement membrane, glial scar, and perineuronal net.

(collagen)
Collagen is a major fibrous component that supports the structure of biological tissues such as skin, bones, and tendons, and is an essential protein found in large amounts in the body of animals.

(Hyaluronic acid)
Hyaluronic acid is a polysaccharide that has a structure in which the disaccharide unit of glucuronic acid and N-acetylglucosamine is linked.In addition, the salt of hyaluronic acid is not particularly limited, and it can be any salt that is acceptable for food or pharmacy, for example, sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt, etc.The molecular weight of hyaluronic acid is not particularly limited.

(Promotes hyaluronic acid production)
In the present invention, "promoting hyaluronic acid production" is not limited to a specific mechanism, as long as it primarily promotes the synthesis of hyaluronic acid in keratinocytes of the epidermis (particularly the cornified layer). Promoting hyaluronic acid production is preferably achieved, for example, by promoting the expression of genes encoding mammalian hyaluronic acid synthases HAS2 and HAS3. In the present invention, the hyaluronic acid production promoter can be used not only as a hyaluronic acid production promoter, but also as a promoter of hyaluronic acid synthase expression (e.g., a promoter of hyaluronic acid gene expression), particularly as a promoter of HAS2 gene or HAS3 gene expression. The hyaluronic acid production promoter of the present invention can prevent functional decline in various disorders, diseases, and illnesses associated with a decrease in hyaluronic acid in the body, particularly in the skin. For example, it can promote skin moisture, improving skin firmness and elasticity while providing moisture, preventing or improving skin problems such as rough skin, wrinkles, and dryness, and preventing or improving joint disorders, joint damage, and the like.

(Improves skin elasticity)
"Improving skin elasticity" not only means "suppressing a decrease in skin elasticity (preventing a decrease in skin elasticity, etc.)," but also includes, for example, "improving the symptoms of decreased skin elasticity (increasing skin thickness, etc.) by administering a specified composition (e.g., orally) after a decrease in skin elasticity has occurred."

(Oral Composition)
The oral composition according to the present invention is, for example, a food or drink (including functional foods, foods for specified health uses, supplements, etc.), a pharmaceutical product, or the like.

For example, when the oral composition is a food or beverage, the food or beverage may take the form of various foods and beverages such as bread, cakes, noodles, confectioneries, jellies, frozen foods, ice cream, dairy products, and beverages, as well as forms similar to those of the oral dosage formulations described above (tablets, capsules, syrups, etc.). Foods in various forms can be prepared by using the active ingredient of the present invention alone or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, humectants, thickeners, etc.

For example, when the oral composition is a pharmaceutical, the pharmaceutical may be in the form of a solid or liquid, although the pharmaceutical is generally amenable to a convenient daily dosage regimen that can be adjusted according to the severity of the pain. Solid forms include, for example, powders, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules. For example, in powders, the carrier is generally a finely divided solid that is a mixture with the finely divided active ingredient. For example, in tablets, the active ingredient is generally mixed with a carrier having the necessary binding capacity in appropriate proportions and compacted in the shape and size desired. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low-melting wax, cocoa butter, and the like. The pharmaceutical may also contain ingredients such as excipients, stabilizers, preservatives, binders, disintegrants, hydrocarbons, fatty acids, alcohols, esters, pH adjusters, and preservatives as needed, within limits that do not impair the desired effects.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way. In the following examples, the unit % used to indicate the amount of each component added means % by mass.

Examples of the present invention will be described below. The samples used in the experiments are as follows:
Proteoglycan: Proteoglycan F (proteoglycan content 20.0% or more, Ichimaru Falcos). In the following experiments, proteoglycan F was used to match the specified amount of proteoglycan.
Normal human adult skin fibroblasts: fibroblasts derived from neonatal foreskin skin, Kurabo, KF-4009
D-MEM medium: Fujifilm Wako Pure Chemical Industries, 041-29775, low glucose (contains L-glutamine and phenol red)

[Experiment 1]: Evaluation of the ability to promote elastin production This evaluation was carried out by confirming whether the addition of proteoglycan promotes elastin production in normal adult human skin fibroblasts.

(Experimental Method)
1. Cell Preparation D-MEM medium (Fujifilm Wako Pure Chemical Industries, product number 187-02705) containing 5% FBS and normal human adult dermal fibroblasts were added. After addition, the medium was replaced with fresh medium and cultured at 5% CO ₂ and 37°C until the cells became 70-80% confluent. After culture, the cells were washed with PBS and the normal human adult dermal fibroblasts were detached from the flask using TrypLE ^™ Select Enzyme (1x), no phenol red (Gibco, product number: 12563011) and subcultured.

After this subculture, normal human adult dermal fibroblasts were seeded into a 24-well cell culture plate (containing D-MEM medium containing 5% FBS) at 4 x 10 ⁴ cells/well. The plate was cultured until 100% confluent, with the medium being replaced with fresh medium. After this culture, the medium in the plate was replaced with D-MEM medium containing 0.1% FBS. After this replacement, the cells were cultured for 24 hours under conditions of 5% CO ₂ and 37°C.

2. Addition of Sample to Cells After the 24-hour culture, the medium was replaced with the medium (culture conditions) shown in Table 1, and the cells were cultured under conditions of 5% CO ₂ and 37° C. for 24 hours.

3. Collection of culture medium and evaluation by ELISA After the culture in 2, the culture medium supernatant was collected. The amount of elastin in the supernatant was evaluated using a Human Elastin ELISA kit (CUSABIO, CSB-E09338h).

In Experiment 1, the control group was a group to which no proteoglycan was added (0 μg/mL). This experiment was conducted with three experimental groups: the control group, a group to which 10 μg/mL of proteoglycan was added (PG10), and a group to which 100 μg/mL of proteoglycan was added (PG100). Three samples (n=3) were prepared for each of the three experimental groups, and Experiment 1 was conducted. The experimental results (elastin production amount) of Experiment 1 are as follows:

[Experiment 2]: Evaluation of Collagen Production Promoting Ability This evaluation was carried out by confirming whether the addition of proteoglycan promotes collagen production in normal adult human skin fibroblasts.

(Experimental Method)
1. Cell Preparation D-MEM medium (Fujifilm Wako Pure Chemical Industries, product number 187-02705) containing 5% FBS and normal human adult dermal fibroblasts were added. After addition, the medium was replaced with fresh medium and cultured at 5% CO ₂ and 37°C until the cells became 70-80% confluent. After culture, the cells were washed with PBS and the normal human adult dermal fibroblasts were detached from the flask using TrypLE ^™ Select Enzyme (1x), no phenol red (Gibco, product number: 12563011) and subcultured.

After this subculture, normal human adult dermal fibroblasts were seeded into a 24-well cell culture plate (containing D-MEM medium containing 5% FBS) at 4 x 10 ⁴ cells/well. The plate was cultured until 100% confluent, with the medium being replaced with fresh medium. After this culture, the medium in the plate was replaced with D-MEM medium containing 0.1% FBS. After this replacement, the cells were cultured for 24 hours under conditions of 5% CO ₂ and 37°C.

2. Addition of Sample to Cells After the 24-hour culture, the medium was replaced with the medium (culture conditions) shown in Table 1, and the cells were cultured under conditions of 5% CO ₂ and 37° C. for 24 hours.

3. Recovery of culture medium and evaluation by ELISA After the culture in 2, the supernatant of the culture medium was recovered. The concentration of type III collagen in the supernatant was evaluated using a Procollagen type I C-peptide (PIP) EIA Kit (MK101, Cosmo Bio).

In Experiment 2, the control group was a group to which no proteoglycan was added (0 μg/mL). This experiment was conducted with three experimental groups: the control group, a group to which 10 μg/mL of proteoglycan was added (PG10), and a group to which 100 μg/mL of proteoglycan was added (PG100). Three samples (n=3) were prepared for each of the three experimental groups, and Experiment 1 was conducted using these samples. The results of Experiment 2 (elastin production amount) are as follows:

[Experiment 3]: Evaluation of skin condition improvement effect through human monitor test This monitor test was conducted from February 2021 to April 2021 on 90 healthy men and women (30 people per group, 3 groups) aged 30 to under 60 (average age 48.4 years) who were aware of sagging, dry, and/or rough skin.

(Test Method)
The groups shown in Table 3 were instructed to ingest one bottle of the test drink once a day between meals.

(Measurement of skin viscoelasticity)
The viscoelasticity of the skin of the subjects (left cheek) was measured using a viscoelasticity measuring device (Cutometer, MPA580), and the relative values were calculated compared to the values before intake for each of the R5 and R7 groups, which were set at 100. The elasticity parameters (R5, R7) are as follows:

R5: Net elasticity. Ratio of UR to UE. UR is the height of elastic elongation when relaxed. UE is the height of elastic elongation when sucked into the probe.
R7: Recovery elasticity, ratio of UR to UF, the instantaneous return rate at which elastic properties appear for pulled up skin.

The measurement results for the relative values of R5 and R7 are shown in Table 4 below. Compared to the placebo group, Groups 1 and 2 had better viscoelasticity.

(Measurement of transepidermal water loss)
The transepidermal water loss of the subjects' skin (left cheek) was measured using a Tewameter (TM300, Courage-Khazaka), and the relative value was calculated by comparing the value before intake for each group with 100. The measurement results are shown in Table 5. Compared to the placebo group, transepidermal water loss was lower in Groups 1 and 2.

(Measurement of skin moisture content)
The moisture content of the subjects' skin (left cheek) was measured using a Corneometer (CM825, Courage-Khazaka). The value before intake for each group was set at 100, and a relative value was calculated. The measurement results are shown in Table 6. Compared to the placebo group, Groups 1 and 2 had higher moisture content.

The above describes embodiments of the present invention (including examples) with reference to the drawings, but the specific configuration of the present invention is not limited to this, and design changes and other modifications that do not deviate from the gist of the present invention are also included within the scope of the present invention.

The present invention provides an agent for promoting elastin production that contains proteoglycan.

Claims (2)

An agent for promoting elastin production containing proteoglycan extracted from salmon nasal cartilage with an acetic acid solution . An agent for promoting elastin production and collagen production, which contains proteoglycan extracted from salmon nasal cartilage with an acetic acid solution .

End