Epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, serine palmitoyltransferase mRNA expression promoter, involucrin mRNA expression promoter, and ATP production promoter

Abstract

To provide agents for promoting epidermal keratinocyte growth, filaggrin mRNA expression, serine palmitoyltransferase mRNA expression, involucrin expression or ATP production by finding substances having excellent activity in promoting epidermal keratinocyte growth, filaggrin mRNA expression, serine palmitoyltransferase mRNA expression, involucrin expression or ATP production.SOLUTION: An epidermal keratinocyte growth promoting agent of the invention comprises one or more effective ingredients selected from the group consisting of 2-ketoglutaric acid, 2-isopropyl malate, inosine 5'-monophosphoric acid, succinic acid, malic acid and pyruvic acid. Also, a filaggrin mRNA expression promotor, a serine palmitoyltransferase mRNA expression promotor, an involucrin expression promotor of the invention comprise, as an effective ingredient, 2-ketoglutaric acid. Further an ATP production promotor of the invention comprises 2-isopropyl malate as an effective ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an epidermal keratinocyte proliferation promoter, a filaggrin mRNA expression promoter, a serine palmitoyltransferase mRNA expression promoter, an involucrin mRNA expression promoter, and an ATP production promoter. [Background technology]

[0002] The epidermis functions to buffer external stimuli and control the loss of body components such as water. It is composed of four layers, 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 divide and proliferate in the basal layer, passing through the spinous and granular layers to differentiate into corneocytes, which form the stratum corneum, composed of tightly cross-linked keratin protein fibers. They are eventually shed from the stratum corneum as sebum. The stratum corneum is the outermost layer of the skin and serves as a physical barrier against external stimuli. To maintain this barrier function, the skin undergoes a cycle of epidermal metabolism (keratinization), from keratinocyte production in the basal layer to sebum shedding, usually repeated every four weeks.

[0003] However, when the skin is exposed to external factors such as ultraviolet rays, extremely dry air, excessive skin washing, and smoking, or when aging progresses, the activity and proliferation ability of epidermal cells declines, and the turnover rate of the epidermis slows, causing differentiation disorders 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 changes such as wrinkles, dullness, loss of texture, and loss of elasticity occur as the skin ages.

[0004] Therefore, if the proliferation of keratinocytes can be promoted, skin turnover will be promoted and the metabolic function of the skin will be restored, which is thought to improve skin aging symptoms such as fine wrinkles, dullness, pigmentation, etc. Extracts from Taiso (see Patent Document 1) and extracts from Tsuchigahara Mu (see Patent Document 2) are known to have the effect of promoting the proliferation of epidermal keratinocytes.

[0005] Furthermore, in order to promote cell proliferation, it is important to supply cells with the energy necessary for cell division. ATP is an example of an energy substance in living organisms, and it is thought that increasing the production of this ATP promotes intracellular energy metabolism, leading to cell proliferation. However, as mentioned above, it has been reported that the amount of ATP, an energy substance, is reduced in cells with reduced function or aging cells compared to normal cells (see Patent Document 3).

[0006] Therefore, if ATP production in cells can be promoted, it is believed that the cells can be activated, cell division can be promoted, and the proliferation ability of the cells can be restored. In particular, promoting ATP production in skin cells is important for promoting skin turnover, restoring the skin's metabolic function, and preventing and improving skin aging such as wrinkles, dullness, and loss of texture. Glycogen (see Patent Document 3), extracts from natural products such as peaches (see Patent Document 4), etc. are known to have the effect of promoting ATP production.

[0007] 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 filaggrin function due to gene mutations or the like is associated with the risk of developing atopic diseases, including atopic dermatitis (eczema, skin inflammation, skin itching, etc.), allergies, asthma, etc., and in more severe cases, can lead to skin diseases such as ichthyosis vulgaris (see Non-Patent Document 1).

[0008] Meanwhile, amino acids, the main components of natural moisturizing factors (NMFs), are produced by the degradation of filaggrin derived from keratohyalin granules within 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, accumulates in keratohyalin granules, and is degraded to filaggrin through dephosphorylation and hydrolysis. It then migrates into the stratum corneum, where it increases the aggregation efficiency of keratin filaments and is known to be involved in the internal organization of keratinocytes (see Non-Patent Document 2). In recent years, it has been discovered that filaggrin is extremely important and essential for skin moisture retention, and that conditions such as dryness reduce the ability of filaggrin synthesis, resulting in a decrease in the amount of amino acids in the stratum corneum (see Non-Patent Document 3).

[0009] Therefore, it is believed that promoting the expression of filaggrin (profilaggrin) in epidermal keratinocytes can prevent, treat, or improve atopic diseases, including atopic dermatitis (eczema, skin inflammation, skin itchiness, etc.), allergies, asthma, etc. Furthermore, promoting the expression of filaggrin, thereby increasing the amount of amino acids in the stratum corneum, is expected to essentially improve the moisture environment of the stratum corneum. Extracts of Gai Ying (Patent Document 5) and the like are known to have the effect of promoting filaggrin expression.

[0010] Ceramide is produced from serine and palmitoyl-CoA during the keratinization process of epidermal cells by the action of enzymes including serine palmitoyltransferase (SPT), known as the rate-limiting enzyme in ceramide synthesis. Ceramide is specifically present as the main component of intercellular lipids that cover the outermost layer of the skin, and plays an important role in maintaining the skin's natural barrier membrane function between the body and the outside world.

[0011] The structure of the stratum corneum can be likened to bricks and mortar, with about 15 layers of keratinocytes stacked together by intercellular lipids to form a strong barrier membrane.Keratinocytes retain moisture by containing natural moisturizing factors, primarily amino acids, within the cells, while intercellular lipids are composed primarily of ceramides (about 50%), as well as amphiphilic lipids such as cholesterol and fatty acids, and are characterized by a lamellar structure, in which hydrophobic and hydrophilic regions alternate.

[0012] A decline in skin barrier function due to various internal and external factors increases transepidermal water loss, causing dryness, scaling, itching, and other symptoms, resulting in so-called dry skin. Furthermore, a decline in skin barrier function increases skin inflammation, leading to a vicious cycle in which the skin's defense function against various external stimuli is impaired. Recent studies have reported a decrease and compositional changes in keratinocyte ceramide components (so-called intercellular lipids) due to aging or in patients with atopic dermatitis, known as a barrier disorder (see Non-Patent Document 4), and it has become widely known that ceramides are important for maintaining and improving skin barrier function. Known methods for improving skin barrier function include supplementing ceramides externally (see Non-Patent Document 5) and enhancing ceramide production within the skin (see Non-Patent Document 6).

[0013] Corneocytes are composed primarily of keratin fibers and are surrounded by cornified envelope cells (CE). CEs are formed when multiple CE precursor proteins, produced as epidermal keratinocytes differentiate, are cross-linked and insolubilized by the enzyme transglutaminase. Furthermore, covalent bonding of ceramides and other molecules creates a hydrophobic structure, providing a foundation for the intercellular lipid lamellar structure in part of the CE, thereby forming the basis for the stratum corneum barrier function.

[0014] Involucrin is known as one of these CE precursor proteins, and it is believed that promoting the production of involucrin improves the stratum corneum barrier function and can prevent, treat, or improve skin aging symptoms such as rough skin and dry skin, as well as dry skin diseases such as atopic dermatitis, psoriasis, ichthyosis, and xerosis. Based on this idea, extracts from the seeds of Ceylon wood are known to have the effect of promoting involucrin production (see Patent Document 6). [Prior art documents] [Patent documents]

[0015] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-316028 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-056854 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-321373 [Patent Document 4] Japanese Patent Application Laid-Open No. 2009-256272 [Patent Document 5] Japanese Patent Application Laid-Open No. 2012-219047 [Patent Document 6] Japanese Patent Application Laid-Open No. 2005-213187 [Non-patent literature]

[0016] [Non-Patent Document 1] "Nat Genet.",2006,Vol.38,No.4,p.441-446 [Non-patent document 2] "Fragrance Journal Special Issue", 2000, Vol. 17, pp. 14-19 [Non-patent document 3] "Arch. Dermatol. Res.", 1996, Vol. 288, p. 442-446 [Non-patent document 4] J. Dermatol.,1993,Vol.20,No.1,p.1-6 [Non-patent document 5] "Fragrance Journal", 2004, Vol. 32, No. 11, pp. 23-32 [Non-patent document 6] Br. J. Dermatol.,2000,Vol.143,Issue 3,p.524-531 Summary of the Invention [Problem to be solved by the invention]

[0017] The present invention aims to discover a substance that has excellent effects in promoting epidermal keratinocyte proliferation, promoting filaggrin mRNA expression, promoting SPT mRNA expression, promoting involucrin mRNA expression, or promoting ATP production, and to provide an epidermal keratinocyte proliferation promoter, a filaggrin mRNA expression promoter, a serine palmitoyltransferase mRNA expression promoter, an involucrin mRNA expression promoter, or an ATP production promoter that contains the substance as an active ingredient. [Means for solving the problem]

[0018] In order to solve the above problems, the epidermal keratinocyte proliferation promoter of the present invention is characterized by having one or more active ingredients selected from the group consisting of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid.

[0019] The filaggrin mRNA expression promoter, serine palmitoyltransferase mRNA expression promoter, and involucrin mRNA expression promoter of the present invention are characterized by containing 2-ketoglutaric acid as an active ingredient.Furthermore, the ATP production promoter of the present invention is characterized by containing 2-isopropylmalic acid as an active ingredient. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide an epidermal keratinocyte proliferation promoter, a filaggrin mRNA expression promoter, a serine palmitoyltransferase mRNA expression promoter, an involucrin mRNA expression promoter, and an ATP production promoter that have excellent effects. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, an embodiment of the present invention will be described. [Epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, serine palmitoyltransferase mRNA expression promoter, involucrin mRNA expression promoter, ATP production promoter] The epidermal keratinocyte proliferation promoter of this embodiment contains one or more active ingredients selected from the group consisting of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid. The filaggrin mRNA expression promoter, serine palmitoyltransferase (SPT) mRNA expression promoter, and involucrin mRNA expression promoter of this embodiment contain 2-ketoglutaric acid as an active ingredient, and the ATP production promoter of this embodiment contains 2-isopropylmalic acid as an active ingredient.

[0022] The 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid used in this embodiment are known compounds. 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid may be in the form of a salt. Among these, inosine 5'-monophosphate is preferably in the form of a salt, and particularly preferably in the form of a sodium salt.

[0023] The 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid used in this embodiment are commercially available and may be used. Alternatively, they may be produced by isolating and purifying from a plant extract containing 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid using known methods, or by synthesizing using known methods. The plant extract includes an extract obtained from a plant containing 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, or pyruvic acid as an extracting material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof.

[0024] Plants containing 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid or pyruvic acid include, but are not limited to, rice.

[0025] The plant extract may be subjected to microbial fermentation, for example, by dissolving the concentrated plant extract in water or by using the plant extract as is without concentrating it to dryness and then inoculating it with a fermenting microorganism.

[0026] The microorganisms that carry out the fermentation are not particularly limited, and examples include lactic acid bacteria, yeast, and koji mold, but yeast is preferred, and Saccharomyces verona is particularly preferred.

[0027] The 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid used in this embodiment have excellent epidermal keratinocyte proliferation-promoting properties and can therefore be used as active ingredients in epidermal keratinocyte proliferation promoters. Furthermore, the 2-ketoglutaric acid used in this embodiment has excellent filaggrin mRNA expression-promoting properties, SPT mRNA expression-promoting properties, and involucrin mRNA expression-promoting properties and can therefore be used as active ingredients in filaggrin mRNA expression promoters, SPT mRNA expression promoters, and involucrin mRNA expression promoters, respectively. Furthermore, the 2-isopropylmalic acid used in this embodiment has excellent ATP production-promoting properties and can therefore be used as an active ingredient in ATP production promoters.

[0028] In other words, to produce an epidermal keratinocyte proliferation promoter, one or more selected from the group consisting of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid can be used. Also, to produce a filaggrin mRNA expression promoter, an SPT mRNA expression promoter, or an involucrin mRNA expression promoter, 2-ketoglutaric acid can be used. Furthermore, to produce an ATP production promoter, 2-isopropylmalic acid can be used.

[0029] The epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter and ATP production promoter of this embodiment can be used for a wide range of applications such as pharmaceuticals, quasi-drugs, foods and beverages.

[0030] In this embodiment, any one of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid may be used as the active ingredient of the epidermal keratinocyte proliferation promoter, or two or more of these may be mixed and used as the active ingredient. When a mixture of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid is used as the active ingredient, the blending ratio may be appropriately adjusted depending on the level of the epidermal keratinocyte proliferation-promoting effect of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid.

[0031] Furthermore, instead of isolated 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, or pyruvic acid, or a mixture thereof (hereinafter sometimes referred to as the compound(s) of this embodiment), a composition containing these compound(s) may be used as the active ingredient of the epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter, or ATP production promoter of this embodiment. Herein, compositions containing these compound(s) in this embodiment include extracts obtained using plants containing the compound(s) as the extraction raw material, fermented products containing the compound(s), and extracts obtained using the fermented products as the extraction raw material. Furthermore, the term "extract" includes extracts obtained by extraction treatment, diluted or concentrated solutions of the extracts, and dried products obtained by drying the extracts.

[0032] 2-Ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid can promote skin turnover by promoting epidermal keratinocyte proliferation or ATP production, and therefore may be used as active ingredients in skin turnover promoters. 2-Ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid may also be used as active ingredients in agents for preventing and improving skin elasticity loss, dullness, and other conditions caused by delayed skin turnover. Furthermore, 2-ketoglutaric acid can improve the skin barrier function by its action of promoting filaggrin mRNA expression, SPT mRNA expression, or involucrin mRNA expression, and therefore may be used as an active ingredient in a skin barrier function improving agent.

[0033] The epidermal keratinocyte proliferation promoter of this embodiment may consist solely of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, pyruvic acid, or a mixture thereof, or may be a formulation of these active ingredients. Furthermore, the filaggrin mRNA expression promoter, SPT mRNA expression promoter, and involucrin mRNA expression promoter of this embodiment may consist solely of 2-ketoglutaric acid, or may be a formulation of 2-ketoglutaric acid. Furthermore, the ATP production promoter of this embodiment may consist solely of 2-isopropylmalic acid, or may be a formulation of 2-isopropylmalic acid.

[0034] The epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter, and ATP production promoter of this embodiment can be formulated into any dosage form, such as powder, granules, tablets, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to standard methods. In this case, examples of auxiliary agents that can be used include excipients, binders, disintegrants, lubricants, stabilizers, and flavorings / flavoring agents. The epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter, and ATP production promoter can be incorporated into other compositions (e.g., topical skin preparations, oral compositions, etc., as described below) and used as ointments, topical liquids, patches, etc.

[0035] When the epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter or ATP production promoter of this embodiment is formulated, the contents of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid and pyruvic acid, and mixtures thereof, are not particularly limited and can be set appropriately depending on the purpose.

[0036] The epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter or ATP production promoter of this embodiment can be used as an active ingredient, if necessary, by blending other substances (e.g., natural extracts) having an epidermal keratinocyte proliferation promoting effect, a filaggrin mRNA expression promoting effect, an SPT mRNA expression promoting effect, an involucrin mRNA expression promoting effect or an ATP production promoting effect together with 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, pyruvic acid, or mixtures thereof.

[0037] Methods for administering the epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter or ATP production promoter of this embodiment to patients include transdermal administration and oral administration, and a method suitable for the prevention, treatment, etc. may be selected appropriately depending on the type of disease.

[0038] Furthermore, the dosage of the epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter, or ATP production promoter of this embodiment may be increased or decreased as appropriate depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0039] The epidermal keratinocyte proliferation promoter of this embodiment promotes skin turnover through the epidermal keratinocyte proliferation-promoting activity of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid, thereby preventing and improving skin aging symptoms such as wrinkles, loss of texture, and loss of elasticity. Furthermore, the epidermal keratinocyte proliferation promoter of this embodiment restores skin metabolic functions, such as the shedding of keratinocytes with abnormal melanin accumulation from the stratum corneum, through the epidermal keratinocyte proliferation-promoting activity of 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid, thereby preventing and improving symptoms such as dullness and pigmentation. Furthermore, the epidermal keratinocyte proliferation promoter of this embodiment, due to its excellent epidermal keratinocyte proliferation-promoting activity, can be used in applications such as regenerative medicine, which require the promotion of epidermal keratinocyte proliferation. However, the epidermal keratinocyte proliferation promoter of this embodiment can be used for all purposes other than these, in which it is meaningful to exert an epidermal keratinocyte proliferation promoting effect.

[0040] The filaggrin mRNA expression promoter of this embodiment can promote intracellular filaggrin expression and improve skin moisturizing ability through the filaggrin mRNA expression-promoting effect of 2-ketoglutaric acid, thereby maintaining skin elasticity and preventing, treating, or ameliorating skin aging, rough skin, etc. Furthermore, the filaggrin mRNA expression promoter of this embodiment can promote intracellular filaggrin production through its filaggrin mRNA expression-promoting effect and enhance the skin's barrier function, thereby preventing, treating, or ameliorating atopic diseases including atopic dermatitis (eczema, skin inflammation, skin itchiness, etc.), allergies, asthma, etc. However, in addition to these uses, the filaggrin mRNA expression promoter of this embodiment can be used for all uses in which it is meaningful to exert a filaggrin mRNA expression-promoting effect.

[0041] The SPT mRNA expression promoter of this embodiment strengthens the skin barrier function through the SPT mRNA expression-promoting effect of 2-ketoglutaric acid, and can prevent, treat, or improve rough skin, dry skin, and dry skin diseases (e.g., atopic dermatitis, psoriasis, ichthyosis, etc.). However, in addition to these uses, the SPT mRNA expression promoter of this embodiment can also be used for any other uses where it is meaningful to exert an SPT mRNA expression-promoting effect.

[0042] The involucrin mRNA expression promoter of this embodiment strengthens the skin's barrier function through the involucrin mRNA expression-promoting effect of 2-ketoglutaric acid, and can prevent, treat, or improve skin aging symptoms such as rough skin and dry skin, and dry skin diseases such as ichthyosis, psoriasis, atopic dermatitis, and xerosis. However, in addition to these uses, the involucrin mRNA expression promoter of this embodiment can also be used for any other uses in which exerting the involucrin mRNA expression-promoting effect is meaningful.

[0043] The ATP production promoter of this embodiment can promote cell turnover through the ATP production-promoting activity of 2-isopropyl malic acid, and prevent and improve skin aging symptoms such as wrinkles, loss of texture, and loss of elasticity. Furthermore, the ATP production promoter of this embodiment can prevent and improve skin symptoms such as dullness and pigmentation by restoring skin metabolic functions, such as the shedding of keratinocytes with abnormal melanin accumulation, through the ATP production-promoting activity of 2-isopropyl malic acid. However, the ATP production promoter of this embodiment can also be used for any other applications where it is meaningful to exert an ATP production-promoting activity.

[0044] 2-Ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid have excellent epidermal keratinocyte proliferation-promoting effects and are therefore suitable for incorporation into, for example, topical skin preparations or oral compositions. In this case, 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid may be incorporated as is, or an epidermal keratinocyte proliferation promoter formulated from 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid, or a mixture thereof, may be incorporated.

[0045] 2-ketoglutaric acid has excellent filaggrin mRNA expression promoting effects, SPT mRNA expression promoting effects, and involucrin mRNA expression promoting effects, and is therefore suitable for incorporation into, for example, external skin preparations or oral compositions. In this case, 2-ketoglutaric acid may be incorporated as is, or a filaggrin mRNA expression promoting agent, SPT mRNA expression promoting agent, or involucrin mRNA expression promoting agent formulated from 2-ketoglutaric acid may be incorporated. 2-Isopropylmalic acid has an excellent ATP production promoting effect and is therefore suitable for incorporation into, for example, external skin preparations or oral compositions. In this case, 2-isopropylmalic acid may be incorporated as is, or an ATP production promoter formulated from 2-isopropylmalic acid may be incorporated.

[0046] Here, topical skin preparations are not limited to specific categories and include a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally. Specific examples include ointments, creams, emulsions, skin lotions, beauty serums, lotions, gels, beauty oils, packs, foundations, lip balms, bath additives, hair tonics, hair lotions, shampoos, rinses, soaps, and body shampoos.

[0047] An oral composition refers to a composition that is unlikely to be harmful to human health and that is taken orally or by administration through the digestive tract in normal social life, and is not limited to administrative classifications such as food and drink, medicine, quasi-drug, etc. Therefore, in this embodiment, the "oral composition" broadly includes orally taken general foods, feed, health foods, health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims), quasi-drugs, medicines, etc.

[0048] Furthermore, the epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter and ATP production promoter of this embodiment have excellent epidermal keratinocyte proliferation promoting effects, filaggrin mRNA expression promoting effects, SPT mRNA expression promoting effects, involucrin mRNA expression promoting effects and ATP production promoting effects, and therefore can be suitably used as reagents for research related to the proliferation mechanism of epidermal keratinocytes, the ATP production mechanism, and the mRNA expression mechanism of filaggrin, SPT and involucrin.

[0049] The epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter and ATP production promoter of this embodiment are preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as their respective effects are achieved. [Example]

[0050] The present invention will be specifically explained below by showing test examples, but the present invention is not limited to the following examples in any way.

[0051] In the following test examples, the following samples 1 to 6 were used as 2-ketoglutaric acid, 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, malic acid, and pyruvic acid. Sample 1: 2-ketoglutaric acid (manufactured by Hydras Chemical Co., Ltd.) Sample 2: 2-Isopropylmalic acid (Sigma-Aldrich) Sample 3: Inosine 5'-monophosphate disodium hydrate (Tokyo Chemical Industry Co., Ltd.) Sample 4: Succinic acid (Tokyo Chemical Industry Co., Ltd.) Sample 5: DL-Malic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) Sample 6: Pyruvic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) The concentration of Sample 3 described below is a value calculated by converting the compound into inosine 5'-monophosphate.

[0052] [Test Example 1] Epidermal keratinocyte proliferation promoting activity test Samples 1 to 6 were tested for their epidermal keratinocyte proliferation promoting activity as follows.

[0053] Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 3.0 × 10 4 After diluting with KGM to a cell density of 100 cells / mL, 100 μL of the cells were seeded per well of 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 the test samples (samples 1 to 6, see Table 1 below for final concentrations) or KGM medium without the test samples was added to each well, and the cells were cultured for 3 days.

[0054] The epidermal keratinocyte proliferation-promoting activity 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, the 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 used to determine the amount of blue formazan produced. From the results obtained, the epidermal keratinocyte proliferation promotion rate (%) was calculated using the following formula.

[0055] Epidermal keratinocyte proliferation promotion rate (%) = A / B x 100 In the formula, A represents the "amount of blue formazan produced in cells to which a test sample was added," and B represents the "amount of blue formazan produced in cells to which no sample was added." The results are shown in Table 1.

[0056] [Table 1]

[0057] As shown in Table 1, Samples 1 to 6 were found to have an excellent effect of promoting the proliferation of epidermal keratinocytes.

[0058] [Test Example 2] Filaggrin (FLG) mRNA expression promoting effect test Sample 1 was tested for its FLG mRNA expression promoting effect as follows.

[0059] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 15 × 10 4 After diluting with KGM medium to a cell density of 30 × 10 cells / mL, 2 mL of the medium was seeded into a 6-well plate. 4The cells were cultured overnight at 37°C in 5% CO2 at 1000 x 1000 cells / well. After culturing, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0060] After 24 hours of incubation, the medium was removed, and 2 mL of the test sample (Sample 1, see Table 2 below for final concentration) dissolved in KBM medium was added to each well and incubated at 37°C in 5% CO2 for 24 hours. As a control, KBM medium without the sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.). The amount of RNA in each sample was measured using a spectrophotometer, and total RNA was adjusted to 200 ng / μL.

[0061] Using this total RNA as a template, the mRNA expression levels of FLG and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device, Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.), with PrimeScript TM RT Master Mix (Perfect Real Time) (Takara Bio) TB Green (R) The assay was performed using a two-step real-time RT-PCR reaction with Fast qPCR Mix (Takara Bio). Primers were from Takara Bio. The expression level of FLG mRNA was calculated based on the total RNA samples prepared from cells cultured with and without the test sample, and the normalized value was calculated using the GAPDH value. The FLG mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0062] FLG mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value with the test sample added" and B represents the "corrected value with no sample added." The results are shown in Table 2.

[0063] [Table 2]

[0064] As shown in Table 2, Sample 1 was found to have an excellent effect of promoting FLG mRNA expression.

[0065] [Test Example 3] Serine palmitoyltransferase (SPT) mRNA expression promoting effect test Sample 1 was tested for its ability to promote SPT mRNA expression as follows.

[0066] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 15 × 10 4 After diluting with KGM medium to a cell density of 30 × 10 cells / mL, 2 mL of the medium was seeded into a 6-well plate. 4 The cells were cultured overnight at 37°C in 5% CO2 at 1000 x 1000 cells / well. After culturing, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0067] After 24 hours of incubation, the medium was removed, and 2 mL of the test sample (Sample 1, see Table 3 below for final concentration) dissolved in KBM medium was added to each well and incubated at 37°C in 5% CO2 for 24 hours. As a control, KBM medium without the sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.). The amount of RNA in each sample was measured using a spectrophotometer, and total RNA was adjusted to 200 ng / μL.

[0068] Using this total RNA as a template, the mRNA expression levels of SPT and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device, Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.), with PrimeScript TMRT Master Mix (Perfect Real Time) (Takara Bio) TB Green (R) The assay was performed using a two-step real-time RT-PCR reaction with Fast qPCR Mix (Takara Bio). Primers were from Takara Bio. The expression level of SPT mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and the normalized value was calculated using the GAPDH value. The SPT mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0069] SPT mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value with the test sample added" and B represents the "corrected value with no sample added." The results are shown in Table 3.

[0070] [Table 3]

[0071] As shown in Table 3, Sample 1 was found to have an excellent effect of promoting SPT mRNA expression.

[0072] [Test Example 4] Involucrin (IVL) mRNA expression promoting effect test Sample 1 was tested for its ability to promote IVL mRNA expression as follows.

[0073] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 15 × 10 4 After diluting with KGM medium to a cell density of 30 × 10 cells / mL, 2 mL of the medium was seeded into a 6-well plate. 4 The cells were cultured overnight at 37°C in 5% CO2 at 1000 x 1000 cells / well. After culturing, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0074] After 24 hours of incubation, the medium was removed, and 2 mL of the test sample (Sample 1, see Table 4 below for final concentration) dissolved in KBM medium was added to each well and incubated at 37°C in 5% CO2 for 24 hours. As a control, KBM medium without the sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.). The amount of RNA in each sample was measured using a spectrophotometer, and total RNA was adjusted to 200 ng / μL.

[0075] Using this total RNA as a template, the mRNA expression levels of IVL and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device, Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.), with PrimeScript TM RT Master Mix (Perfect Real Time) (Takara Bio) TB Green (R) The analysis was performed using a two-step real-time RT-PCR reaction with Fast qPCR Mix (Takara Bio). Primers from Takara Bio were used. The expression level of IVL mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and the corrected value was calculated using the GAPDH value. The IVL mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula.

[0076] IVL mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value with the test sample added" and B represents the "corrected value with no sample added." The results are shown in Table 4.

[0077] [Table 4]

[0078] As shown in Table 4, Sample 1 was found to have an excellent effect of promoting IVL mRNA expression.

[0079] [Test Example 5] ATP production promoting effect test Sample 2 was tested for its ATP production promoting effect as follows.

[0080] Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsinization. The harvested cells were collected at a concentration of 2.0 × 10 5 After diluting with KGM to a cell density of 100 cells / mL, 100 μL of the cells were seeded into each well of a collagen-coated 96-well plate and cultured overnight. After the culture was completed, the medium was removed, and 100 μL of KGM containing the test sample (Sample 2, see Table 5 below for final concentration) or KGM without the test sample was added to each well, followed by 2 hours of culture.

[0081] The ATP production promotion effect was measured using the firefly luciferase luminescence method to measure the amount of ATP in the cells. After 2 hours of incubation, 100 μL of ATP measurement reagent (manufactured by Toyo B-Net Co., Ltd., product name "Cellular ATP Measurement Reagent") was added to each well, and a luciferase-mediated chemiluminescence reaction was carried out. After the reaction, the amount of chemiluminescence, which was proportional to the amount of ATP in the cells, was measured using a chemiluminescence measurement device (manufactured by Thermo Fisher Scientific, product name: Varioskan LUX Multimode Microplate Reader). From the results obtained, the ATP production promotion rate (%) was calculated using the following formula:

[0082] ATP production promotion rate (%)=A / B×100 In the formula, A represents the "amount of chemiluminescence in cells to which a test sample has been added," and B represents the "amount of chemiluminescence in cells to which no sample has been added." The results are shown in Table 5.

[0083] [Table 5]

[0084] As shown in Table 5, Sample 2 was found to have an excellent ATP production promoting effect.

[0085] [Formulation example 1] A cream having the following composition was prepared by a conventional method. 2-ketoglutaric acid 0.02g 2-Isopropylmalic acid 0.02g Inosine 5'-monophosphate 0.01 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 parahydroxybenzoate 1.5g Fragrance 0.1g Purified water Rest (total amount is 100g)

[0086] [Formulation example 2] An emulsion was prepared in a conventional manner according to the following composition. Succinic acid 0.01g Malic acid 0.01g 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 Cetyl alcohol 2.00g Glyceryl monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhetinate 0.10g Phellodendron bark extract 0.10g Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g Conchiolin 0.10g Phellodendron bark extract 0.10g Chamomile extract 0.10g Fragrance 0.05g Purified water Rest (total amount is 100g)

[0087] [Formulation example 3] A cosmetic essence having the following composition was prepared by a conventional method. Pyruvate 0.01g 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 parahydroxybenzoate) 0.15g Ethanol 2.0g Purified water Rest (total amount is 100g)

[0088] [Formulation example 4] Tablets having the following composition were prepared by a conventional method. 2-ketoglutaric acid 2.0mg 2-Isopropylmalic acid 1.0mg Inosine 5'-monophosphate 1.0mg Succinic acid 1.0mg Dolomite (contains 20% calcium and 10% magnesium) 83.4mg Casein phosphopeptide 16.7mg Vitamin C 33.4mg Maltitol 136.8mg Collagen 12.7mg Sucrose fatty acid ester 12.0mg

[0089] [Formulation example 5] An oral liquid preparation having the following composition was prepared by a conventional method. <Composition in 1 ampoule (100 mL)> Malic acid 0.2% by mass Pyruvic acid 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) [Industrial Applicability]

[0090] The epidermal keratinocyte proliferation promoter, filaggrin mRNA expression promoter, SPT mRNA expression promoter, involucrin mRNA expression promoter and ATP production promoter of the present invention can significantly contribute to the prevention and improvement of skin aging symptoms such as wrinkles, loss of texture and loss of elasticity; the prevention and improvement of symptoms such as dullness and pigmentation in the skin; and the prevention and improvement of symptoms of decreased skin barrier function such as dry skin diseases.

Claims

1. An epidermal keratinocyte proliferation promoter, characterized in that the active ingredient is one or more selected from the group consisting of 2-isopropylmalic acid, inosine 5'-monophosphate, succinic acid, and malic acid. (however: An epidermal keratinocyte proliferation promoter comprising an extract from kiwi fruit containing malic acid; and an epidermal keratinocyte proliferation promoter comprising a yuzu extract containing succinic acid or malic acid; (Except).

2. Filaggrin mRNA expression promoter characterized by containing 2-ketoglutaric acid as an active ingredient (However, this does not include filaggrin mRNA expression promoters containing yuzu extract containing 2-ketoglutaric acid).

3. A serine palmitoyltransferase mRNA expression promoter characterized by containing 2-ketoglutaric acid as an active ingredient.

4. Involucrin mRNA expression promoter characterized by containing 2-ketoglutaric acid as an active ingredient (However, this does not include the involucrin mRNA expression promoter containing a yuzu extract containing 2-ketoglutaric acid).

5. An ATP production promoter characterized by containing 2-isopropylmalic acid as an active ingredient.

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