Aging process inhibitors

Abstract

An aging process inhibitor comprising peptide Gly-Pro and / or peptide Glu-Hyp-Gly, their salts or chemically modified products thereof.

Description

Technical Field

[0001] The present invention relates to an aging process inhibitor, and food or drink containing the same. Background Art

[0002] One of the causes of aging may be oxidative stress given to various cells by active oxygen species, peroxides, etc. For example, the following non-patent document 1 reports that hair turns white (hereinafter also referred to as "bleaching") due to accumulation of active oxygen species or peroxides in cells forming hair follicles. In addition, the following non-patent documents 2 and 3 report that the reduction of type 17 collagen promotes hair loss and bleaching accompanying aging. Japanese Patent Laid-Open No. 2009-161509 (Patent Document 1) discloses that type 17 collagen has the function of inhibiting hair loss and bleaching.

[0003] Citation List

[0004] Patent Literature

[0005] PTL1: Japanese Patent Publication No. 2009-161509

[0006] Non-patent literature

[0007] NPL1: JM Wood et al.,FASEB J,2009,Vol 23,No.7,pp.2065-2075

[0008] NPL2: Matsumura H et al.,Science,2016,Vol 351,pp.575,add4395-1,2

[0009] NPL3: Tanimura S et al., Cell Stem Cell, 2011, Vol 8, pp.177-187 Summary of the invention

[0010] Technical issues

[0011] On the other hand, a collagen peptide mixture obtained by hydrolyzing collagen or gelatin using a known proteolytic enzyme is known. The collagen peptide mixture is reported to have a variety of physiological activities in joints, bones, cartilage, skin, etc. in living organisms. However, it has not been reported so far that the collagen peptide mixture has an inhibitory effect on hair loss and decolorization. Glutathione is known to be a peptide with a so-called antioxidant effect of removing active oxygen species and peroxides from living organisms, but it has not been reported that the collagen peptide mixture involves glutathione synthesis. Therefore, extensive research has been conducted to explore the aging process inhibitory effect, especially the inhibitory effect on hair loss and decolorization, the glutathione synthesis promotion effect, etc., as new physiological activities of collagen peptide mixtures and collagen-derived peptides contained in collagen peptide mixtures.

[0012] In view of the above situation, the object of the present invention is to provide an aging process inhibitor and a food or drink containing the aging process inhibitor, wherein the aging process inhibitor includes a peptide, etc., and the peptide has at least one of a promoting effect on type 17 collagen gene expression and a promoting effect on glutathione synthetase gene expression, thereby being able to inhibit hair loss and depigmentation or enhance the antioxidant effect.

[0013] Solution to the Problem

[0014] While exploring new physiological activities of the collagen peptide mixture, the present inventors found that the predetermined peptide contained in the collagen peptide mixture exhibits at least one of a promoting effect on type 17 collagen gene expression and a promoting effect on glutathione synthetase gene expression. Based on this finding, an aging process inhibitor containing a peptide to provide an inhibitory effect on hair loss and decolorization or an antioxidant effect enhancement effect was obtained, thereby completing the present invention.

[0015] Specifically, the present invention is as follows.

[0016] The aging process inhibitor according to the present invention includes both or one of the peptides Gly-Pro and Glu-Hyp-Gly, or a salt thereof or a chemically modified product thereof.

[0017] Preferably, the peptide is derived from collagen.

[0018] Preferably, the aging process inhibitor is a collagen peptide mixture comprising any peptides.

[0019] Preferably, the weight average molecular weight of the collagen peptide is greater than or equal to 100 Da and less than or equal to 5000 Da.

[0020] Preferably, the aging process inhibitor is a type 17 collagen gene expression promoter or a glutathione synthetase gene expression promoter.

[0021] The food or drink according to the present invention contains the aging process inhibitor.

[0022] Beneficial Effects of the Invention

[0023] According to the present invention, an aging process inhibitor capable of producing an inhibitory effect on hair loss and discoloration or an antioxidant effect enhancing effect, and a food or drink containing the same can be provided. DETAILED DESCRIPTION

[0024] In the following, embodiments of the present invention will be described in more detail. As used herein, descriptions in the form of "A to B" represent the upper and lower limits of a range (i.e., above A and below B), and when no unit is described for A, and only a unit is described for B, the unit of A is the same as the unit of B.

[0025] [Aging process inhibitors]

[0026] The aging process inhibitor according to the present invention includes both or one of the peptides Gly-Pro and Glu-Hyp-Gly, their salts or chemically modified products thereof. The aging process inhibitor having such characteristics can have a promoting effect on the expression of type 17 collagen gene or a promoting effect on the expression of glutathione synthetase gene, thereby obtaining an inhibitory effect on hair loss and depigmentation or an antioxidant effect enhancement effect.

[0027] [Both or one of the peptides Gly-Pro and Glu-Hyp-Gly, their salts or chemically modified products thereof]

[0028] As described above, the aging process inhibitor comprises both or one of the peptides Gly-Pro and Glu-Hyp-Gly, a salt thereof or a chemically modified product thereof. In the present specification, unless otherwise specified, the "amino acid" forming the peptide is represented by a three-character abbreviation. In addition, unless otherwise specified, "amino acid" refers to an L-type amino acid. In addition, for the "peptide" in the present specification, for example, "Gly-Pro" refers to a peptide (dipeptide) in which glycine and proline are arranged in the order from the N-terminal side to the C-terminal side, and "Glu-Hyp-Gly" refers to a peptide (tripeptide) in which glutamic acid, hydroxyproline and glycine are arranged in the order from the N-terminal side to the C-terminal side. The same applies to the description of peptides other than "Gly-Pro" and "Glu-Hyp-Gly".

[0029] Preferably, the aging process inhibitor comprises both or one of the peptides Gly-Pro and Glu-Hyp-Gly, or a salt thereof or a chemically modified product thereof. In this case, the aging process inhibitor can more significantly exhibit a promoting effect on the expression of type 17 collagen gene or a promoting effect on the expression of glutathione synthetase gene.

[0030] The term "salt" of a peptide is, for example, an inorganic acid salt such as hydrochloride, sulfate or phosphate; an organic acid salt such as methanesulfonate, benzenesulfonate, succinate or oxalate; an inorganic base salt such as sodium, potassium or calcium salt; an organic base salt such as triethylammonium salt of the peptide.

[0031] The "chemically modified product" of a peptide refers to a compound in which the free functional groups of the amino acid residues as the constituent units are chemically modified. Chemical modification can be performed, for example, on the hydroxyl group of hydroxyproline, on the amino group of the amino acid on the N-terminal (amino terminal) side, and on the carboxyl group of the amino acid on the C-terminal (carboxyl terminal) side. For the specific manner and treatment conditions of chemical modification, known conventional chemical modification techniques for amino acids and peptides are applied. The chemically modified product of each amino acid and peptide obtained by this chemical modification can enhance solubility under weakly acidic to neutral conditions, enhance compatibility with other active ingredients, etc.

[0032] For example, as a chemical modification of the hydroxyl group in hydroxyproline, the tripeptide Glu-Hyp-Gly can be O-acetylated. The O-acetylation can be performed by applying acetic anhydride to the peptide in an aqueous solvent or a non-aqueous solvent. As a chemical modification of the carboxyl group in glycine, esterification, amidation, etc. can be performed. The esterification can be performed by suspending the peptide in methanol and then passing dry hydrogen chloride gas through the resulting suspension. The amidation can be performed by applying carbodiimide, etc. to the peptide.

[0033] As chemical modification of the free amino group in the peptide, methylation may be performed, and as chemical modification of the free hydroxyl group in the peptide, at least one of phosphorylation and sulfation may be performed.

[0034] Preferably, the peptide is derived from collagen. In this article, the collagen as a raw material can be obtained in the following manner, for example, on the skin, dermis, bone, cartilage, tendon, etc. of an animal (usually a cow, pig, sheep, chicken or ostrich) or on the bone, skin, scale, etc. of a fish, known conventional defatting or decalcification treatment, extraction treatment, etc. are carried out. In addition, gelatin can be used as a raw material for peptides. The gelatin can be obtained by treating the collagen thus obtained by a known conventional method (e.g., extraction with hot water). For collagen and gelatin, commercially available products can be used as raw materials.

[0035] Peptides can be obtained by hydrolyzing collagen and / or gelatin with two or more endoproteases and exoproteases in combination. By hydrolysis, peptides can be obtained as a collagen peptide mixture, in which the peptides are present together with other collagen peptides. The collagen peptide mixture itself and the mixture obtained by partially purifying the collagen peptide mixture can be used as an aging process inhibitor according to the present invention. That is, the aging process inhibitor is preferably a collagen peptide mixture. In addition, by further purifying the collagen peptide mixture, a purified product containing the peptide in high purity can be obtained. When the peptide is derived from collagen, it is preferably obtained by using a method as described below in which collagen or gelatin is enzymatically treated in two stages.

[0036] In addition, the weight average molecular weight of the collagen peptide mixture is preferably 100Da or more and 5000Da or less. The weight average molecular weight of the collagen peptide mixture is more preferably 120Da or more and 3500Da or less, and more preferably 150Da or more and 3000Da or less. When the weight average molecular weight of the collagen peptide mixture is within the above range, the aging process inhibitor can fully produce the promoting effect on the expression of type 17 collagen gene or the promoting effect on the expression of glutathione synthetase gene. If the weight average molecular weight is greater than 5000Da, the above-mentioned effect of the aging process inhibitor may be insufficient.

[0037] The weight average molecular weight of the collagen peptide mixture can be determined by performing size exclusion chromatography (SEC) under the following measurement conditions.

[0038] Instrument: High Performance Liquid Chromatography (HPLC) (manufactured by TOSOH CORPORATION)

[0039] Column: TSKGel (registered trademark) G2000SW XL

[0040] Column temperature: 40°C

[0041] Column size: 7.8mm (ID) × 30cm, 5μm

[0042] Eluent: 45% by mass acetonitrile (containing 0.1% by mass trifluoroacetic acid)

[0043] Flow rate: 1.0mL / min

[0044] Injection volume: 10 μL

[0045] Detection: UV 214nm

[0046] Molecular weight markers: The following five types are used

[0047]

[0048] Specifically, a sample containing about 0.2 g of the collagen peptide mixture is added to about 100 ml of distilled water, the mixture is stirred, and then filtered with a 0.2 μm filter to prepare a sample for measuring the weight average molecular weight (measurement sample). By using size exclusion chromatography on the measurement sample, the weight average molecular weight of the collagen peptide mixture can be determined.

[0049] [Method for producing an aging process inhibitor]

[0050] The peptide contained in the aging process inhibitor can be obtained by a known conventional method. For example, the peptide can be obtained by purchasing commercially available amino acids. The peptide can also be obtained by using a method involving hydrolyzing collagen or gelatin.

[0051] The peptides (both or one of Gly-Pro and Glu-Hyp-Gly) can be obtained by known conventional liquid phase or solid phase peptide synthesis methods, or methods involving hydrolysis of collagen or gelatin. From the viewpoint of efficiency, the peptide is preferably prepared by using a chemical synthesis method using amino acids as described below, or a method comprising enzymatic treatment of collagen or gelatin in two stages as described below. In addition, the peptide can be produced by a method of enzymatic treatment using only a secondary enzyme without a primary enzyme, or a method of enzymatic treatment using both a primary enzyme and a secondary enzyme, rather than a method of enzymatic treatment of collagen or gelatin in two stages. Below, as an example of a method for producing a peptide contained in an aging process inhibitor, a method for producing a peptide contained in an aging process inhibitor, especially "Glu-Hyp-Gly", is described.

[0052] <Chemical Synthesis Method>

[0053] The peptide can be obtained using a conventional peptide synthesis method. As a peptide synthesis method, a solid phase synthesis method and a liquid phase synthesis method are known. As a solid phase synthesis method, an Fmoc method and a Boc method are known. The peptide can be obtained using any of the Fmoc method and the Boc method. As a solid phase peptide synthesis method, a method for synthesizing a tripeptide represented by Glu-Hyp-Gly can be performed as follows.

[0054] First, beads of a polystyrene polymer gel having a diameter of about 0.1 mm and a surface modified with amino groups are provided as a solid phase. In addition, diisopropylcarbodiimide is provided as a condensing agent. Next, the amino group of glycine is protected with an Fmoc (fluorenyl-methoxy-carbonyl) group, which is an amino group on the C-terminal (carboxyl terminal) side of the amino acid sequence, and the carboxyl peptide of glycine is bound to the amino group as a solid phase by a dehydration reaction using a condensing agent. In addition, the solid phase is washed with a solvent to remove the residual condensing agent and amino acid, and then the protective group (deprotection) of the amino group of glycine bound to the solid phase peptide is removed.

[0055] Subsequently, hydroxyproline in which the amino group is protected by an Fmoc group is provided, and the carboxyl peptide of hydroxyproline is bound to the deprotected amino group of glycine using a condensing agent. Thereafter, in the same manner as above, the amino group of hydroxyproline is deprotected, glutamic acid protected by an Fmoc group is provided, and a reaction of binding glutamic acid to the hydroxyproline peptide is performed, thereby synthesizing a tripeptide represented by Glu-Hyp-Gly as a solid phase.

[0056] Finally, the tripeptide can be prepared by deprotecting the amino group of glutamic acid and separating the tripeptide from the solid phase by immersion in trifluoroacetic acid under heating.

[0057] <Production method using collagen and gelatin>

[0058] Furthermore, a method of enzymatically treating collagen or gelatin in two stages to produce a tripeptide represented by Glu-Hyp-Gly can be performed as follows.

[0059] The term "two-stage enzyme treatment (collagen or gelatin)" refers to the following. That is, primary enzyme treatment is carried out by a known conventional method for destroying the peptide bonds of collagen or gelatin, and then secondary enzyme treatment is carried out with an enzyme having aminopeptidase N activity, an enzyme having both aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity, or an enzyme having aminopeptidase N activity and an enzyme having prolyl tripeptidyl aminopeptidase activity. By carrying out the primary enzyme treatment, a collagen peptide mixture precursor can be obtained. By further carrying out the secondary enzyme treatment, a collagen peptide mixture containing Glu-Hyp-Gly can be obtained from the collagen peptide mixture precursor. The method for two-stage enzyme treatment of collagen or gelatin will be described in more detail below.

[0060] (Primary enzyme treatment)

[0061] The enzyme used in the primary enzyme treatment is not particularly limited as long as it is an enzyme that can destroy the peptide bonds of collagen or gelatin, and any proteolytic enzyme can be used. Specifically, examples include collagenase, thiol protease, serine protease, acid protease, alkaline protease and metalloprotease. One selected from the group consisting of these enzymes can be used alone, or two or more thereof can be used in combination. As thiol protease, chymopapain, papain, bromelain and ficin derived from plants, tissue proteases and calcium-dependent proteases derived from animals, etc. can be used. As serine protease, trypsin, cathepsin D, etc. can be used. As acid protease, pepsin, chymotrypsin, etc. can be used. Considering that the aging process inhibitor of the present invention is used for drugs, specific health foods, etc., it is preferred that an enzyme other than an enzyme derived from a pathogenic microorganism is used as the enzyme used in the primary enzyme treatment.

[0062] Based on 100 parts by mass of collagen or gelatin, the amount of enzyme in the primary enzyme treatment is, for example, preferably 0.1 to 5 parts by mass of the above enzyme. Preferably, the treatment temperature and treatment time in the primary enzyme treatment are 30 to 65 ° C and 10 minutes to 72 hours, respectively. The weight average molecular weight of the collagen peptide mixture precursor obtained by the primary enzyme treatment is preferably 500 to 20000Da, more preferably 500 to 10000Da, and even more preferably 500 to 8000Da. It can be said that when the weight average molecular weight is within the above range, peptides with appropriate molecular weight can be appropriately produced. If necessary, the enzyme can be inactivated after the primary enzyme treatment. In this case, the inactivation temperature is, for example, preferably 70 to 100 ° C. The weight average molecular weight of the collagen peptide mixture precursor can be determined by using the SEC method.

[0063] (Secondary enzyme treatment)

[0064] Examples of enzymes used in the secondary enzyme treatment include enzymes having aminopeptidase N activity, enzymes having both aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity, and combinations of enzymes having aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity. As used herein, the term "enzyme having aminopeptidase N activity" is a peptidase having the function of releasing an amino acid from the N-terminal side of a peptide chain, wherein the enzyme acts when an amino acid other than proline or hydroxyproline is present at the second position on the N-terminal side. As used herein, the term "enzyme having prolyl tripeptidyl aminopeptidase activity" is a peptidase that releases only three amino acid residues from the N-terminal side of a peptide having proline or hydroxyproline at the third position on the N-terminal side. Considering that the aging process inhibitor of the present invention is used for drugs, specific health foods, etc., preferably, as the enzyme used in the secondary enzyme treatment, an enzyme other than an enzyme derived from a pathogenic microorganism is used.

[0065] Examples of enzymes having aminopeptidase N activity include aminopeptidase N (EC 3.4.11.2.; T. Yoshimoto et al., Agric. Biol. Chem., 52: 217-225 (1988)), and enzymes having aminopeptidase N activity derived from Aspergillus. Examples of enzymes having prolyl tripeptidyl aminopeptidase activity include prolyl tripeptidyl aminopeptidase (EC 3.4.14.; A. Banbula et al., J. Biol. Chem., 274: 9246-9252 (1999)).

[0066] By performing the secondary enzyme treatment, a collagen peptide mixture containing peptides not contained in the collagen peptide mixture precursor can be obtained. Specifically, a collagen peptide mixture containing Glu-Hyp-Gly can be obtained.

[0067] Based on 100 parts by mass of the collagen peptide mixture precursor, the amount of the enzyme in the secondary enzyme treatment is, for example, preferably 0.01 to 5 parts by mass of the above enzyme. Preferably, the treatment temperature and treatment time in the secondary enzyme treatment are 30 to 65°C and 10 minutes to 72 hours, respectively. The weight-average molecular weight of the collagen peptide mixture obtained by the secondary enzyme treatment is preferably 100 to 5000Da, more preferably 120 to 3500Da, and even more preferably 150 to 3000Da. As described above, the weight-average molecular weight of the collagen peptide mixture can also be determined by using the SEC method.

[0068] The secondary enzyme treatment is mainly carried out to produce Glu-Hyp-Gly peptides. Therefore, it is preferred to adjust the amount of enzyme, treatment temperature, treatment time and pH in the secondary enzyme treatment so that the peptides contained in the collagen peptide mixture precursor are not over-hydrolyzed. Therefore, the weight-average molecular weight of the collagen peptide mixture is preferably within the above range. The enzyme must be inactivated after the secondary enzyme treatment. In this case, the inactivation temperature is preferably 70 to 100°C, for example. In addition, it is preferred to perform a sterilization treatment at 120°C for several seconds or longer. In addition, the collagen peptide mixture can be spray-dried by heating at 200°C or higher.

[0069] In the secondary enzyme treatment, not only can use the enzyme with aminopeptidase N activity and the enzyme with prolyl tripeptidyl aminopeptidase activity, but also can use enzymes with different activities, and can use two or more enzymes with different activities respectively in combination.Therefore, by-products can be digested and removed.Preferably, according to the type of collagen used as raw material and the type of enzyme used in the primary enzyme treatment, the enzyme used in this case is appropriately selected.The example of different activities includes dipeptidase activity, such as proline activity and hydroxyproline activity.Therefore, by-products (such as dipeptides) can be digested and removed.

[0070] In addition, aminopeptidase N activity is basically an activity that causes the amino acids on the N-terminal side to be released one by one. Therefore, in the case where the collagen peptide mixture precursor obtained by the primary enzyme treatment contains a very large peptide of molecular weight, when only the enzyme with aminopeptidase N activity is used to treat the secondary enzyme, the duration of the secondary enzyme treatment is significantly increased. In order to cope with this situation, for example, prolyl oligopeptidase can be used in the secondary enzyme treatment, and the proline oligopeptidase is an endopeptidase with the activity (proline enzyme activity) that causes the hydrolysis of carboxyl side proline. Therefore, the secondary enzyme treatment can be effectively carried out.

[0071] In a method including enzymatic treatment of collagen or gelatin in two stages, the primary enzyme treatment can produce a peptide with a relatively large molecular weight. The peptide can have, for example, an amino acid sequence represented by [X1-Gly-X2-Glu-Hyp-Gly] (X1 and X2≠Hyp). In the subsequent secondary enzyme treatment, an enzyme with aminopeptidase N activity acts on the peptide represented by [X1-Gly-X2-Glu-Hyp-Gly] to release the X1 at the N-terminus to obtain a peptide with an amino acid sequence represented by [Gly-X2-Glu-Hyp-Gly]. Next, an enzyme with aminopeptidase N activity acts twice on the peptide represented by [Gly-X2-Glu-Hyp-Gly] to release glycine and X2 to obtain a peptide represented by [Glu-Hyp-Gly].

[0072] (Purification of collagen peptide mixture)

[0073] By performing enzyme treatment in two stages as described above, a collagen peptide mixture containing Glu-Hyp-Gly can be prepared. Since the collagen peptide mixture contains peptides other than the tripeptide represented by Glu-Hyp-Gly, if necessary, the collagen peptide mixture is preferably purified. As a purification method in this case, known conventional methods can be used, examples of which include ultrafiltration and various types of liquid chromatography, such as size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and affinity chromatography.

[0074] Specifically, the collagen peptide mixture can be purified according to the following steps. That is, about 2g / 10ml of the collagen peptide mixture is loaded into an ion exchange column (e.g., "TOYOPEARL" (registered trademark) DEAE-650" (trade name) manufactured by TOSOH CORPORATION), and then the first pore volume fraction eluted with distilled water is collected. Subsequently, the first pore volume fraction is loaded into a column having an ion exchange group opposite to that of the above-mentioned ion exchange column (e.g., "TOYOPEARL" (registered trademark) SP-650 manufactured by TOSOH CORPORATION), and then the second pore volume fraction eluted with distilled water is collected.

[0075] Next, the second pore volume fraction is loaded into a gel filtration column (e.g., "SEPHADEX LH-20" (trade name) manufactured by GE Healthcare Japan Corporation), and eluted with a 30% by mass methanol aqueous solution to collect a fraction containing the Glu-Hyp-Gly peptide. Finally, the pore volume fraction is separated using a reverse phase column (e.g., "μBondasphere 5μC18 The fractions were fractionated by high performance liquid chromatography (HPLC) using a HPLC Column (trade name) according to a linear concentration gradient of an acetonitrile aqueous solution of 32 mass % or less containing 0.1 mass % of trifluoroacetic acid. In this way, Glu-Hyp-Gly can be obtained with high purity.

[0076] [Promoter of type 17 collagen gene expression or promoter of glutathione synthetase gene expression]

[0077] Preferably, the aging process inhibitor according to the present invention is a promoter of type 17 collagen gene expression or a promoter of glutathione synthetase gene expression. As described above, the aging process inhibitor comprises both or one of the peptides Gly-Pro and Glu-Hyp-Gly, their salts or chemically modified products thereof. This makes it possible to exhibit a promoting effect on type 17 collagen gene expression. Therefore, the aging process inhibitor, as a promoter of type 17 collagen gene expression, promotes type 17 collagen gene expression, thereby suppressing hair loss and decolorization. Type 17 collagen gene expression promoters promote type 17 collagen gene expression, and therefore can be expected to have an inhibitory effect on the progression of age-related thinning hair, hair loss and gray hair, a skin beautification promoting effect, and the like.

[0078] In addition, the aging process inhibitor contains a peptide, a salt thereof, or a chemically modified product thereof, and thus has a promoting effect on glutathione synthetase gene expression. Therefore, the aging process inhibitor, as a promoter of glutathione synthetase gene expression, promotes glutathione synthetase gene expression, and thus can remove active oxygen species, peroxides, etc. from living organisms. The promoter of glutathione synthetase gene expression can remove active oxygen species, peroxides, etc. from living organisms, and thus can also be expected to have the following effects: such as skin whitening based on the inhibition of pigmentation caused by inflammation, skin beautification based on the inhibition of eczema, promotion of corneal injury healing, improvement of liver function, and improvement of Parkinson's disease.

[0079] The aging process inhibitor can be administered orally or parenterally in a variety of forms. For these forms, the aging process inhibitor can take the following dosage forms when administered orally, such as tablets, granules, capsules, powders, liquids, suspensions, and emulsions. In addition, the aging process inhibitor in any of the above dosage forms can also be mixed with food or drink. The aging process inhibitor includes any peptide that is rapidly absorbed in the intestine and can therefore be administered orally.

[0080] When administered parenterally, the aging process inhibitor may be in the form of an external preparation such as an ointment, cream and lotion, and a transdermal preparation, etc. In addition, the aging process inhibitor may be directly applied to the head skin in the form of a solution or a coating. When the aging process inhibitor is used as a coating, the concentration of the peptide or the like contained in the coating is preferably 0.001 to 5% by mass.

[0081] The dosage of the aging process inhibitor varies according to the age, sex, weight and sensitivity differences of the subject, the method of administration, the interval between administrations, the type of preparation, etc. When the aging process inhibitor is administered orally, the dosage per adult is, for example, preferably 0.0001 to 2500 mg / kg, more preferably 0.0001 to 500 mg / kg. When the dosage form of the aging process inhibitor is, for example, a tablet, the amount of the aging process inhibitor contained in the tablet may be 0.001 to 80% by mass per tablet, and when the dosage form of the aging process inhibitor is, for example, a powder, the amount of the aging process inhibitor contained in the powder may be 0.001 to 100% by mass. When the aging process inhibitor is administered parenterally or by another form of preparation, the dosage may be appropriately determined with reference to the dosage for oral administration. The aging process inhibitor may be administered once a day or in several doses, or once a day or once every few days.

[0082] The aging process inhibitor may appropriately include other active ingredients, formulation carriers, etc., as long as the effect of the present invention is not adversely affected. The examples of other active ingredients include inulin, caffeic acid, quinic acid, its derivatives, marjoram extract, crude drugs such as Kinfukan, motherwort (polygala), Hakubiso and Desmos chinensis Lour, royal jelly, Echinacea extract, acai berry extract, and Cupuacu extract, etc. In addition, the example of a pharmaceutically acceptable carrier for being formulated into a pharmaceutical preparation includes a diluent, a binder (syrup, gum arabic, gelatin, sorbitol, tragacanth and polyvinyl alkanoate), an excipient (lactose, sucrose, corn starch, potassium phosphate, sorbitol and glycine), a lubricant (magnesium stearate, talc, polyethylene glycol and silicon dioxide), a disintegrant (potato starch) and a wetting agent (sodium lauryl sulfate).

[0083] [Use invention]

[0084] As described above, the aging process inhibitor according to the present invention includes both or one of the peptides Gly-Pro and Glu-Hyp-Gly, their salts or chemically modified products thereof. As a property of the peptide, the aging process inhibitor may have at least one of a promoting effect on the expression of type 17 collagen gene or a promoting effect on the expression of glutathione synthetase gene. In other words, based on the property, the present invention is a peptide, its salt or its chemically modified product that has newly found a use in inhibiting the aging process.

[0085] [Food or Drinks]

[0086] The food or drink according to the present invention contains the aging process inhibitor. For example, as described above, the peptide preferably contained in the aging process inhibitor is rapidly absorbed in the intestine and can therefore be administered orally. Therefore, the aging process inhibitor of the present invention can be used as a food or drink, wherein the aging process inhibitor is mixed with a food or drink. In addition, the aging process inhibitor according to the present invention can be used as a specific health food or a food with functional requirements. The concentration of the aging process inhibitor contained in the food or drink is preferably 0.001 to 100% by mass.

[0087] Example

[0088] Hereinafter, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.

[0089] [Example 1]

[0090] [Sample preparation]

[0091] <Preparation of peptide and collagen peptide mixture>

[0092] The peptides and collagen peptide mixtures shown in Tables 1 to 4 below were produced by using the above-mentioned method or purchased from the manufacturer described later. The peptides and collagen peptide mixtures were used as samples to determine whether they have an effect on the messenger RNA level (mRNA level) of the type 17 collagen gene and the mRNA level of the glutathione synthetase gene in epidermal cells described later.

[0093] Here, abbreviations are used for the peptides shown in Tables 1 and 2, wherein the amino acids forming the peptides are each represented by one character. In Table 1, "EO" represents a glutamic acid-hydroxyproline dipeptide (manufactured by PH Japan Co., Ltd.). "GP" represents a glycine-proline dipeptide (trade name: "G-3015", manufactured by BACHEM Co.). "EOG" represents a glutamic acid-hydroxyproline-glycine tripeptide (manufactured by PH Japan Co., Ltd.).

[0094] Furthermore, in the quantitative analysis by LC-MS / MS under the conditions described later, the collagen peptide mixture A (trade name: "COLLAPEP PU", manufactured by Nitta Gelatin Inc., weight average molecular weight (Mw): about 630 Da) shown in Table 3 was found to contain the following amounts of "EOG" and "GP".

[0095] Glu-Hyp-Gly: 4ppm, Gly-Pro: 2,379ppm, Total: 2,383ppm.

[0096] Next, in the quantitative analysis by LC-MS / MS under the conditions described later, the collagen peptide mixture B (trade name: "TYPE-S", manufactured by Nitta Gelatin Inc., weight average molecular weight (Mw): about 750 Da) shown in Table 4 was found to contain the following amounts of "EOG" and "GP".

[0097] Glu-Hyp-Gly: 9ppm, Gly-Pro: 1,159ppm, Total: 1,168ppm.

[0098] In quantitative analysis by LC-MS / MS under the conditions described later, the collagen peptide mixture C shown in Table 4 (collagen peptide mixture developed by Nitta Gelatin Inc, weight average molecular weight (Mw): about 450 Da) was found to contain the following amounts of "EOG" and "GP".

[0099] Glu-Hyp-Gly: 24ppm, Gly-Pro: 26,387ppm, Total: 26,411ppm.

[0100] Quantitative analysis by LC-MS / MS was performed under the following conditions.

[0101] HPLC instrument: "ACQUITY UPLC H-Class Bio" (manufactured by Waters Corporation)

[0102] Column: "Hypersil GOLD PFP 2.1×150 mm, 5 μm" (manufactured by Thermo Fisher Scientific. Inc.) Column temperature: 40° C. (linear gradient)

[0103] Mobile phase: (A) aqueous solution containing 0.2% formic acid and 2 mM ammonium acetate

[0104] (B) 100% ethanol (gradient setting)

[0105]

[0106]

[0107] Injection volume: 0.5 μl

[0108] MS / MS instrument: "Xevo TQ-XS", manufactured by Waters Corporation Ionization method: Positive ESI

[0109] Capillary voltage(kV):1

[0110] Desolventization temperature (℃) 500

[0111] Source temperature (℃) 150

[0112] MRM conditions:

[0113] Peptide (abbreviation) Precursor ion (m / z) Product ion (m / z)

[0114] Gly-Pro(GP) 173 116

[0115] Glu-Hyp-Gly(EOG) 318 225

[0116] <Preparation of epidermal cells>

[0117] First, normal human epidermal keratinocytes NHEK (NB) (manufactured by KURABO INDUSTRIES LTD.) were obtained as epidermal cells. The cells were cultured at 1.25×10 4 cells / dish (concentration 0.25×10 4 5 mL of cell dispersion containing 10 cells / mL) was inoculated in a necessary number of φ60 mm commercial culture dishes and cultured in a serum-free medium (trade name: "HuMedia KG-2", manufactured by KURABO INDUSTRIES LTD.) for 2 days. Then, it was confirmed that the cells were sub-confluent in the culture dish, and then the culture medium in the culture dish was replaced with a basal medium (trade name: "HuMedia KB-2", manufactured by KURABO INDUSTRIES LTD). In this way, epidermal cells for evaluating the mRNA level of type 17 collagen gene and the mRNA level of glutathione synthetase gene were prepared.

[0118] <Gene expression test>

[0119] Peptides or collagen peptide mixtures were added to the basal medium in the culture dish to the concentrations shown in Tables 1 to 4, and the cells were cultured at 37°C in an atmosphere of 5% by volume of carbon dioxide for 72 hours to prepare samples for gene expression testing. In addition, a control sample (hereinafter also referred to as "blank") obtained by adding only ion-exchanged water to the basal medium in the culture dish was prepared. This control sample was also cultured at 37°C in an atmosphere of 5% by volume of carbon dioxide for 72 hours.

[0120] Next, using an RNA extraction kit (trade name: "TRIzol" (registered trademark) reagent, manufactured by Life Technologies Japan Ltd.), total RNA was extracted from the epidermal cells in the culture dish according to the protocol attached to the kit to obtain an extract containing the total RNA of each sample. Subsequently, using a cDNA preparation kit (trade name (product number): "High Capacity RNA-to-cDNA Kit (4387406)", manufactured by Life Technologies Japan Ltd.), the RNA in the extract was reverse transcribed according to the protocol attached to the kit to obtain cDNA from the RNA in the extract. In addition, real-time (RT)-PCR was performed on the cDNA using a DNA amplification device (trade name: "Step One Plus (TM) Real-Time PCR System", manufactured by Applied Biosystems Inc.).

[0121] In RT-PCR, the mRNA levels of type 17 collagen (manufactured by Life Technologies Japan Ltd., primer: Hs009900361_ml) and glutathione synthetase (GSS, manufactured by Life Technologies Japan Ltd., primer: Hs01547656_ml) as target genes were measured. GAPDH was selected as an internal standard (correction gene). For the calculation of mRNA levels, a calibration curve method was used. The primers and probes attached to the kit (trade name: "TaqMan (registered trademark) Gene Expression Assays, manufactured by Applied Biosystems Inc.) were used as primers and probes for RT-PCR.

[0122] The data obtained from RT-PCR were analyzed as follows. First, the mRNA levels (gene expression levels) of two target genes (type 17 collagen and glutathione synthetase) were calculated in the sample and the control sample, respectively. Next, the mRNA levels of the two target genes were corrected using the mRNA level of GAPDH as a correction gene to obtain the correction values ​​of the sample and the control sample. Specifically, the values ​​(relative values) obtained by dividing the mRNA levels of the two target genes by the mRNA level of GAPDH were determined, respectively.

[0123] Then, the ratio of the corrected value obtained for each sample to the corrected value of the control sample (defined as 100) was determined (gene expression increase rate (%)). The effect of the addition of the peptide and collagen peptide mixture on the mRNA level of the type 17 collagen gene and the mRNA level of the glutathione synthetase gene in epidermal cells was evaluated (whether it showed a promoting effect on gene expression).

[0124] In addition, the gene expression increase rate (%) was statistically processed to evaluate the significance of the promotion of the gene expression of type 17 collagen genes and glutathione synthetase genes in each sample. For significance assessment, software ("Excel (Ver 2016)" (trade name), manufactured by Social Survey Research Information Co., Ltd) was used for statistical processing, Smirnov-Grubbs (two-sided test) was performed, and the significance level (P value) was set to 0.01 as a threshold. Thereafter, a student's t-test (t-test) was performed to evaluate significance. Tables 1 to 4 show the results. In Tables 1 and 4, samples with "++" were determined to have significance for the promotion of gene expression. In samples with "+", the gene expression increase rate (%) exceeded 100. Samples with "-" were determined to have no significance for the promotion of gene expression.

[0125] Here, Table 1 shows the gene expression increase rate of the type 17 collagen gene when the peptides "EO", "GP" and "EOG" are added to the epidermal cells, respectively. Table 2 shows the gene expression increase rate of the glutathione synthetase gene when the peptides "GP" and "EOG" are added to the epidermal cells, respectively. Table 3 shows the gene expression increase rate of the type 17 collagen gene when "Collagen Peptide Mixture A" is added to the epidermal cells. Table 4 shows the increase in the gene expression increase rate of the glutathione synthetase gene when "Collagen Peptide Mixture B" and "Collagen Peptide Mixture C" are added to the epidermal cells, respectively.

[0126] [Table 1]

[0127] Table 1

[0128]

[0129] [Table 2] Table 2

[0130]

[0131] [Table 3] Table 3

[0132]

[0133] [Table 4] Table 4

[0134]

[0135] [discuss]

[0136] As can be seen from Tables 1-4, samples containing both or one of the peptides Gly-Pro (GP) and Glu-Hyp-Gly (EOG) have at least one of the effects of promoting type 17 collagen gene expression and promoting glutathione synthetase gene expression. Collagen peptide mixtures A to C containing these peptides also have at least one of the effects of promoting type 17 collagen gene expression and promoting glutathione synthetase gene expression. On the other hand, samples containing the peptide Glu-Hyp (EO) did not show a significant promoting effect on type 17 collagen gene expression. This indicates that, as inhibitors of the aging process, the peptides Gly-Pro and Glu-Hyp-Gly and the collagen peptide mixture containing these peptides have the effect of inhibiting hair loss and decolorization by promoting type 17 collagen gene expression. This also indicates that, as an inhibitor of the aging process, the above peptides and the collagen peptide mixture containing these peptides have an antioxidant effect of removing active oxygen species, peroxides, etc. from living organisms by promoting the expression of glutathione synthetase gene and thus promoting glutathione synthesis.

[0137] [Example 2]

[0138] [Sample preparation]

[0139] <Preparation of collagen peptide mixture>

[0140] As a collagen peptide mixture containing both or one of the peptides Gly-Pro (GP) and Glu-Hyp-Gly (EOG), a collagen peptide mixture D (trade name: "COLLAGENAID", manufactured by Nitta Gelatin Inc., weight average molecular weight (Mw): about 4000 Da) was prepared. In the quantitative analysis by LC-MS / MS under the same conditions as in [Example 1] above, the collagen peptide mixture D contained a total of 132 ppm of "EOG" and "GP".

[0141] [Testing the inhibition of aging process in humans]

[0142] A total of 95 subjects (2 males and 92 females) in their teens to 70s were administered collagen peptide mixture D, and it was examined whether the subjects perceived the aging process inhibition effect. Specifically, collagen peptide mixture D was orally administered to 95 subjects at a dose of 4-6g per day for 10-20 days (average 14 days), without specifying the administration time. Afterwards, the subjects who perceived the aging process inhibition effect were interviewed (surveyed) about the relevant parts and details (specific contents) of the effect.

[0143] Tables 5 to 10 show the results. Table 5 shows the parts where the aging process inhibitory effect is perceived, and the number of subjects who perceived the aging process inhibitory effect in each part (multiple selections are allowed). Table 6 shows the specific contents when the aging process inhibitory effect is perceived on the skin, and the number of subjects who provided these contents (multiple selections are allowed). Table 7 shows the specific contents when the aging process inhibitory effect is perceived on the hair, and the number of subjects who provided these contents (multiple selections are allowed). Table 8 shows the specific contents when the aging process inhibitory effect is perceived on the nails, and the number of subjects who provided these contents (multiple selections are allowed). Table 9 shows the specific contents when the aging process inhibitory effect is perceived on the joints, and the number of subjects who provided these contents (multiple selections are allowed). Table 10 shows the specific contents when the aging process inhibitory effect is perceived in other parts, and the number of subjects who provided these contents (multiple selections are allowed).

[0144] [Table 5]

[0145] Table 5

[0146]

[0147] [Table 6] Table 6

[0148] Skin: Effect Details (Content) Number of subjects Improved elasticity 8 Sagging improvement 2 Drying improvement / fluffiness 15 Improvement of stratum corneum roughness 2 Texture Improvement 2 Improves makeup smoothness 3 Wrinkle improvement 4 Hair follicle improvement 1 Improvement of dry hands 3 Improved skin brightness 2 elasticity 2 Glossiness 5 Soft / springy feel 3 Smoothness / slippery feel 3 Full face firming 1 Reduce acne 1 Spot Improvement 1 total 58

[0149] [Table 7] Table 7

[0150] Hair: Effect Details (Content) Number of subjects Gloss improvement 4 Dryness (fluffiness) improvement 4 Reduced hair loss 4 Settlement Improvement 2 Improved combing properties 2 Dry feeling 2 Improved hair thickness 2 Reduced gray hair 1 Improved softness 1 Improved hair stiffness 1 Increased hair volume 1 Hair Growth 1 total 25

[0151] [Table 8] Table 8

[0152] Nails: Effect Details (Content) Number of subjects Improve vulnerability 3 Glossiness 1 toughness 1 total 5

[0153] [Table 9] Table 9

[0154] Joints: Effect Details (Content) Number of subjects Improved joint pain 3 Snapping joints 1 Improvement of unfamiliarity 1 total 5

[0155] [Table 10]

[0156] Table 10

[0157] Other: Effect details (content) Number of subjects Improve bowel movements 3 Chest elasticity 1 total 4

[0158] [discuss]

[0159] As can be seen from Tables 5-10, collagen peptide mixture D (aging process inhibitor) containing both or one of the peptides Gly-Pro (GP) and Glu-Hyp-Gly (EOG) has an aging process inhibitory effect in the skin, hair, nails, joints and other parts.

[0160] Although the embodiments and examples of the present invention have been described above, the configurations of the above-described embodiments and examples may be appropriately combined as originally conceived.

[0161] The embodiments and examples disclosed herein should be considered illustrative rather than limiting in any way.The scope of the invention is given by the appended claims rather than the foregoing description, and all changes that fall within the scope of the appended claims and their equivalents are embraced therein.

Claims

1. Use of an agent for preparing a non-medical product for inhibiting hair loss or hair depigmentation, wherein the agent comprises both or one of the peptides Gly-Pro and Glu-Hyp-Gly or a salt thereof.

2. The use according to claim 1, wherein the peptide is derived from collagen.

3. The use according to claim 1, wherein the agent is a collagen peptide mixture.

4. The use according to claim 3, wherein the weight average molecular weight of the collagen peptide mixture is greater than or equal to 100 Da and less than or equal to 5000 Da. 5 . The use according to claim 1 , wherein the agent is an agent for promoting the expression of type 17 collagen gene or an agent for promoting the expression of glutathione synthetase gene.

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