JP2018039751A - Epidermal cell-to-cell function enhancing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an epidermal cell-to-cell function enhancing agent that contains a specific peptide and thereby enhances an epidermal cell-to-cell function.SOLUTION: An epidermal cell-to-cell function enhancing agent contains at least one peptide selected from the group consisting of Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly), Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly, a derivative thereof or a salt thereof.SELECTED DRAWING: None

Description

  The present invention relates to an agent for enhancing function between epidermal cells.

  Collagen and hydrolysates thereof, and peptides and mixtures thereof contained in the hydrolysates have been conventionally applied to cosmetics for the purpose of beauty and the like, and are contained in foods and the like. For example, Japanese Patent Application Laid-Open No. 2001-131084 (Patent Document 1) discloses a collagen synthesis promoter that dramatically increases the synthesis activity of collagen protein in vivo using the peptide mixture. Japanese Patent Application Laid-Open No. 2004-1223637 (Patent Document 2) discloses a hyaluronic acid production promoter that promotes hyaluronic acid production in vivo by using a similar peptide mixture. Japanese Unexamined Patent Application Publication No. 2010-024200 (Patent Document 3) discloses a biological collagen synthesis promoter, and a cosmetic, a quasi-drug, and a therapeutic agent containing a biopeptide synthesis promoting action dipeptide or tripeptide. International Publication No. 2014/175001 (Patent Document 4) discloses a whitening accelerator and an atopic dermatitis ameliorating agent using the above peptide mixture.

  Further, Non-Patent Documents 1 to 3 below describe improvement of skin barrier function by collagen hydrolyzate. Specifically, skin barrier dysfunction mice orally ingested with a hydrolyzate of collagen showed that the abnormality was improved by improving the water retention of the skin and reducing the amount of water transpiration. ing.

Japanese Patent Laid-Open No. 2001-131084 Japanese Patent Laid-Open No. 2004-123637 JP 2010-024200 A International Publication No. 2014/175001

Shimizu J. et al., Oral collagen-derived dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine, ameliorate skin barrier dysfunction and alter gene expression profile in the skin, Biochem. Biophys. Res. Commun, 456 (2015) 626-630 Oba C. et al., Collagen hydrolysate intake improves the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice, Photodermatol Photoimmunol Photomed, 29 (2013) 204-211 Oba C. et al., Effect of administered collagen hydrolysate on gene expression profiles in mouse skin: a DNA microarray analysis, Physiol Genomics, 47 (2015) 355-363

  Here, the skin barrier function refers to the function of retaining moisture and heat in the skin composed of epidermis of epithelial system (derived from ectoderm) and dermis of mesenchymal system (derived from mesoderm), and the entry of foreign substances from outside This refers to the protective function against external stimuli. When this skin barrier function is reduced, there is an increased risk of skin dryness due to reduced water retention, bacterial or viral invasion and infection, and the development of inflammation and allergies (so-called atopic dermatitis). Therefore, in view of the improvement of the skin barrier function, the above-mentioned Patent Documents 1 to 3 and Non-Patent Documents 1 to 3 are considered to be most effective when the epidermis located on the outermost side of the living body is targeted. Neither of them discloses whether the above-described peptide mixture or the like improves the skin barrier function of the epidermis.

  Furthermore, considering the improvement of the skin barrier function of the epidermis, not only the cells that make up each layer of the epidermis (basal layer, spiny layer, granule layer, stratum corneum), but also between the cells (between cells) Although it is preferable to consider functional enhancement, Patent Document 4 does not mention functional enhancement between cells. Therefore, a peptide mixture as described above that enhances the function between cells in the epidermis is not yet known.

  This invention is made | formed in view of the said situation, and it aims at providing the function enhancer between epidermal cells which can strengthen the function between the cells in an epidermis by containing a specific peptide at least.

As a result of intensive studies, the present inventors have found that Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu- One or more peptides selected from the group consisting of Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly, their derivatives or their salts are intercellular It has been found that it has an action of promoting the expression of a gene that contributes to the enhancement of the function, and has completed the present invention. That is, the present invention is as follows.

[1] The present invention relates to Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, An interepidermal cell function comprising one or more peptides selected from the group consisting of Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly, derivatives thereof or salts thereof Strengthening agent.

[2] Furthermore, the present invention relates to one or more peptides selected from the group consisting of Pro-Hyp, Hyp-Gly and Glu-Hyp, derivatives thereof or salts thereof, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly An epidermal cell function-enhancing agent, which comprises one or more peptides, derivatives thereof, or salts thereof.

  [3] The epidermal intercellular function-enhancing agent described in [1] or [2] is preferably an oral agent or a transdermal agent.

  [4] It is preferable that the interdermal cell function-enhancing agent according to any one of [1] to [3] acts on epidermal cells.

  [5] The epidermal intercellular function enhancer according to any one of [1] to [4] is preferably a ceramide synthesis accelerator or a tight junction formation accelerator.

ADVANTAGE OF THE INVENTION According to this invention, the epidermis cell function enhancement agent which can strengthen the function between the cells in an epidermis can be provided by containing a specific peptide at least. Specifically, Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, Ser- An epidermal cell function-enhancing agent containing one or more peptides selected from the group consisting of Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp, and Ala-Hyp-Gly, derivatives thereof or salts thereof, Increase the expression level of mRNA (messenger RNA) encoding serine palmitoyltransferase, claudin 1 and occludin. By increasing the expression level, it is possible to enhance ceramide synthesis between cells in the epidermis and formation of tight junctions (tight junctions), thereby enhancing the function between the epidermis cells, that is, enhancing the skin barrier function in the epidermis. Become.

  Hereinafter, the interdermal cell function enhancing agent according to the present invention will be described in detail. In the present specification, the notation of the form “X to Y” means the upper and lower limits of the range (ie, X or more and Y or less), and there is no unit description in X, and the unit is described only in Y The unit of X and the unit of Y are the same.

≪Skin-cell function enhancer≫
The epidermal cell function-enhancing agent according to the present invention includes Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu. -Containing at least one peptide selected from the group consisting of Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly, its derivatives or their salts .

<Peptide>
Peptides contained in the epidermal intercellular function enhancer according to the present invention are Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, and (Pro-Hyp-Gly) _{2 as described above.} , Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly , Also referred to as “the peptide”). In addition to the present peptide, the epidermal cell function-enhancing agent can include a derivative thereof, a salt of the present peptide, and a salt of the derivative thereof as described later. The epidermal intercellular function enhancer contains at least one of the present peptide, its derivative, the salt of the present peptide and the salt of its derivative, thereby increasing the expression level of mRNA encoding serine palmitoyltransferase, claudin 1 and occludin. Can be increased. It is possible to enhance the function between epidermal cells by promoting the synthesis of ceramide between epidermal cells and the formation of tight junctions through the enhancement of these expression levels.

  Here, it is known that serine palmitoyltransferase is involved in the biosynthesis process of ceramide and is the rate-limiting enzyme of the process. By increasing the expression level of mRNA encoding serine palmitoyltransferase, it is considered that the synthesis of ceramide is promoted to increase the amount of ceramide between cells in the epidermis.

  Claudin is one of the proteins constituting a tight junction, and at least 24 isoforms have been found. Since claudin-1 is the main in the skin, and its gene-deficient mice die one day after birth due to excessive water loss from the skin, it is speculated that claudin-1 is an important factor for tight junction formation. ing. Furthermore, claudin 1 is thought to contribute to the localization of tight junctions on the side of the cell membrane by binding to actin filaments, which are the cytoskeleton, via the membrane-lining protein ZO-1. If the expression level of mRNA encoding claudin 1 increases, the amount of claudin 1 increases in epidermal cells, and it is considered that the formation of tight junctions between epidermal cells is promoted. Occludin is known as one of the transmembrane proteins that form a tight junction like claudin. Although the detailed mechanism related to the formation of tight junction by occludin is still under study, the expression of mRNA encoding it is considered to be an indicator of the amount of occludin in epidermal cells, that is, an indicator of tight junction formation.

  The peptide contained in the epidermal cell function enhancer is preferably at least one of Pro-Hyp, Hyp-Gly and Glu-Hyp, more preferably at least one of Pro-Hyp and Hyp-Gly. . Furthermore, it is also preferable to use this peptide combining 2 or more types selected from the said group.

When two or more kinds of peptides are used in combination, this peptide is selected from the group consisting of Pro-Hyp, Hyp-Gly and Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro -Hyp-Gly) ₂ , 1 selected from the group consisting of Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly It is preferable that the mixture is a combination of more than one species (hereinafter also referred to as “peptide mixture”). By containing a peptide mixture of such a combination, the epidermal intercellular function enhancer can further increase the expression level of mRNA encoding serine palmitoyltransferase, claudin 1 and occludin.

  This peptide can be synthesized from amino acids using, for example, a conventionally known solid phase synthesis method or liquid phase synthesis method. In the case of the solid-phase synthesis method, the Fmoc method and the Boc method are further known, and this peptide can be synthesized by any method. A peptide mixture can also be produced by mixing different types of peptides synthesized using these methods.

  Furthermore, this peptide can also be produced by hydrolyzing gelatin in combination with two or more conventionally known endo-type proteases and exo-type proteases. The present peptide is preferably obtained by hydrolyzing with the above protease and then purifying the hydrolyzate. When this peptide is hydrolyzed using the above protease, it can also produce one type of peptide selected from the above group, and can also produce a peptide mixture consisting of two or more types selected from the above group. it can.

<Derivative>
The epidermal intercellular function-enhancing agent according to the present invention may contain one or more peptide derivatives selected from the above group. Peptide derivatives are compounds that have been modified to such an extent that the structure and properties of the peptide are not changed significantly by oxidizing, reducing, or replacing atoms or substituents by using so-called chemical modification techniques. Say. In order to make this peptide a derivative of a peptide, a conventionally known technique relating to chemical modification can be used, and conventionally known means and conditions may be used as specific means and treatment conditions. Hereinafter, a derivative of one or more peptides selected from the above group may be referred to as a “derivative of the present peptide”.

  Chemical modification can be performed for each amino acid unit in the peptide. Examples of the site of chemical modification include a hydroxyl group, an amino group and a carboxyl group contained in an amino acid, an amino group located at the N terminus of the peptide, a hydroxyl group or a carboxyl group located at the C terminus of the peptide, and the like. By chemical modification of these functional groups, for example, the charge can be changed, whereby effects such as improved solubility in an aqueous solvent and improved compatibility with other components can be obtained.

  Acetylation is illustrated as a chemical modification of the hydroxyl group of hydroxyproline. Chemical modification of the amino group located at the N-terminus of the peptide includes acylation such as succinylation, maleylation, acetylation, polypeptidylation, deamination, benzoylation, alkylsulfonylation, allylsulfonylation, dinitrophenyl , Trinitrophenylation, carbamylation, phenylcarbamylation, thiolation and the like. Examples of chemical modification of the carboxyl group located at the C-terminus of the peptide include esterification and amidation. Furthermore, the present peptide can be cationized, and in this case, ethylenediamine or spermine can be used.

  Specific methods for the above-described chemical modification are exemplified below. First, for example, acetylation of a hydroxyl group and an amino group can be performed by allowing acetic anhydride to act in an aqueous solvent or a non-aqueous solvent. The esterification with respect to the carboxyl group can be carried out by passing dry hydrogen chloride gas after suspension in methanol. Furthermore, amidation with respect to a carboxyl group can be performed by acting carbodiimide or the like.

<Salt of peptide or derivative thereof>
The interepidermal cell function-enhancing agent according to the present invention can contain a salt of one or more peptides selected from the group described above or a derivative thereof. The “peptide salt or peptide derivative salt” may be a pharmaceutically acceptable salt, for example, an inorganic acid salt such as hydrochloride, sulfate, phosphate, hydrobromide, acetate, Metal salts such as methanesulfonate, benzenesulfonate, p-toluenesulfonate, succinate, oxalate, fumarate, maleate and other organic acid salts, sodium salt, potassium salt, calcium salt And organic or inorganic ammonium salts such as trimethylammonium salt, triethylammonium salt and ammonium salt. In order to convert the peptide into a peptide salt or a peptide derivative salt, a conventionally known method may be used. Hereinafter, a salt of one or more peptides selected from the above group or a derivative thereof may be referred to as “a salt of the peptide or a derivative of the peptide”.

  The epidermal intercellular function-enhancing agent according to the present invention preferably contains 0.001% by mass or more, more preferably 0.01% by mass or more, of the present peptide, a derivative thereof, or a salt thereof. Furthermore, it is most preferable to contain the present peptide, its derivative or a salt thereof in a total of 0.1% by mass or 1% by mass or more. In terms of molar concentration, the epidermal intercellular function-enhancing agent preferably contains 0.1 mM or more, more preferably 0.5 mM or more of the present peptide, its derivative or a salt thereof. A preferred example of the interepidermal cell function enhancing agent according to the present invention is, for example, the use of “TYPE-S” (manufactured by Nitta Gelatin Co., Ltd.) as a peptide mixture in which two or more of the present peptides are combined. “TYPE-S” includes a total of 3.2% by mass as the peptide mixture described above. Further, “LCP” (manufactured by Nitta Gelatin Co., Ltd.) contains a total of 1.6 mass% as the peptide mixture described above.

  Here, the composition and content of the peptide, derivative thereof or salt thereof contained in the epidermal cell function-enhancing agent according to the present invention are measured using, for example, a liquid chromatograph-tandem mass spectrometer (LC / MS / MS). It can be measured by using.

  Specific measurement conditions for the present peptide using a liquid chromatograph-tandem mass spectrometer (LC / MS / MS) are as follows.

  First, an aqueous sample solution is filtered, and then diluted with an ammonium bicarbonate buffer as necessary to obtain a sample for measurement. In the liquid chromatograph, an ODS column (trade name: “Hypersil GOLD PFP”, manufactured by Thermo Fisher Scientific) is used as the solid phase, and a 2 mM ammonium acetate aqueous solution containing methanol and 0.2% by mass formic acid is used as the mobile phase. . After primary separation of the measurement sample by this liquid chromatograph, the target peptide is quantified by analysis using a mass spectrometer (trade name: “TSQ Vantage”, manufactured by Thermo Fisher Scientific).

<Oral and transdermal agents>
It is preferable that the interepidermal cell function enhancing agent according to the present invention is an oral agent or a transdermal agent. That is, the epidermal intercellular function enhancer can be administered orally or transdermally in various forms. Examples of the dosage form include oral tablets, tablets, granules, capsules, powders, liquids, suspension preparations, emulsion preparations and the like. Further, the oral preparation includes a form in which the present peptide (including a derivative thereof or a salt thereof) contained in the agent for enhancing the function between epidermal cells is mixed with a meal or beverage. Since this peptide hardly decomposes into amino acids in the digestive tract and is rapidly absorbed in the intestine, it is suitable for oral administration.

  When administered transdermally, the epidermal intercellular function-enhancing agent is a transdermal agent, and in this case, the administration forms are application to the skin, injection, transdermal absorption agent, suppository, nasal drop and inhalant. Etc. are exemplified. Examples of the dosage form of the transdermal agent include a liquid agent, a film agent, an ointment, a cream agent, and a cataplasm that are directly applied to the skin.

  The dose of the interepidermal cell function-enhancing agent according to the present invention varies depending on the user's condition, body weight, type of the peptide, route of administration, etc., but in the case of an oral preparation per day for an adult, the content of the peptide is about 0. 0.01 to 2000 mg, preferably about 0.1 to 1000 mg, more preferably about 0.5 to 500 mg, and particularly preferably about 1 to 200 mg. In the case of a transdermal agent, the content of the peptide relative to the entire transdermal agent is about 0.00001 to 20% by mass, preferably about 0.0001 to 10% by mass, more preferably about 0.001 to 5% by mass. . These preparations can be administered in one to several times a day or once to several days. What is necessary is just to adjust so that the sum total of this peptide mixture may become the said dosage or the said content, when it is a peptide mixture which combined 2 or more types of this peptide.

  Furthermore, the carrier when formulating this peptide to be an epidermal intercellular function enhancer includes diluent, binder (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone), excipient (lactose, sucrose) , Corn starch, potassium phosphate, sorbit, glycine), lubricant (magnesium stearate, talc, polyethylene glycol, silica), disintegrant (potato starch), wetting agent (sodium lauryl sulfate), and the like. When applying an epidermal intercellular function enhancer as an oral or transdermal drug or quasi-drug, according to a conventionally known method, the present peptide, a component for formulation (for example, the above-mentioned carrier) and other below-described agents An agent for enhancing the function between epidermal cells can be produced by mixing an active ingredient.

  The epidermal intercellular function-enhancing agent according to the present invention may appropriately contain other active ingredients as long as the effects thereof are not impaired. Examples of other active ingredients include hyaluronic acid. The blending amount of other active ingredients can be appropriately changed within a range that does not impair the effects of the present invention.

  The epidermal intercellular function enhancer according to the present invention is specifically applied to pharmaceuticals, quasi-drugs, cosmetics, foods for specified health use, health foods, etc. It can be administered to animals.

<Action between cells in stratum corneum and granule layer>
The agent for enhancing function between interepidermal cells according to the present invention preferably acts on epidermal cells. Specifically, the epidermal intercellular function enhancer acts on epidermal keratinocytes and contributes to intercellular functions in the stratum corneum or granule layer. The intercellular function in the stratum corneum is the water retention function in the stratum corneum layer of the epidermis formed by the intercellular lipids mainly composed of ceramide, cholesterol, fatty acids, etc., and the entry of foreign substances from the outside This refers to a protective function against external stimuli such as prevention. The intercellular function in the granule layer refers to the moisture retention function in the granule layer of the epidermis that is achieved by the close contact of cells by the formation of tight junctions, and the protection function against external stimuli such as prevention of foreign invasion from the outside. Say. Therefore, the epidermal intercellular function enhancer contributes to the intercellular function in the stratum corneum through, for example, ceramide synthesis promoting action, thereby increasing the amount of the intercellular lipid (specifically, the amount of ceramide). Since the function is enhanced, the barrier function between cells of the epidermis can be enhanced. Furthermore, the function enhancer between epidermal cells enhances the barrier function between cells of the epidermis because, for example, the functions described above are enhanced by increasing the formation density of tight junctions in the granule layer through the action of promoting the formation of tight junctions. it can.

<Ceramide synthesis accelerator>
The interepidermal cell function-enhancing agent according to the present invention has an action of enhancing the synthesis of serine palmitoyltransferase in epidermal cells. Serine palmitoyltransferase is known as the rate-limiting enzyme in the ceramide biosynthesis process in the epidermis as described above. Therefore, it can be used as an index for grasping ceramide synthesis activity in vivo. Therefore, the present invention has the effect of enhancing the synthesis of serine palmitoyltransferase in epidermal cells, so that the amount of ceramide can be maintained or increased between cells in the stratum corneum as a ceramide synthesis promoter.

<Tight junction formation accelerator>
The interepidermal cell function-enhancing agent according to the present invention has an action of enhancing the synthesis of claudin 1 and occludin in epidermal cells. Claudin 1 and occludin are each known as a protein constituting a tight junction in the granule layer in the epidermis as described above. For this reason, it can be used as an index for grasping the formation of a tight junction in a living body. Therefore, the present invention has the effect of enhancing the synthesis of claudin 1 and occludin in epidermal cells, thereby maintaining or increasing the density of tight junctions between cells in the granule layer as an accelerator for tight junction formation. Can be made.

  The administration form and dosage form of the ceramide synthesis promoter and tight junction formation promoter are as described above as the function enhancer between epidermal cells. That is, oral and transdermal agents are preferable. Ceramide synthesis promoters and tight junction formation promoters can be applied to pharmaceuticals, quasi-drugs, cosmetics, foods for specified health use, health foods, etc., or included in various foods, in the same manner as the epidermal intercellular function enhancer. Therefore, it can be administered to humans, mammals and the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

≪Peptide preparation≫
The peptides of Examples 1 to 13 and Comparative Example 1 below were synthesized by the peptide solid phase synthesis method described above. Furthermore, by using the above-mentioned mass spectrometer, it was confirmed that the peptides synthesized in each Example and Comparative Example from the mass were as follows.

<Example 1> Pro-Hyp [PO]
<Example 2> Hyp-Gly [OG]
<Example 3> Glu-Hyp [EO]
<Example 4> Gly-Pro [GP]
<Example 5> Pro-Hyp-Gly [POG]
<Example 6> (Pro-Hyp-Gly) ₂ [(POG) ₂ ]
<Example 7> Pro-Ala-Gly [PAG]
<Example 8> Glu-Hyp-Gly [EOG]
<Example 9> Ser-Hyp-Gly [SOG]
<Example 10> Pro-Ala [PA]
<Example 11> Phe-Hyp [FO]
<Example 12> Ala-Hyp [AO]
<Example 13> Ala-Hyp-Gly [AOG]
<Comparative example 1> Gly-Pro-Hyp [GPO].

  Furthermore, the peptides of Examples 1 to 13 synthesized as described above were combined to prepare peptide mixtures of Examples 14 to 46. In the peptide mixtures of Examples 14 to 46, equal amounts of each peptide were mixed so that the total amount was 0.1 mM (each 0.05 mM).

<Example 14> PO + OG
<Example 15> PO + EO
<Example 16> PO + GP
<Example 17> PO + POG
Example 18 PO + (POG) ₂
<Example 19> PO + PAG
<Example 20> PO + EOG
<Example 21> PO + SOG
<Example 22> PO + PA
<Example 23> PO + FO
<Example 24> PO + AO
<Example 25> PO + AOG
<Example 26> OG + EO
<Example 27> OG + GP
<Example 28> OG + POG
<Example 29> OG + (POG) ₂
<Example 30> OG + PAG
<Example 31> OG + EOG
<Example 32> OG + SOG
<Example 33> OG + PA
<Example 34> OG + FO
<Example 35> OG + AO
<Example 36> OG + AOG
<Example 37> EO + GP
<Example 38> EO + POG
Example 39 EO + (POG) ₂
<Example 40> EO + PAG
<Example 41> EO + EOG
<Example 42> EO + SOG
<Example 43> EO + PA
<Example 44> EO + FO
<Example 45> EO + AO
Example 46 EO + AOG.

<Example 47>
As Example 47, a commercially available peptide mixture (trade name: “TYPE-S”, manufactured by Nitta Gelatin Co., Ltd.) was prepared as a peptide mixture obtained by hydrolyzing collagen.

  When this “TYPE-S” was analyzed using LC / MS / MS (trade name: “TSQ Vantage”, manufactured by Thermo Fisher Scientific) under the above conditions, this peptide mixture contained the following peptides: It was.

One or more peptides selected from the group consisting of Pro-Hyp, Hyp-Gly and Glu-Hyp contain PO: 422 ppm, OG: 14581 ppm, EO: 58 ppm Gly-Pro, Pro-Hyp-Gly, (Pro- Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly As the above peptides, GP: 672 ppm, POG: 340 ppm, (POG) ₂ : 489 ppm, PAG: 12520 ppm, EOG: 308 ppm, SOG: 283 ppm, PA: 2141 ppm, FO: 283 ppm, AO: 120 ppm, AOG: 331 ppm.

<Example 48>
As Example 48, a commercially available peptide mixture (trade name: “LCP”, manufactured by Nitta Gelatin Co., Ltd.) was prepared as a peptide mixture obtained by hydrolyzing collagen.

  This “LCP” was analyzed using the above LC / MS / MS under the same conditions as in the above example. As a result, this peptide mixture contained the following peptides.

One or more peptides selected from the group consisting of Pro-Hyp, Hyp-Gly and Glu-Hyp contain PO: 167 ppm, OG: 545 ppm, EO: 14 ppm Gly-Pro, Pro-Hyp-Gly, (Pro- Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly As the above peptides, GP: 249 ppm, POG: not detected, (POG) ₂ : 56 ppm, PAG: 15568 ppm, EOG: 126 ppm, SOG: 3 ppm, PA: 105 ppm, FO: not detected, AO: 50 ppm, AOG: 29 ppm.

  In the analysis by LC / MS / MS, a peptide whose content is described as “non-detected” means that the content is below the detection limit or not contained.

≪RNA expression level confirmation test≫
In this test, it was examined whether or not the RNA expression level of the target gene described later in epidermal cells was increased by the addition of the peptides or peptide mixtures of the above-described Examples and Comparative Examples. The results are shown in Tables 1-6.

In this test, human normal epidermal keratinocytes NHEK (NB) (manufactured by Kurashiki Boseki Co., Ltd.) were used as epidermal cells. The cells were seeded in a commercial petri dish at 3.6 × 10 ⁴ cells / cm ² and cultured in a serum-free medium (trade name: “HuMedia KG-2”, manufactured by Kurashiki Boseki Co., Ltd.) for 7 days. . After confirming that the cells were subconfluent in the petri dish, the medium in the petri dish was replaced with a serum-free medium that does not contain growth factors (trade name: “MCDB153 medium”, manufactured by Functional Peptide Institute, Inc.) The peptides or peptide mixtures of the above examples and comparative examples were added in three samples at a concentration as shown in Tables 1 to 6 and cultured for 72 hours in an atmosphere of 37 ° C. and a carbon dioxide concentration of 5% by volume. Thereafter, total RNA was extracted from the cells for each sample. Subsequently, this extract was subjected to reverse transcription using a conventionally known method to obtain cDNA. Real-time RT-PCR was performed on the obtained cDNA with an apparatus (trade name: “Step One Plus”, manufactured by Applied Biosystems).

  In real-time RT-PCR, the amount of mRNA of serine palmitoyltransferase (primer: Hs00272311_ml), claudin 1 (primer: Hs00221623_ml) and occludin (primer: Hs00170162_ml) as target genes was measured for each sample of the above Examples and Comparative Examples. GAPDH was selected as the internal standard (corrected gene). The calibration curve method was used for the calculation. For real-time RT-PCR, a reagent kit (trade name: “TaqMan (registered trademark) Gene Expression Assays”, manufactured by Applied Biosystems) was used.

  Data obtained from RT-PCR was analyzed as follows. First, mRNA amounts (gene expression levels) of three target genes were calculated. Next, GAPDH mRNA expression levels were corrected using the gene expression levels of the three target genes as correction genes. Specifically, the values (relative values) obtained by dividing the gene expression levels of the three target genes by the GAPDH mRNA expression levels were determined.

  Tables 1 and 2 show the relative values of serine palmitoyltransferase gene expression levels in each Example and Comparative Example. Tables 3 and 4 show the relative values of claudin 1 gene expression levels in the examples and comparative examples. Tables 5 and 6 show the relative values of the occludin gene expression levels in the examples and comparative examples. The values in each table are the average value ± standard deviation of three samples in each example and comparative example. * And ** in each table indicate that there is a significant difference at the level of P <0.05 and P <0.01, respectively, with respect to the control in Paired-t-test.

≪Evaluation results≫
As understood from Tables 1 and 2, the peptides of this example, namely Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro -One or more peptides selected from the group consisting of Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly are serine palmitoyl Increased the gene expression of transferase. As a result, the interdermal function enhancer containing this peptide can maintain or increase the amount of ceramide between cells in the stratum corneum in the epidermis by promoting ceramide synthesis as a ceramide synthesis promoter. You can see that

As understood from Tables 3 to 6, the peptides of this example, namely Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro One or more peptides selected from the group consisting of Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly are claudin 1 and occludin gene expression levels were increased. As a result, the epidermal intercellular function-enhancing agent containing this peptide can maintain the tight junction density by promoting the formation of tight junctions between the cells of the granule layer in the epidermis, or the tight junction formation promoter. It can be seen that the density can be increased.

≪Sensory test≫
Based on the above evaluation results, the peptides of this example, namely Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly Enhancement of interepidermal cells containing one or more peptides selected from the group consisting of Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly The agent was formulated for cosmetics (cosmetics) and food / beverage products and subjected to a sensory test, which will be described below. In each formulation example, each product used for this is manufactured by a conventional method. For this reason, only the content of each product was shown in each prescription example. Furthermore, in each formulation example, “appropriate amount” means within a range that does not impair the effects of the present invention, and a conventionally known content range, specifically, from 0.001 to 10% by mass. The amount selected.

<Use effect test using lotion>
In this test, 10 healthy women who were aware of dry skin of 33-35 years old were subjects, and lotions (Example 49 and Comparative Example 2) having the compositions shown in Table 7 (the unit of numerical values is mass%) were used. ) Twice a day (morning and evening) by applying to the face after washing. Specifically, 5 of the 10 female subjects were allowed to use the lotion of Example 49, and the remaining 5 subjects were allowed to use the lotion of Comparative Example 2. For these 10 test subjects, 4 weeks after the start of the test, the skin moist feeling, skin texture, and makeup paste were evaluated by a 5-point score (1 point: worsened), with 3 points at the start of the test. 2 points: slightly deteriorated, 3: unchanged, 4 points: slightly improved, 5 points: improved). Table 8 shows the average score as a result of hearing. None of the subjects complained of health or skin abnormalities during the period of use.

  As can be seen from Table 8, the skin lotion to which the peptide mixture of this example was applied was superior to the comparative example in each evaluation of moisturizing skin, skin texture, and makeup paste. Therefore, the interepidermal cell function-enhancing agent according to the present invention contributes to the intercellular function in the stratum corneum or granule layer through the action on the percutaneous epidermal keratinocytes, thereby improving the skin barrier function of the epidermis. It is suggested that there is.

<Use effect test using beauty drinks>
In this test, 10 healthy women who are aware of dry skin of 31 to 56 years old were subjects, and cosmetic drinks (Example 50 and Comparative Example 3) having the compositions shown in Table 9 (the unit of numerical values is% by mass). ) Was taken once a day (20 g). Specifically, 5 of 10 female subjects were allowed to drink the beauty drink of Example 50, and the remaining 5 subjects were allowed to drink the beauty drink of Comparative Example 3. For these 10 test subjects, 4 weeks after the start of the test, the skin moist feeling, skin texture, and makeup paste were evaluated by a 5-point score (1 point: worsened), with 3 points at the start of the test. 2 points: slightly deteriorated, 3: unchanged, 4 points: slightly improved, 5 points: improved). Table 10 shows the number of points interviewed as the average value. None of the subjects complained of health or skin abnormalities during the period of use.

  As can be seen from Table 10, the beauty drink to which the peptide mixture of this example was applied was superior to the comparative example in each evaluation of moisturizing skin, skin texture, and makeup paste. Therefore, the epidermal intercellular function enhancer according to the present invention contributes to the intercellular function in the stratum corneum or granule layer through the action on oral epidermal keratinocytes, thereby improving the skin barrier function of the epidermis. It is suggested that there is.

≪Other prescription examples≫
Based on the above evaluation results, the peptides of this example, namely Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly Enhancement of interepidermal cells containing one or more peptides selected from the group consisting of Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly Other prescription examples as cosmetics (cosmetics) and food / beverage products are shown below. In each formulation example, each product used for this is manufactured by a conventional method. For this reason, only the content of each product was shown in each prescription example. Furthermore, in each formulation example, “appropriate amount” means within a range that does not impair the effects of the present invention, and a conventionally known content range, specifically, from 0.001 to 10% by mass. The amount selected.

<Prescription Example 1> Creamy serum
1. Water balance Methyl Gurces-10 5
3. Glycerin 10
4.1,3-Butylene glycol 10
5. Arginine 0.8
6). Carbomer 0.3
7). Preservative appropriate amount 8. Peptide mixture of Example 48 0.1.

<Prescription Example 2> Shampoo Mass%
1. Water balance Lauryl sulfate TEA 10
3. Cocoyl glutamic acid K 2
4). Lauramidopropyl betaine 1.5
5. Cocamide DEA 3
6). Guar hydroxytrimonium chloride 0.1
7). Preservative appropriate amount 8. pH adjuster appropriate amount9. Perfume appropriate amount10. Peptide mixture of Example 48 0.1.

<Prescription Example 3> Hair Treatment Mass%
1. Water balance Stearyl trimethyl ammonium chloride 3
3. Cetanol 5
4). Glycerol monostearate 1
5. Cetyl ethylhexanoate 1
6). Olive oil 2
7). Preservative appropriate amount 8. pH adjuster appropriate amount9. Perfume appropriate amount10. Peptide mixture of Example 48 0.1.

<Prescription Example 4> Jelly mass%
1. Water balance Sugar 2
3. Fructose glucose liquid sugar 10
4). Apple juice 2
5. Malic acid 0.1
6). Tartaric acid 0.1
7). Trisodium citrate 0.1
8). Gelling agent 1
9. Perfume appropriate amount10. Peptide mixture of Example 48

<Prescription Example 5> Tablet mass%
1. Sorbitol remainder 2. Sucrose fatty acid ester 2
3. Sucralose 0.1
4). Malic acid 2
5. Grapefruit flavor appropriate amount 6. Lemon flavor appropriate amount 7. Peptide mixture of Example 48

<Formulation example 6> Cookie mass%
1. Flour 40
2. Edible oils and fats 10
3. Sugar 20
4). Whole egg 15
5. Nonfat dry milk 10
6). Cornstarch 3
7). Salt 0.2
8). Perfume appropriate amount 9. Peptide mixture of Example 48

  Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined and variously modified. .

  The embodiments and examples disclosed herein are illustrative in all respects and should not be construed as being restrictive. The scope of the present invention is shown not by the above-described embodiment but by the scope of claims, and is intended to include meanings equivalent to the scope of claims and all modifications within the scope.

Claims (5)

Pro-Hyp, Hyp-Gly, Glu-Hyp, Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro -An epidermal intercellular function enhancer containing one or more peptides selected from the group consisting of Ala, Phe-Hyp, Ala-Hyp and Ala-Hyp-Gly, derivatives thereof or salts thereof. One or more peptides selected from the group consisting of Pro-Hyp, Hyp-Gly and Glu-Hyp, derivatives thereof or salts thereof;
Gly-Pro, Pro-Hyp-Gly, (Pro-Hyp-Gly) ₂ , Pro-Ala-Gly, Glu-Hyp-Gly, Ser-Hyp-Gly, Pro-Ala, Phe-Hyp, Ala-Hyp and Ala -An epidermal intercellular function-enhancing agent containing at least one peptide selected from the group consisting of Hyp-Gly, a derivative thereof, or a salt thereof.   The interepidermal cell function enhancing agent according to claim 1 or 2, wherein the interepidermal cell function enhancing agent is an oral agent or a transdermal agent.   The interepidermal cell function enhancing agent according to any one of claims 1 to 3, wherein the interepidermal cell function enhancing agent acts on epidermal cells.   The interepidermal cell function enhancer according to any one of claims 1 to 4, wherein the interepidermal cell function enhancer is a ceramide synthesis promoter or a tight junction formation promoter.