JP2025035496A - Skin barrier function improver - Google Patents

Abstract

To find out a component having a skin barrier function ameliorating effect from natural product-derived components, and provide a skin barrier function improver having the component as an active ingredient.SOLUTION: As an active ingredient of a skin barrier function improver of the present invention, a combination of a yeast fermentation product of rice and one or more kinds selected from a group consisting of artemisia extract, aloe vera extract, Morinda citritolia juice, Coix seed extract, and citrus unshiu marcow extract is used. According to the present invention, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, loricrin mRNA expression promoter, kallikrein 5 mRNA expression promoter, kallikrein 7 mRNA expression promoter, serine peptidase inhibitory factor Kazal-type 5 (SPINK5) mRNA expression promoter, caspase 14 mRNA expression promoter, ZO-1 mRNA expression promoter, semaphorin 3A mRNA expression promoter, epidermal keratinocyte proliferation promoter, and sirtuin 1 mRNA expression promoter are further provided.SELECTED DRAWING: None

Description

The present invention relates to a skin barrier function improving agent, and in particular to a skin barrier function improving agent that contains a specific combination of naturally occurring ingredients as active ingredients.

The epidermis, which functions to mitigate external stimuli and control the loss of water and other body components, is composed of a four-layer structure, starting from the basal layer at the bottom, followed by the spinous layer, granular layer, and stratum corneum. Most of the cells in each layer are keratinocytes differentiated from the basal layer. Keratinocytes that divide and proliferate in the basal layer differentiate into keratinocytes as they pass through the spinous and granular layers, forming the stratum corneum, which is made up of keratin protein fibers with strong cross-linking, and are eventually shed from the stratum corneum as scum. This stratum corneum is the outermost shell of the skin and serves as a physical barrier against stimuli from the outside world. To provide this barrier function, the skin repeats a cycle (keratinization) from when keratinocytes are produced in the basal layer until they become scum and fall off, usually in a four-week cycle, to renew the epidermis.

However, when the skin is exposed to external factors such as ultraviolet rays, extremely dry air, excessive skin washing, smoking, or as a result of aging, the activity and proliferation ability of epidermal cells declines, and the turnover rate of the epidermis slows, causing differentiation failure such as thinning of the epidermis and thickening of the stratum corneum. As a result, the skin's moisturizing function and elasticity decline, abnormal peeling of the stratum corneum occurs, and the skin ages, causing changes such as wrinkles, dullness, loss of texture, and reduced elasticity.

Therefore, if the proliferation of keratinocytes can be promoted, skin turnover will be promoted and the skin's metabolic function will be restored, which is thought to improve skin aging such as fine wrinkles, dullness, and pigmentation. Extracts from Taiso (see Patent Document 1) and extracts from Tsuchigai Mo (see Patent Document 2) are known to have the effect of promoting the proliferation of epidermal keratinocytes.

The structure of the stratum corneum is likened to bricks and mortar, with 15 or so layers of keratinocytes stacked together by intercellular lipids, forming a strong barrier membrane. A strong structure called the cornified envelope (CE) is formed inside the cell membrane of keratinocytes, and moisture is maintained by the natural moisturizing factors, mainly composed of amino acids, contained within the cells. Meanwhile, the intercellular lipids of keratinocytes are mainly composed of about 50% ceramide, and are composed of amphipathic lipids such as cholesterol and fatty acids, and are characterized by a layered structure in which hydrophobic and hydrophilic parts are alternately repeated, a so-called lamellar structure.

The peripheral zone (CE) is formed when multiple CE component proteins, produced as epidermal keratinocytes differentiate, are cross-linked and insolubilized by the enzyme transglutaminase. Furthermore, ceramides and other molecules are covalently bonded to form a hydrophobic structure, providing a base for the lamellar structure of intercellular lipids in part of the CE, thereby forming the basis for the stratum corneum barrier function.

Involucrin is known as one of the CE component proteins expressed in the early stages of CE formation, and loricrin is known as one of the main component proteins of CE. It is believed that promoting the production of involucrin and loricrin improves the stratum corneum barrier function and can prevent, treat or ameliorate skin aging symptoms such as rough skin and dry skin, and dry skin diseases such as atopic dermatitis, psoriasis, ichthyosis and xerosis. 2-ketoglutaric acid and the like have been known to promote involucrin mRNA expression (see Patent Document 3), and cordyceps extracts and the like have been known to promote loricrin production (see Patent Document 4).

Filaggrin is a component of the skin and is thought to be involved in the barrier function of the skin, preventing the invasion of allergens, toxins, and infectious organisms. It is known that a decrease in the function of filaggrin due to gene mutations or other factors is associated with the risk of developing atopic diseases, including atopic dermatitis (eczema, skin inflammation, skin itchiness, etc.), allergies, asthma, etc., and in more severe cases, can lead to skin diseases such as ichthyosis vulgaris (see Non-Patent Document 1).

On the other hand, amino acids, which are the main components of natural moisturizing factors (NMF), are produced by the decomposition of filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilaggrin in epidermal keratinocytes present in the granular layer just below the stratum corneum. It is then immediately phosphorylated and accumulated in keratohyalin granules, dephosphorylated and hydrolyzed to filaggrin, which migrates to the stratum corneum, increases the aggregation efficiency of keratin filaments, and is known to be involved in the internal construction of keratinocytes (see Non-Patent Document 2). In recent years, it has been known that this filaggrin is very important and essential for maintaining moisture in the skin, and that the synthetic ability of filaggrin decreases due to conditions such as dryness, resulting in a decrease in the amount of amino acids in the stratum corneum (see Non-Patent Document 3).

Therefore, it is believed that by promoting the expression of filaggrin (profilaggrin) in epidermal keratinocytes, it is possible to prevent, treat or improve atopic diseases including atopic dermatitis (eczema, skin inflammation, skin itchiness, etc.), allergies, asthma, etc. In addition, it is expected that by promoting the expression of filaggrin and thereby increasing the amount of amino acids in the stratum corneum, it is possible to essentially improve the moisture environment of the stratum corneum. Extracts of Gaiyo (Patent Document 5) and the like are known to have the effect of promoting filaggrin expression.

Caspase is an abbreviation of cysteine-aspartic-acid-protease, and is a type of cysteine protease. To date, 14 types of caspases have been discovered, from caspase-1 to caspase-14. Of these, caspase-14 is known to have the ability to break down filaggrin, a basic protein present in the epidermis, to produce natural moisturizing factor (NMF).

Therefore, it is believed that by promoting the expression of caspase-14, the production of natural moisturizing factors (NMFs) can be promoted, and the decline in the skin's barrier function and water retention function can be prevented or improved. In addition, it is believed that by preventing or improving the decline in the skin's barrier function through the promotion of caspase-14 expression, it is possible to prevent, treat or improve skin diseases such as atopic dermatitis and ichthyosis, which are believed to be caused in part by a decline in the skin's barrier function. Yeast extracts (Patent Document 6) and the like are known to have the effect of promoting caspase-14 expression.

In healthy skin, keratinocytes (epidermal keratinocytes) differentiate to continually produce keratinocytes, and at the same time, old, unnecessary keratinocytes become dandruff and are shed, maintaining the stratum corneum at a constant thickness through repeated skin turnover. However, it is known that the barrier function of the skin can be weakened by external factors such as dryness, ultraviolet rays, and chemicals, as well as internal factors such as oxidative stress and aging, and that these factors can cause abnormalities in skin turnover.

The stratum corneum is formed by adhesion between keratinocytes via adhesive structures called corneodesmosomes. Corneodesmosomes are composed of multiple proteins including desmoglein 1 (DSG1) and desmocollin 1 (DSC1). Corneodesmosomes are known to be cleaved by proteases such as kallikrein 5 (KLK5), a trypsin-like serine protease, and kallikrein 7 (KLK7), a chymotrypsin-like serine protease (see Non-Patent Documents 4 and 5).

In healthy skin, normal stratum corneum delamination occurs when corneodesmosomes are degraded by the serine protease. In normal stratum corneum delamination, the delamination is rarely noticeable to the naked eye. However, when the activity of the serine protease decreases due to skin problems such as rough skin or aging, the delamination of the corneodesmosomes is delayed, causing the keratinocytes to accumulate and the keratinocytes to delaminate smoothly, and are recognized as scales because they are exfoliated in clumps (see Non-Patent Document 6). Many skin diseases accompanied by abnormalities in the stratum corneum delamination function have been reported, such as senile xerosis, dandruff, ichthyosis vulgaris, sex-linked ichthyosis, and psoriasis (see Non-Patent Documents 7 to 9).

The serine protease activity in the epidermis is tightly regulated by serine protease inhibitors such as lymphoepithelial Kazal-type inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 (SPINK5) gene. In patients with a severe congenital ichthyosa-like erythroderma known as Netherton syndrome, the SPINK5 gene has been identified as the defective gene, and the pathology is known to be one in which the activity of serine proteases such as KLK7 is increased, resulting in enhanced protein degradation.

Thus, promoting the production of KLK5 and KLK7 is important for promoting skin turnover. However, excessive production of KLK5 and KLK7 may lead to abnormal desquamation. Therefore, in normal stratum corneum desquamation, serine proteases such as KLK5 and KLK7 act on corneodesmosomes to degrade them, promoting stratum corneum desquamation, and at the same time, serine protease inhibitors such as LEKTI regulate and balance the activity of the serine proteases, thereby maintaining the homeostasis of protease activity in the skin.

Therefore, by adjusting the balance between the activity of serine proteases such as KLK5 and KLK7 in the skin and the activity of serine proteases such as LEKTI, it is expected that the homeostasis of protease activity in the skin can be maintained, improving the barrier function of the skin and also improving skin turnover.

In addition, it was previously believed that only the stratum corneum was responsible for the barrier function of the skin, but since the barrier function of the skin collapses when the constituent proteins of tight junctions (hereinafter sometimes abbreviated as TJ) present in the granular layer of the epidermis are genetically deleted, TJs are now believed to play an important role in the barrier function of the skin (see Non-Patent Document 10). TJs are binding devices that not only bring adjacent cells into close contact with each other, but also control the permeation of substances by sealing the gaps between cells. Proteins that make up this TJ include claudins, occludin, ZO-1, and ZO-2, and these proteins are thought to form the skeleton of the TJ strand and control the barrier function of the TJ (see Non-Patent Document 11). From the above, if the expression of claudins, occludin, ZO-1, and ZO-2 is reduced for some reason, structural destruction of TJs occurs, and they no longer function as a barrier to permeation of substances, which is expected to cause skin symptoms such as dry skin, rough skin, atopic dermatitis, and various infectious diseases.

Therefore, it is believed that by promoting the production of ZO-1 in the epidermis and encouraging TJ formation in epidermal keratinocytes, the barrier function and moisture retention function of the skin can be enhanced, and the above-mentioned skin symptoms can be prevented or improved. Based on this idea, natural products such as coptis jasmine extract (see Patent Document 7) and spruce extract (see Patent Document 8) have been disclosed as substances that improve skin barrier function through the promotion of TJ formation.

Itching can occur for a variety of reasons, including dryness, irritation from foreign objects, and atopic dermatitis, and is known to involve chemical mediators such as histamine, and antihistamines and other drugs are used to suppress these chemical mediators. However, there are also cases of itching that do not respond to antihistamines (intractable itching), and it is believed that there are also mechanisms of itching that do not involve histamine and other drugs.

In normal skin, the nerve fibers (C fibers) that transmit itching and pain terminate at the dermal-epidermal junction, but in recent years, it has been found that in skin with atopic dermatitis and dry skin, the nerve fibers extend into the epidermis, particularly to just below the stratum corneum. This condition is thought to be deeply involved in the symptoms of so-called sensitive skin, in which nerve fibers are easily activated by external stimuli such as mechanical, chemical, and physical stimuli, and which are easily sensitive to itching and pain.

It is known that the intraepidermal extension of nerve fibers involves the nerve growth factor (NGF), which is a nerve extension factor, and semaphorin 3A (Sema3A), which is a nerve repulsive factor. In normal skin, Sema3A is dominant over NGF, so nerve fibers cannot extend into the epidermis, but in atopic dermatitis, dry skin, etc., NGF is dominant over Sema3A, and it is believed that this is why nerve fibers penetrate into the epidermis (see Non-Patent Document 12). It has also been reported that intradermal administration of Sema3A protein to dermatitis model mice improves skin rash and reduces pruritus (see Patent Document 9).
Therefore, if the nerve repulsion action of Sema3A can be promoted, it is thought that pain and itching in atopic dermatitis, dry skin, sensitive skin, etc. can be suppressed.

Furthermore, it has recently been revealed that Sema3A increases bone mass (bone-protecting effect) by simultaneously inhibiting osteoclast differentiation and promoting osteoblast differentiation, and it is expected that Sema3A will be used as a drug that combines the dual aspects of inhibiting bone resorption and promoting bone formation (Non-Patent Document 13).

Conventionally, Scutellaria root extract and other ingredients have been proposed as ingredients that promote the expression of Sema3A (see Patent Document 10).

SIRT1 is a protein that belongs to a family called sirtuin, and is known to exist in the cell nucleus and cytoplasm and function as an NAD-dependent protein deacetylase. In recent years, it has been revealed that SIRT1 plays an important role in suppressing cellular aging, and is particularly expressed in skin tissue and is involved in suppressing skin aging (see Non-Patent Document 14). In addition to cellular aging, SIRT1 has been shown to have various functions such as diabetes improvement, cardiovascular protection, renal disease improvement, inhibition of inflammatory cytokine production, and neuroprotection. Therefore, if SIRT1 can be activated, it is thought to be useful for preventing, treating, or improving various diseases such as aging such as skin aging, diabetes, cardiovascular disease, nervous system disease, and inflammatory disease.

JP 2006-316028 A JP 2006-056854 A JP 2021-127344 A JP 2014-040398 A JP 2012-219047 A JP 2022-144014 A JP 2007-176830 A JP 2007-176835 A JP 2008-297243 A JP 2016-193863 A

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The present invention aims to find ingredients derived from natural products that have the effect of improving skin barrier function, and to provide a skin barrier function improver that contains such ingredients as active ingredients.

In order to solve the above problems, the skin barrier function improving agent of the present invention is characterized in that it contains as active ingredients a combination of yeast fermented rice and one or more selected from the group consisting of chinese ginger extract, aloe vera extract, noni juice, coix seed extract, and satsuma mandarin extract.
In addition, the filaggrin mRNA expression promoter, involucrin mRNA expression promoter, loricrin mRNA expression promoter, kallikrein 5 mRNA expression promoter, kallikrein 7 mRNA expression promoter, serine peptidase inhibitor Kazal type 5 (SPINK5) mRNA expression promoter, caspase 14 mRNA expression promoter, ZO-1 mRNA expression promoter, semaphorin 3A mRNA expression promoter, epidermal keratinocyte proliferation promoter, and sirtuin 1 mRNA expression promoter of the present invention are characterized in that they contain a further specific combination of the above combinations as active ingredients.
In the above invention, the yeast fermentation product of rice is preferably a Saccharomyces verona fermentation product.

According to the present invention, by using a combination of rice yeast fermentation product and specific ingredients as active ingredients, it is possible to provide a skin barrier function improving agent, a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, a loricrin mRNA expression promoter, a kallikrein 5 mRNA expression promoter, a kallikrein 7 mRNA expression promoter, a serine peptidase inhibitor Kazal type 5 (SPINK5) mRNA expression promoter, a caspase 14 mRNA expression promoter, a ZO-1 mRNA expression promoter, a semaphorin 3A mRNA expression promoter, an epidermal keratinocyte proliferation promoter, and a sirtuin 1 mRNA expression promoter, all of which have excellent effects.

Hereinafter, an embodiment of the present invention will be described.
The skin barrier function improving agent of this embodiment contains as an active ingredient a combination of yeast fermented rice and one or more selected from the group consisting of chinese ginger extract, aloe vera extract, noni juice, coix seed extract, and satsuma mandarin extract.

[Rice fermented with yeast]
In this embodiment, a fermented product obtained by fermenting rice with yeast (hereinafter referred to as "yeast fermented rice product") is used as one of the active ingredients.

In the yeast-fermented rice product according to this embodiment, the rice used as the fermentation raw material is the fruit of rice (scientific name: Oryza sativa), a plant of the Poaceae family. Rice is cultivated throughout Japan and is easily available from these regions. There are no particular limitations on the fermentation raw material, and it can be appropriately selected depending on the purpose. Examples of the fermentation raw material include unhulled rice, brown rice, polished rice, and mixtures thereof, and among these, polished rice is preferred.

The yeast fermented rice product according to this embodiment is obtained by fermenting rice with yeast. The fermentation process can be carried out, for example, by dispersing rice in water and inoculating it with yeast that will carry out fermentation.

In order to increase the fermentation efficiency, it is preferable to decompose the starch contained in the rice and/or the cell walls of the rice before inoculating the yeast.

Examples of starch decomposition treatment include enzyme treatment and inoculation with koji mold, with enzyme treatment being particularly preferred. Examples of starch decomposition enzymes include amylases such as α-amylase, β-amylase, glucoamylase, and isoamylase, with α-amylase and glucoamylase being particularly preferred from the viewpoint of increasing fermentation efficiency, with glucoamylase being even more preferred. On the other hand, cell wall decomposition treatment is preferably performed using enzymes such as cellulase and xylanase.

The resulting treated liquid may be subjected to the desired means such as cooling, heating, pH adjustment, or sterilization to inactivate the enzymes and koji mold, or it may be inoculated with yeast for fermentation as is.

Examples of yeasts that perform fermentation include those of the genus Saccharomyces such as Saccharomyces cerevisiae (baker's yeast) and Saccharomyces veronae; those of the genus Schizosaccharomyces such as Schizosaccharomyces pombe; those of the genus Kluyveromyces such as Kluyveromyces thermotolerans; those of the genus Zygosaccharomyces such as Zygosaccharomyces thermotolerans; those of the genus Lachancea such as Lachancea thermotolerans; those of the genus Candida utilis; Preferred examples include the genus Candida, such as Candida utilis and Torula yeast.
In the present embodiment, these yeasts can be used alone or in combination of two or more. When two or more types are used in combination, fermentation may be performed simultaneously with two or more types of yeast, or primary fermentation with koji mold may be performed first, followed by secondary fermentation with yeast.

Of these, yeasts used in this embodiment are particularly preferably yeasts of the genus Saccharomyces or Lachancea, from the viewpoint of safety when used in skin cosmetics or foods and beverages, and from the viewpoint of the effects of the fermented rice product, and Saccharomyces veronae (also known as Lachancea thermotolerans) is even more preferable. Here, Saccharomyces veronae (also known as Lachancea thermotolerans) differs from other yeasts such as Saccharomyces cerevisiae in that it does not substantially carry out alcoholic fermentation.

The fermentation process using the above yeast can be carried out, for example, by placing the fermentation raw material and the above yeast in a fermenter and treating at a temperature range of 20 to 40°C, preferably 25 to 35°C, for 12 to 72 hours, preferably 18 to 48 hours. Furthermore, if the pH of the reaction liquid is adjusted to 5.0 to 7.0, preferably 6.0 to 7.0, at the start of the fermentation process, the fermentation process will proceed smoothly. The fermentation process is terminated by subjecting the fermentation liquid to a desired means such as cooling, heat sterilization, or filtration to inactivate the fermenting bacteria.

The fermentation liquid thus obtained may be used as the fermented product as it is, or may be appropriately diluted or concentrated before use as the fermented product. Furthermore, the concentrate may be further dried or subjected to crude purification.
In this specification, unless otherwise specified, the term "yeast fermented rice product" includes a fermentation liquid obtained by fermenting rice with yeast, a diluted liquid or concentrated liquid of the fermentation liquid, a dried product obtained by drying the fermentation liquid, or a crude or purified product thereof. Hereinafter, the yeast fermented rice product may be simply referred to as "rice fermented product".

The yeast fermented rice product obtained in the above manner can exert an excellent effect of improving the skin barrier function, and can be used alone as an active ingredient in a skin barrier function improving agent, but when used in combination with the below-mentioned mussel extract, aloe vera extract, noni fruit juice, coix seed extract, and satsuma mandarin extract, it exerts an especially excellent effect of improving the skin barrier function.

[Ginger extract, aloe vera extract, coix seed extract, satsuma mandarin extract]
In this embodiment, the extraction raw materials used to produce the Gaiyo extract, the Aloe vera extract, the Coix seed extract, and the Satsuma mandarin extract are Gaiyo (herbal medicine name), Aloe vera (scientific name: Aloe vera), Coix seed (herbal medicine name) and Satsuma mandarin (scientific name: Citrus unshiu Marcow), respectively.

Gaiyo (herbal medicine name) is the leaf of mugwort (scientific name: Artemisia princeps Pampanini), a perennial herb belonging to the Asteraceae family that is distributed throughout Japan and can be easily obtained from these areas. Gaiyo has traditionally been used to stop bleeding in the gynecological area, prevent miscarriage, and prevent abdominal pain caused by cold symptoms.

Aloe vera is a succulent plant belonging to the genus Aloe in the family Liliaceae. It is cultivated in Africa, India, etc., and can be easily obtained from these regions. The constituent parts of Aloe vera that can be used as the extraction raw material can be appropriately selected depending on the purpose, and examples include leaves, mesophyll, stems, flowers, fruits, peels, stalks, seeds, roots, mixtures of these, or the whole plant, or juice (squeezed liquid) obtained by squeezing these, or concentrates or dried products thereof, etc., but it is preferable to use concentrates or dried products of the squeezed liquid of the mesophyll.

Job's tears (coix lachrymal-jobi L.) is the seed of Job's tears. Job's tears is a plant native to China and Indochina, and is an annual plant of the Poaceae family that is cultivated in the warm southwestern part of Japan, and can be easily obtained from these regions. Job's tears has traditionally been used for irrigation, draining dampness, clearing heat, draining pus, removing paralysis, strengthening the spleen and relieving pain.

Satsuma mandarin (Citrus unshiu Marcow) is a plant of the Rutaceae family, which is mainly cultivated in coastal areas south of the Kanto region and can be easily obtained from these areas. The constituent parts of Satsuma mandarin that can be used as the extraction raw material can be appropriately selected depending on the purpose, and examples include leaves, stems, branches, flowers, buds, fruit, peel, pit, seeds, roots, and mixtures of these, with the peel being preferred.

Extracts from the above plants can be obtained by drying the extraction raw material, pulverizing it using a crusher or as is, and then subjecting it to extraction using an extraction solvent. Drying can be done in the sun, or using a commonly used dryer. The raw material can also be used after pre-treatment such as degreasing with a non-polar solvent such as hexane. Pre-treatment such as degreasing allows for efficient extraction using a polar solvent.

As the extraction solvent, it is preferable to use a polar solvent, such as water or a hydrophilic organic solvent, which is preferably used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent.

Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has been subjected to various treatments. Treatments that can be applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, water that can be used as an extraction solvent in this embodiment also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered physiological saline, etc.

Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols with 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; polyhydric alcohols with 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin; and lower aliphatic ketones with 3 to 4 carbon atoms, such as acetone and methyl ethyl ketone.

When a mixture of two or more polar solvents is used as an extraction solvent, the mixture ratio is arbitrary and can be adjusted appropriately. For example, when a mixture of water and a hydrophilic organic solvent is used as an extraction solvent, the mixture ratio can be arbitrarily mixed and used, that is, between more than 0:100 and less than 100:0 (volume ratio, hereinafter the same), and can be adjusted appropriately.
For example, when a mixture of water and a lower aliphatic alcohol is used as an extracting solvent, the ratio (volume %) of the lower aliphatic alcohol in the mixture can be 10% or more, or even 30% or more, or the ratio (volume %) of the lower aliphatic alcohol in the mixture can be 90% or less, or even 80% or less. When a mixture of water and a polyhydric alcohol is used, the ratio (volume %) of the polyhydric alcohol in the mixture can be 20% or more, or 90% or less, and when a mixture of water and a lower aliphatic ketone is used, the ratio (volume %) of the lower aliphatic ketone in the mixture can be 10% or more, or 80% or less.

The extraction process is not particularly limited as long as the soluble components contained in the extraction raw material can be dissolved in the extraction solvent, and can be carried out according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent in an amount 5 to 15 times (mass ratio) the amount of the extraction raw material, and the soluble components are extracted at room temperature or under reflux heating, and then the extraction residue is removed by filtration to obtain an extract. The solvent is distilled off from the obtained extract to obtain a paste-like concentrate, and the concentrate is further dried to obtain a dry product.
In addition, the "extract" in this embodiment includes an extract obtained using the above-mentioned plant as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or any of these crude or purified products.

The thus obtained ginger extract, aloe vera extract, coix seed extract and satsuma mandarin extract can exert excellent effects such as improving the skin barrier function, and can be used alone as an active ingredient in a skin barrier function improving agent, but when used in combination with the aforementioned rice yeast fermentation product, they exert particularly excellent effects such as improving the skin barrier function.

[Noni juice]
In this embodiment, the raw material for obtaining noni juice is noni (scientific name: Morinda citrifolia).

Noni (Morinda citrifolia), known as Yaeyama Aoki in Japanese, is a plant of the Rubiaceae family, and is distributed in areas such as Okinawa to the Ogasawara Islands, Taiwan to China, and the coastal areas of India, and is easily available from these areas. The part used in this embodiment is the fruit.
Noni juice can be obtained by squeezing the juice from the noni fruit using a juicer or similar device.

The obtained noni juice may be used as it is, or may be concentrated or dried to be used as a concentrate or a dried product. When dried, a freeze-dried product is preferred.
In addition, the obtained noni fruit juice may be further subjected to an extraction process and used as an extract. The extraction solvent and extraction process described above for the plant extract can be suitably applied to the extraction of noni fruit juice.

The noni juice obtained in the manner described above can exert an excellent effect of improving the skin barrier function, and can be used alone as an active ingredient in a skin barrier function improving agent, but when used in combination with the aforementioned rice yeast fermentation product, it exerts an especially excellent effect of improving the skin barrier function, etc.

[Skin barrier function improver, FLG mRNA expression promoter, etc.]
The yeast fermented rice product obtained as described above can exhibit excellent skin barrier function improving effects, etc., and can be used alone as an active ingredient of a skin barrier function improving agent, etc. In addition, the chinese ginger extract, aloe vera extract, noni fruit juice, coix seed extract, and satsuma mandarin extract (hereinafter, these may be abbreviated as "five ingredients") can also exhibit excellent skin barrier function improving effects, etc., and can be used alone as an active ingredient of a skin barrier function improving agent, etc.
However, in this embodiment, by using the yeast fermented rice in combination with the above five components, the skin barrier function improving effect can be dramatically improved.

The skin barrier function improving effect of the combination of the yeast fermented rice and the above five ingredients is exerted based on one or more actions selected from the group consisting of, for example, filaggrin (FLG) mRNA expression promoting action, involucrin (IVL) mRNA expression promoting action, loricrin (LOR) mRNA expression promoting action, kallikrein 5 (KLK5) mRNA expression promoting action, kallikrein 7 (KLK7) mRNA expression promoting action, serine peptidase inhibitor Kazal type 5 (SPINK5) mRNA expression promoting action, caspase 14 mRNA expression promoting action, ZO-1 mRNA expression promoting action, semaphorin 3A (Sema3A) mRNA expression promoting action, and epidermal keratinocyte proliferation promoting action. However, the skin barrier function improving effect of the combination of the yeast fermented rice and the above five ingredients is not limited to the skin barrier function improving action exerted based on the above actions.

Specific combinations of the yeast fermented rice and the above five ingredients may also be used as active ingredients of FLG mRNA expression promoters, IVL mRNA expression promoters, LOR mRNA expression promoters, KLK5 mRNA expression promoters, KLK7 mRNA expression promoters, SPINK5 mRNA expression promoters, caspase 14 mRNA expression promoters, ZO-1 mRNA expression promoters, Sema3A mRNA expression promoters, or epidermal keratinocyte proliferation promoters through the action of each combination.

The above specific combination will now be described in detail.
A combination of yeast fermented rice and a Gaiyo extract can be used as an active ingredient of an FLG mRNA expression promoter.
A combination of rice fermented with yeast and a Gaiyo extract and/or a Coix seed extract can be used as an active ingredient of an IVL mRNA expression promoter.
A combination of yeast fermented rice and one or more selected from the group consisting of chinese ginger extract, aloe vera extract, noni juice, and coix seed extract can be used as an active ingredient of an LOR mRNA expression promoter.

A combination of rice fermented with yeast and a Gaiyo extract can be used as an active ingredient of a KLK5 mRNA expression promoter.
A combination of yeast fermented rice, and Gaiyo extract and/or Noni juice can be used as an active ingredient of a KLK7 mRNA expression promoter.
A combination of yeast fermented rice and one or more selected from the group consisting of chinese ginger extract, noni juice, coix seed extract, and satsuma mandarin extract can be used as an active ingredient of a SPINK5 mRNA expression promoter.

A combination of yeast fermented rice, and Gaiyo extract and/or Noni juice can be used as an active ingredient of an agent for promoting caspase 14 mRNA expression.
A combination of yeast fermented rice and one or more selected from the group consisting of chinese ginger extract, aloe vera extract, coix seed extract, and satsuma mandarin extract can be used as an active ingredient of a ZO-1 mRNA expression promoter.

A combination of yeast fermented rice and one or more selected from the group consisting of cockle extract, aloe vera extract, noni juice, and satsuma mandarin extract can be used as an active ingredient of a Sema3A mRNA expression promoter.
A combination of yeast fermented rice and one or more selected from the group consisting of chinese ginger extract, aloe vera extract, noni juice, and satsuma mandarin extract can be used as an active ingredient of an epidermal keratinocyte proliferation promoter.

Furthermore, the combination of yeast fermented rice and extract of Gaiyo has an effect of promoting the expression of Sirtuin 1 (SIRT1) mRNA, and therefore can be used as an active ingredient of an agent for promoting the expression of SIRT1 mRNA.
Hereinafter, these agents (agents other than skin barrier function improving agents) may be collectively referred to as "FLG mRNA expression promoting agents, etc.", and their actions and uses may similarly be collectively referred to as "FLG mRNA expression promoting action, etc." and "Uses of promoting FLG mRNA expression, etc."

According to this embodiment, a combination of rice yeast fermentation product and the above five components can be used to produce a skin barrier function improvement agent, and the above specific combination can be used to produce an FLG mRNA expression promoter, etc.
The skin barrier function improving agent and FLG mRNA expression promoting agent of the present embodiment can be used in a wide range of applications, such as medicines, quasi-drugs, cosmetics, and oral compositions.

Of the five ingredients, chinese ginger extract, aloe vera extract, noni juice, coix seed extract, and satsuma mandarin extract, one of each may be combined with the rice fermentation product, or two or more of the five ingredients may be combined with the rice fermentation product.

In the skin barrier function improving agent and FLG mRNA expression promoter of this embodiment, the blending ratio of the rice yeast fermentation product to the above five components can be set appropriately depending on their activity, but for example, the total of the above five components can be 2 parts by mass or more, 5 parts by mass or more, 10 parts by mass or more, or 20 parts by mass or more per 100 parts by mass of the rice yeast fermentation product (solid content equivalent, the same applies below). Also, the total of the above five components can be 1000 parts by mass or less, 500 parts by mass or less, 150 parts by mass or less, or 100 parts by mass or less per 100 parts by mass of the rice yeast fermentation product.

The skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment may consist of only a combination of the yeast fermented product of rice and the above five components, or may be a formulation of the combination of the yeast fermented product of rice and the above five components.

The skin barrier function improving agent and FLG mRNA expression promoting agent of the present embodiment can be formulated into any dosage form such as powder, granules, tablets, liquid, etc., using pharma- ceutically acceptable carriers such as dextrin and cyclodextrin, and any other auxiliary agent, according to a conventional method. In this case, examples of auxiliary agents that can be used include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, and odorants. The skin barrier function improving agent and FLG mRNA expression promoting agent can be used by blending them with other compositions (e.g., skin cosmetics, oral compositions, etc.), and can also be used as ointments, external liquids, patches, etc.

When the skin barrier function improving agent and FLG mRNA expression promoter of this embodiment are formulated, the content of the combination of the yeast fermented rice product and the above five components is not particularly limited and can be set appropriately depending on the purpose.

The skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment can be used as active ingredients by blending other natural extracts having a skin barrier function improving effect or FLG mRNA expression promoting effect, etc., together with the combination of the rice yeast fermentation product and the above five components, as necessary.

Methods of administering the skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment to patients include transdermal administration and oral administration, and a method suitable for prevention, treatment, etc. may be appropriately selected depending on the type of disease. In addition, the dosage of the skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual differences between patients, administration method, administration period, etc.

The skin barrier function improving agent of this embodiment can improve the barrier function of the skin and can also improve the moisture retention function of the skin through one or more actions selected from the group consisting of FLG mRNA expression promoting action, IVL mRNA expression promoting action, LOR mRNA expression promoting action, KLK5 mRNA expression promoting action, KLK7 mRNA expression promoting action, SPINK5 mRNA expression promoting action, caspase 14 mRNA expression promoting action, ZO-1 mRNA expression promoting action, Sema3A mRNA expression promoting action, and epidermal keratinocyte proliferation promoting action, which are possessed by the combination of the active ingredient, yeast fermentation product of rice, and the above five ingredients. The improvement of the barrier function and moisture retention function of the skin is effective in preventing, treating, or improving not only rough skin and dry skin, but also dry skin diseases (e.g., atopic dermatitis, psoriasis, ichthyosis, dandruff, etc.) and various infectious diseases transmitted through the skin.

The skin barrier function improving agent, KLK5 mRNA expression promoter, KLK7 mRNA expression promoter, SPINK5 mRNA expression promoter, epidermal keratinocyte proliferation promoter, and SIRT1 mRNA expression promoter of this embodiment can improve epidermal turnover through their KLK5 mRNA expression promoting effect, KLK7 mRNA expression promoting effect, SPINK5 mRNA expression promoting effect, epidermal keratinocyte proliferation promoting effect, or SIRT1 mRNA expression promoting effect.
In addition, the skin barrier function improving agent, KLK5 mRNA expression promoter, KLK7 mRNA expression promoter, and SPINK5 mRNA expression promoter of this embodiment can normalize stratum corneum peeling through their KLK5 mRNA expression promoting effect, KLK7 mRNA expression promoting effect, or SPINK5 mRNA expression promoting effect.
The skin barrier function improving agent or ZO-1 mRNA expression promoting agent of this embodiment can promote the formation of tight junctions through its ZO-1 mRNA expression promoting action.

The Sema3A mRNA expression promoter of this embodiment promotes the expression of Sema3A mRNA in the skin (particularly in the epidermis) through its Sema3A mRNA expression promoting action, thereby suppressing the intraepidermal extension of nerve fibers. The suppression of intraepidermal extension of nerve fibers is effective in suppressing skin itching and pain (particularly itching and pain that are not dependent on chemical mediators such as histamine), for example, itching and pain in atopic dermatitis, dry skin, sensitive skin, etc. In addition, the Sema3A mRNA expression promoter of this embodiment suppresses osteoclast differentiation and promotes osteoblast differentiation through its Sema3A mRNA expression promoting action, and can be used for increasing bone mass (bone protection).
The epidermal keratinocyte proliferation promoter of the present embodiment, through its epidermal keratinocyte proliferation promoting action, improves the barrier function and moisture retention function of the skin, improves epidermal turnover, and also restores the metabolic function of the skin, such as shedding of keratinocytes with abnormal accumulation of melanin from the stratum corneum, thereby preventing and improving symptoms such as dullness and pigmentation of the skin. It can also be used in applications such as regenerative medicine that require the promotion of epidermal keratinocyte proliferation.
The SIRT1 mRNA expression promoter of this embodiment, by virtue of its SIRT1 mRNA expression promoting effect, can be used for the prevention, treatment, or amelioration of various diseases such as metabolic diseases, inflammatory diseases, cellular aging, diabetes, cardiovascular diseases, renal diseases, and neurological diseases, as well as for various phenomena involving Sirtuin 1, such as anti-aging and prolongation of lifespan.

However, in addition to the uses described above, the skin barrier function improving agent, FLG mRNA expression promoting agent, IVL mRNA expression promoting agent, LOR mRNA expression promoting agent, KLK5 mRNA expression promoting agent, KLK7 mRNA expression promoting agent, SPINK5 mRNA expression promoting agent, caspase 14 mRNA expression promoting agent, ZO-1 mRNA expression promoting agent, Sema3A mRNA expression promoting agent, epidermal keratinocyte proliferation promoting agent, and SIRT1 mRNA expression promoting agent of this embodiment each have the following effects: improving skin barrier function, promoting FLG mRNA expression, promoting IVL mRNA expression, promoting LOR mRNA expression, promoting KLK5 mRNA expression, promoting KLK7 mRNA expression, promoting SPINK5 mRNA expression, promoting caspase 14 mRNA expression, promoting ZO-1 mRNA expression, promoting Sema3A It can be used for all applications where it is meaningful to exert the effects of promoting mRNA expression, promoting epidermal keratinocyte proliferation, and promoting SIRT1 mRNA expression.

The skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment each have an excellent skin barrier function improving effect and FLG mRNA expression promoting effect, and are therefore suitable for incorporation into, for example, a skin topical preparation. In this case, the combination of the rice yeast fermentation product and the above five components may be incorporated as is, or the combination of the rice yeast fermentation product and the above five components may be formulated and then incorporated. Here, there is no restriction on the classification of the skin topical preparation, and in addition to the skin cosmetics described below, it broadly includes quasi-drugs and pharmaceuticals that are used transdermally.

[Incorporation into skin cosmetics]
The combination of the rice yeast fermentation product and the above five components is suitable for incorporation into skin cosmetics because it has excellent effects of improving skin barrier function, promoting FLG mRNA expression, etc. In this case, the combination of the rice yeast fermentation product and the above five components may be incorporated as is, or the combination of the rice yeast fermentation product and the above five components may be formulated and then incorporated.

By blending a combination of the yeast fermented rice and the above five ingredients, a skin cosmetic suitable for improving skin barrier function can be obtained. In addition, by blending the above specific combinations, a skin cosmetic suitable for promoting FLG mRNA expression, promoting IVL mRNA expression, promoting LOR mRNA expression, promoting KLK5 mRNA expression, promoting KLK7 mRNA expression, promoting SPINK5 mRNA expression, promoting caspase 14 mRNA expression, promoting ZO-1 mRNA expression, promoting Sema3A mRNA expression, promoting epidermal keratinocyte proliferation, or promoting SIRT1 mRNA expression can be obtained. In other words, blending into a skin topical preparation is one of the suitable uses for skin barrier function improving agents, FLG mRNA expression promoters, etc.

There are no particular limitations on the types of skin cosmetics that can be formulated with a combination of the yeast fermented rice and the above five ingredients, and examples of skin cosmetics include ointments, creams, milky lotions, skin toners, lotions, gels, beauty oils, packs, foundations, shampoos, and rinses.

When the combination of the yeast fermented rice product and the above five components is blended into a skin cosmetic, the blending amount can be adjusted as appropriate depending on the type of skin cosmetic, but a suitable blending ratio is approximately 0.0005 to 0.3 mass% converted to the solid content of the combination of the yeast fermented rice product and the above five components, and a particularly suitable blending ratio is approximately 0.001 to 0.3 mass%.

The skin cosmetic of this embodiment can be used in combination with main agents, auxiliaries, or other ingredients used in the manufacture of ordinary skin cosmetic products, such as astringents, germicides/antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory/anti-allergic agents, antioxidants/active oxygen removers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc., so long as the combination of the rice yeast fermentation product and the above five ingredients does not interfere with the skin barrier function improving effect and FLG mRNA expression promoting effect, etc. By using the ingredients in combination in this way, a more general product can be obtained, and the synergistic effect with the other active ingredients used in combination can bring about a superior effect than would normally be expected.

Incorporation into oral compositions
The combination of the rice yeast fermentation product and the above five components has an excellent effect of improving the skin barrier function, and is therefore suitable for incorporation into an oral composition. In this case, the combination of the rice yeast fermentation product and the above five components may be incorporated as is, or the combination of the rice yeast fermentation product and the above five components may be formulated and then incorporated.

By blending a combination of the yeast fermented rice and the above five components, an oral composition suitable for improving skin barrier function can be obtained. In addition, by blending the above specific combinations, an oral composition suitable for promoting FLG mRNA expression, promoting IVL mRNA expression, promoting LOR mRNA expression, promoting KLK5 mRNA expression, promoting KLK7 mRNA expression, promoting SPINK5 mRNA expression, promoting caspase 14 mRNA expression, promoting ZO-1 mRNA expression, promoting Sema3A mRNA expression, promoting epidermal keratinocyte proliferation, or promoting SIRT1 mRNA expression can be obtained. In other words, blending into an oral composition is one of the suitable uses for skin barrier function improving agents, FLG mRNA expression promoters, etc.

Here, the oral composition refers to a composition that is unlikely to cause harm to human health and is taken orally or by administration through the digestive tract in normal social life, and is not limited to administrative classifications such as food, medicine, and quasi-drug. Therefore, the "oral composition" in this embodiment broadly includes general foods, health foods, health functional foods (foods for specified health uses, foods with nutritional functions, foods with functional claims), quasi-drugs, medicines, etc. that are taken orally. The oral composition according to this embodiment is preferably an oral composition that can display the favorable effects of the combination of the yeast fermented rice and the above five components on the oral composition or its packaging, and is particularly preferably a health functional food (foods for specified health uses, foods with functional claims, foods with nutritional functions), quasi-drugs, and medicines.

When the combination of rice yeast fermentation product and the above five components is incorporated into an oral composition, the amount of active ingredient can be appropriately changed taking into consideration the purpose of use, symptoms, gender, etc., but taking into consideration the general intake of the oral composition to which it is added, it is preferable to set the daily intake of the extract for an adult, converted into the solid content of the combination of rice yeast fermentation product and the above five components, to about 1 to 1000 mg. Note that when the oral composition to which it is added is a granular, tablet, or capsule-shaped oral composition, the amount of the combination of rice yeast fermentation product and the above five components added is usually 0.1 to 100% by mass, preferably 5 to 100% by mass, converted into the solid content of the combination of rice yeast fermentation product and the above five components, relative to the oral composition to which it is added.

The oral composition of this embodiment may be any oral composition that does not interfere with the activity of the combination of the yeast fermented rice and the above five ingredients, or may be a dietary supplement that contains as its main ingredient a combination of the yeast fermented rice and the above five ingredients.

When producing the oral composition of this embodiment, any auxiliary agent can be added, such as sugars such as dextrin and starch; proteins such as gelatin, soy protein, and corn protein; amino acids such as alanine, glutamine, and isoleucine; polysaccharides such as cellulose and gum arabic; and fats and oils such as soybean oil and medium-chain fatty acid triglycerides, to form an oral composition of any shape.

There are no particular limitations on the oral composition that may contain a combination of the yeast fermented rice and the above five ingredients, but specific examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (including concentrated concentrates and powders for adjusting these beverages); frozen desserts such as ice cream, ice sorbet, and shaved ice; noodles such as soba, udon, harusame, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; candies, chewing gum, candies, gum, chocolate, tablet sweets, and snacks. These include confectioneries such as sweets, biscuits, jellies, jams, creams, baked goods, etc.; fishery and livestock processed foods such as kamaboko, ham, sausages, etc.; dairy products such as processed milk and fermented milk; oils and fats and oil-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressings, etc.; seasonings such as sauces and sauces; soups, stews, salads, side dishes, pickles; other health and nutritional supplements in various forms; tablets, capsules, drinks, etc. When the combination of the yeast fermented rice and the above five components is formulated into these oral compositions, commonly used auxiliary ingredients and additives can be used in combination.

Other embodiments
The skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment each have excellent skin barrier function improving effects and FLG mRNA expression promoting effects, and therefore can also be suitably used as reagents for research into the mechanisms of these actions.

In addition, the skin barrier function improving agent and FLG mRNA expression promoting agent of this embodiment are preferably applied to humans, but as long as their respective action effects are exerted, they can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.).

The present invention will be specifically explained below with the following manufacturing examples, test examples, and compounding examples, but the present invention is not limited to the following examples in any way.

[Production Example 1] Production of fermented rice

0.1 kg of polished white rice flour was dispersed in 1 kg of water and sterilized at 110°C for 5 minutes, after which glucoamylase was added and reacted at 60°C for 1 hour.

Then, Saccharomyces verona (Akita Konno Shoten) was inoculated into the resulting enzyme-treated liquid and fermented at 30°C for 24 hours. The resulting fermented liquid was sterilized at 110°C for 5 minutes. The resulting culture liquid was filtered through diatomaceous earth, and the filtrate was concentrated and dried to obtain a rice fermented product (approximately 64 g).

[Production Example 2] Production of Gaiyo extract 1500 mL of 80% by volume ethanol solution was added to 100 g of dried mugwort leaves, and extraction was carried out for 2 hours at 80 to 90°C using a reflux extractor, followed by hot filtration. The resulting extract was dried to obtain Gaiyo extract (7.5 g).

[Production Example 3] Production of Satsuma mandarin extract 100 g of dried Satsuma mandarin peel was mixed with 80% by volume ethanol, extracted for 2 hours at 80 to 90°C using a reflux extractor, and filtered while hot. The resulting extract was dried to obtain a Satsuma mandarin extract (25 g).

[Production Example 4] Production of Aloe vera extract 100 mL of 30% by volume 1,3-butylene glycol solution was added to 1 g of solid obtained by concentrating the squeezed liquid of Aloe vera mesophyll, and extraction was performed by heating at 50°C for 1 hour, followed by filtration. The obtained extract was dried to obtain an Aloe vera extract (0.8 g).

[Production Example 5] Production of noni juice 1,3-butylene glycol and purified water were added to 100 g of noni juice to prepare a liquid. The obtained liquid was dried to obtain a solid content (5 g) of noni juice.

[Production Example 6] Production of Job's Tears Extract 1500 mL of 50% by volume 1,3-butylene glycol solution was added to 100 g of crushed Job's Tears seeds, followed by extraction with heating and filtration. The resulting extract was dried to obtain Job's Tears extract (6 g).

[Test Example 1] Test for promoting effect of filaggrin (FLG) mRNA expression The fermented rice product (Production Example 1) and the Gaiyo extract (Production Example 2) were tested for their promoting effect of FLG mRNA expression as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were precultured in normal human epidermal keratinocyte growth medium (KGM) and the cells were harvested by trypsinization. The harvested cells were diluted with KGM to a cell density of 1.5 x ¹⁰⁵ cells/mL, and then 2 mL of the cells were seeded onto a 6-well plate (3.0 x ¹⁰⁵ cells/well) and cultured overnight at 37°C and 5% _CO2 . After culturing, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM without the addition of growth additives) and cultured for another 24 hours.

After 24 hours of incubation, the medium was removed, and 2 mL of the test sample dissolved in KBM (see Table 1 below for sample concentration (converted to solid content)) was added to each well, and incubation was continued for 24 hours at 37°C and 5% _CO2 . As a control, incubation was performed in the same manner using KBM without sample. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd., Cat. No. 311-07361). The amount of each RNA was measured using a spectrophotometer (measurement wavelength: 260 nm), and total RNA was adjusted to 200 ng/μL.

Using this total RNA as a template, the expression levels of mRNA for FLG and GAPDH, an internal standard, were measured. Detection was performed by a two-step real-time RT-PCR reaction using PrimeScript ^TM RT Master Mix (Perfect Real Time) and TB Green ^(R) Fast qPCR Mix (both manufactured by Takara Bio) using a real-time PCR device Thermal Cycler Dice (R ⁾ Real Time System III (manufactured by Takara Bio). Perfect Real Time Primer (manufactured by Takara Bio) was used as the primer. The expression level of FLG mRNA was calculated based on the total RNA preparation prepared from cells cultured with "test sample addition" and "no sample addition", and the correction value was calculated with the value of GAPDH. From the obtained value, the FLG mRNA expression promotion rate (%) was calculated using the following formula.

FLG mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following:
A: Corrected value with test sample added B: Corrected value with no sample added The results are shown in Table 1.

As shown in Table 1, it was confirmed that the combined use of fermented rice and Gaiyo extract significantly improved the FLG mRNA expression promoting effect.

[Test Example 2] Test on the promotion of involucrin (IVL) mRNA expression

The IVL mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were changed to rice fermentation product (Production Example 1), Spirea Extract (Production Example 2), and Coix Seed Extract (Production Example 6) (see Tables 2 and 3 below for sample concentrations (converted to solids)) and the detection target was changed to IVL mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Tables 2 and 3.

As shown in Tables 2 and 3, it was confirmed that the combined use of rice fermentation product and Gaiyo extract or Coix seed extract significantly improved the IVL mRNA expression promoting effect.

[Test Example 3] Loricrin (LOR) mRNA expression promotion test

The LOR mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were changed to rice fermentation product (Production Example 1), Spiral Root Extract (Production Example 2), Aloe Vera Extract (Production Example 4), Noni Juice (Production Example 5), and Coix Seed Extract (Production Example 6) (see Tables 4 to 7 below for sample concentrations (converted to solid content)), and the detection target was changed to LOR mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Tables 4 to 7.

As shown in Tables 4 to 7, it was confirmed that the LOR mRNA expression promoting effect was significantly improved by combining the fermented rice product with ginger extract, aloe vera extract, noni juice, or coix seed extract.

[Test Example 4] Kallikrein 5 (KLK5) mRNA expression promotion test

The KLK5 mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were rice fermentation product (Production Example 1) and Spirea Extract (Production Example 2) (see Table 8 below for sample concentrations (converted to solids)) and the detection target was KLK5 mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Table 8.

As shown in Table 8, it was confirmed that the combined use of fermented rice and Gaiyo extract significantly improved the effect of promoting KLK5 mRNA expression.

[Test Example 5] Kallikrein 7 (KLK7) mRNA expression promotion test

The KLK7 mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were changed to rice fermentation product (Production Example 1), Snap Root Extract (Production Example 2) and Noni Juice (Production Example 5) (see Tables 9 to 10 below for sample concentrations (converted to solids)) and the detection target was changed to KLK7 mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Tables 9 and 10.

As shown in Tables 9 to 10, it was confirmed that the combined use of fermented rice with Gaiyo extract or Noni juice significantly improved the effect of promoting KLK7 mRNA expression.

[Test Example 6] Serine peptidase inhibitor Kazal type 5 (SPINK5) mRNA expression promotion test

The SPINK5 mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were changed to rice fermentation product (Production Example 1), Snap Root extract (Production Example 2), Satsuma mandarin extract (Production Example 3), Noni juice (Production Example 5) and Coix seed extract (Production Example 6) (see Tables 11 to 14 below for sample concentrations (converted into solid content)) and the detection target was changed to SPINK5 mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Tables 11 to 14.

As shown in Tables 11 to 14, it was confirmed that the combined use of fermented rice with mussel extract, satsuma mandarin extract, noni juice, or coix seed extract significantly improved the promotion of SPINK5 mRNA expression.

[Test Example 7] Caspase 14 mRNA expression promotion test

The caspase 14 mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were changed to rice fermentation product (Production Example 1), Snap Root Extract (Production Example 2) and Noni Juice (Production Example 5) (see Tables 15 and 16 below for sample concentrations (converted to solid content)) and the detection target was changed to caspase 14 mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Tables 15-16.

As shown in Tables 15 and 16, it was confirmed that the combined use of fermented rice with Gaiyo extract or Noni juice significantly improved the caspase 14 mRNA expression promoting effect.

[Test Example 8] ZO-1 mRNA expression promotion test

The ZO-1 mRNA expression promoting effect was tested in the same manner as in Test Example 1, except that the test samples were changed to rice fermentation product (Production Example 1), Snap Root extract (Production Example 2), Satsuma mandarin extract (Production Example 3), Aloe vera extract (Production Example 4) and Coix seed extract (Production Example 6) (see Tables 17 to 20 below for sample concentrations (converted into solid content)) and the detection target was changed to ZO-1 mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)).
The results are shown in Tables 17 to 20.

As shown in Tables 17 to 20, it was confirmed that the combined use of rice fermentation product with ginger extract, satsuma mandarin extract, aloe vera extract, or coix seed extract significantly improved the ZO-1 mRNA expression promoting effect.

[Test Example 9] Sirtuin 1 (SIRT1) mRNA expression promotion test

The test samples were rice fermentation product (Production Example 1) and Cockle extract (Production Example 2) (see Table 21 below for sample concentration (converted to solid content)), and the detection target was changed to Sirtuin 1 (SIRT1) mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)), and the SIRT1 mRNA expression promoting effect was tested in the same manner as in Test Example 1.
The results are shown in Table 21.

As shown in Table 21, it was confirmed that the combined use of fermented rice and Gaiyo extract significantly improved the SIRT1 mRNA expression promoting effect.

[Test Example 10] Semaphorin 3A (Sema3A) mRNA expression promotion test

The test samples were changed to rice fermentation product (Production Example 1), cockle extract (Production Example 2), satsuma mandarin extract (Production Example 3), aloe vera extract (Production Example 4) and noni juice (Production Example 5) (see Tables 22 to 25 below for sample concentrations (converted to solids)) and the detection target was changed to semaphorin 3A (Sema3A) mRNA (the primer used was Perfect Real Time Primer (manufactured by Takara Bio Inc.)), but the effect of promoting Sema3A mRNA expression was tested in the same manner as in Test Example 1.
The results are shown in Tables 22 to 25.

As shown in Tables 22 to 25, it was confirmed that the combined use of fermented rice with mussel extract, satsuma mandarin extract, aloe vera extract, or noni juice significantly improved the effect of promoting Sema3A mRNA expression.

[Test Example 11] Test for epidermal keratinocyte proliferation-promoting effect The epidermal keratinocyte proliferation-promoting effect of rice fermentation product (Production Example 1), cockle extract (Production Example 2), satsuma mandarin extract (Production Example 3), aloe vera extract (Production Example 4) and noni juice (Production Example 5) was tested as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were diluted with KGM to a cell density of 3.0 x ¹⁰⁴ cells/mL, and then seeded at 100 μL per well on a collagen-coated 96-well plate and cultured overnight. After the culture was completed, the medium was removed, and 100 μL of KGM medium containing a test sample (see Tables 26 to 29 below for sample concentration (solid content equivalent)) or KGM medium without a test sample was added to each well, and cultured for 3 days.

The epidermal keratinocyte proliferation-promoting effect was measured using the MTT assay. After 3 days of culture, the medium was removed, and 100 μL of MTT dissolved in PBS buffer at a final concentration of 0.4 mg/mL was added to each well. After 2 hours of culture, blue formazan produced within the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was taken as the amount of blue formazan produced. From the results obtained, the epidermal keratinocyte proliferation promotion rate (%) was calculated using the following formula.

Epidermal keratinocyte proliferation promotion rate (%) = A/B x 100
Each term in the formula represents the following:
A: Amount of blue formazan produced in cells to which a test sample was added. B: Amount of blue formazan produced in cells to which no sample was added. The results are shown in Tables 26 to 29.

As shown in Tables 26 to 29, it was confirmed that the combined use of fermented rice with mussel extract, satsuma mandarin extract, aloe vera extract, or noni juice significantly improved the epidermal keratinocyte proliferation-promoting effect.

[Formulation Example 1]
According to the composition below, a milky lotion was prepared by a conventional method.
Fermented rice 0.1g
Gaiyou extract 0.25g
Jojoba oil 4.00g
1,3-butylene glycol 3.00g
Arbutin 3.00g
Polyoxyethylene cetyl ether (20E.O.) 2.50g
2.00g olive oil
Squalane 2.00g
Cetanol 2.00g
Glyceryl monostearate 2.00g
Polyoxyethylene sorbitan oleate (20E.O.) 2.00g
Methyl paraoxybenzoate 0.15g
Stearyl glycyrrhetinate 0.10g
Phellodendron extract 0.10g
Dipotassium glycyrrhizinate 0.10g
Ginkgo leaf extract 0.10g
Conchiolin 0.10g
Phellodendron bark extract 0.10g
Chamomile extract 0.10g
Fragrance 0.05g
Purified water Remainder (total amount is 100g)

[Formulation Example 2]
A cream having the following composition was prepared by a conventional method.
Fermented rice 0.1g
Unshu mandarin extract 0.25g
Sophora root extract 0.1g
Scutellaria root extract 0.1g
Liquid paraffin 5.0g
White beeswax 4.0g
Squalane 10.0g
Cetanol 3.0g
Lanolin 2.0g
Stearic acid 1.0g
Polyoxyethylene sorbitan oleate (20E.O.) 1.5g
Glyceryl monostearate 3.0g
Oil-soluble licorice extract 0.1g
1,3-butylene glycol 6.0g
Methyl paraoxybenzoate 1.5g
Fragrance 0.1g
Purified water Remainder (total amount is 100g)

[Formulation Example 3]
A beauty essence having the following composition was prepared by a conventional method.
Fermented rice 0.1g
Aloe vera extract 0.25g
Chamomile extract 0.1g
Carrot extract 0.1g
Xanthan gum 0.3g
Hydroxyethyl cellulose 0.1g
Carboxyvinyl polymer 0.1g
1,3-butylene glycol 4.0g
Dipotassium glycyrrhizinate 0.1g
Glycerin 2.0g
Potassium hydroxide 0.25g
Fragrance 0.01g
Preservative (methyl paraoxybenzoate) 0.15g
Ethanol 2.0g
Purified water Remainder (total amount is 100g)

[Formulation Example 4]
Tablets having the following composition were prepared by a conventional method.
Fermented rice 4.0mg
Noni juice (solid content) 1.0 mg
Dolomite (containing 20% calcium and 10% magnesium) 83.4 mg
Casein phosphopeptide 16.7mg
Vitamin C 33.4mg
Maltitol 136.8mg
Collagen 12.7mg
Sucrose fatty acid ester 12.0 mg

[Formulation Example 5]
An oral liquid preparation having the following composition was produced by a conventional method.
<Composition in 1 ampoule (100 mL)>
Fermented rice 0.4% by mass
Coix seed extract 0.1% by mass
Sorbitol 12.0% by mass
Sodium benzoate 0.1% by mass
Fragrance 1.0% by mass
Calcium sulfate 0.5% by mass
Purified water remainder (100% by mass)

Claims (13)

Rice yeast fermentation product,
One or more selected from the group consisting of Gaiyo extract, Aloe vera extract, Noni fruit juice, Coix seed extract, and Satsuma mandarin extract;
A skin barrier function improving agent characterized by having the above combination as active ingredients. Rice yeast fermentation product,
Gaiyou extract,
A filaggrin mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
Gaiyo extract and/or Yokuinin extract,
13. An involucrin mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
One or more selected from the group consisting of Gaiyo extract, Aloe vera extract, Noni juice, and Coix seed extract;
A loricrin mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
Gaiyou extract,
A kallikrein 5 mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
Gaiyo extract and/or Noni juice,
A kallikrein 7 mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
One or more selected from the group consisting of Gaiyo extract, Noni fruit juice, Coix seed extract, and Satsuma mandarin extract;
A serine peptidase inhibitor Kazal type 5 (SPINK5) mRNA expression promoter, characterized in that the above combination is contained as active ingredients. Rice yeast fermentation product,
Gaiyo extract and/or Noni juice,
A caspase 14 mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
One or more selected from the group consisting of a Gaiyo extract, an aloe vera extract, a Coix seed extract, and a Satsuma mandarin extract;
A ZO-1 mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
One or more selected from the group consisting of a ginger extract, an aloe vera extract, a noni juice, and a satsuma mandarin extract;
A semaphorin 3A mRNA expression promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
One or more selected from the group consisting of a ginger extract, an aloe vera extract, a noni juice, and a satsuma mandarin extract;
2. An epidermal keratinocyte proliferation promoter comprising the above combination as active ingredients. Rice yeast fermentation product,
Gaiyou extract,
A sirtuin 1 mRNA expression promoter comprising the above combination as active ingredients. The agent according to any one of claims 1 to 12, wherein the rice yeast fermentation product is a Saccharomyces verona fermentation product.