Hair growth agent

Abstract

A hair growth agent comprising at least one amino acid or peptide selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly and Ser-Hyp-Gly, or a salt thereof, or a chemically modified product thereof.

Description

Technical Field

[0001] The present invention relates to a hair growth agent. Background Art

[0002] It is known that collagen hydrolysate (hereinafter also referred to as "collagen peptide mixture") exhibits various physiological activities in living organisms. For example, WO 2012 / 102308 (PTL 1) discloses that a collagen peptide mixture is used as a therapeutic agent or prophylactic agent for diabetes because the collagen peptide mixture acts on an enzyme that controls insulin secretion. Japanese Patent Laid-Open No. 2009-120512 (PTL 2) discloses that a collagen peptide mixture is used as a promoter for articular cartilage regeneration because the collagen peptide mixture has a cartilage regeneration promoting effect. Japanese Patent Laid-Open No. 2005-029488 (PTL 3) discloses that a collagen peptide mixture is used as an antihypertensive agent because the collagen peptide mixture has an antihypertensive effect.

[0003] Citation List

[0004] Patent Literature

[0005] PTL1: WO 2012 / 102308

[0006] PTL2: Japanese Patent Laid-Open No. 2009-120512

[0007] PTL 3: Japanese Patent Laid-Open No. 2005-029488

[0008] PTL 4: Japanese Patent Laid-Open No. 2009-161509

[0009] Non-Patent Literature

[0010] NPL1: Tanimura S et al., Cell Stem Cell, 2011, Vol 8, pp.177-187 Summary of the Invention

[0011] Technical Problem

[0012] Here, Japanese Patent Laid-Open No. 2009-161509 (PTL 4) and NPL 1 disclose that type XVII collagen has an inhibitory effect on hair loss and an inhibitory effect on hair discoloration. However, heretofore, it has not been known that the above-mentioned collagen peptide mixture has a promoting effect on hair development or hair growth or has an effect of preventing the progression of hair loss. Therefore, extensive research has been conducted to explore a promoting effect on hair development or hair growth or an effect of preventing the progression of hair loss as a new physiological activity of the collagen peptide mixture and amino acids, peptides, etc. derived from collagen contained in the collagen peptide mixture.

[0013] In view of the above circumstances, an object of the present invention is to provide a hair growth agent containing amino acids, peptides, etc., which has at least one of the effects of promoting hair development or hair growth or preventing the progression of hair loss.

[0014] Solution to the problem

[0015] When exploring the new physiological activities of a collagen peptide mixture, the present inventors found that the predetermined amino acids, predetermined peptides, etc. contained in the collagen peptide mixture have at least one of the effects of promoting hair development or hair growth or preventing the progression of hair loss, thus achieving the present invention. Specifically, the present invention is as follows.

[0016] The hair growth agent according to the present invention contains one or more amino acids or peptides selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly, salts thereof, or chemically modified products thereof.

[0017] Preferably, the amino acids and peptides are derived from collagen.

[0018] Preferably, the hair growth agent is a collagen peptide mixture containing at least one of the amino acids or peptides.

[0019] Preferably, the weight-average molecular weight of the collagen peptide is 100 Da or more and 8000 Da or less.

[0020] Preferably, the hair growth agent is a cell growth promoter for dermal papilla cells.

[0021] Preferably, the hair growth agent is a promoter for hair development or hair growth or an agent for preventing the progression of hair loss.

[0022] Advantageous effects of the present invention

[0023] According to the present invention, a hair growth agent can be provided, which contains amino acids, peptides, etc., and has at least one of the effects of promoting hair development or hair growth and preventing the progression of hair loss. Brief description of the drawings

[0024] Figure 1 is a photograph showing the head of an 11-week-old hairless mouse in a control group fed a magnesium-deficient special diet.

[0025] Figure 2is a photograph showing the head of an 11-week-old hairless mouse in the first group fed a magnesium-deficient special diet containing Pro-Hyp. Detailed Description of the Invention

[0026] Hereinafter, embodiments of the present invention will be described in more detail. As used herein, a description in the form of "A to B" represents 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 that of B. As used herein, the term "hair growth" in "hair growth agent" includes not only the meaning of "hair growth" indicating the action of growing hair, but also the meaning of "hair development" indicating the action of developing new hair and promoting hair growth, and the meaning of "preventing the progression of hair loss" indicating the action of reducing the possibility of hair loss.

[0027] [Hair Growth Agent]

[0028] The hair growth agent according to the present invention contains one or more amino acids or peptides selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly, salts thereof, or chemically modified products thereof. A hair growth agent having such characteristics has a promoting effect on the growth of dermal papilla cells, and thus can have at least one of the effects of promoting hair development or hair growth of hair and preventing the progression of hair loss.

[0029] [Predetermined amino acid or predetermined peptide having an effect of promoting hair development or hair growth of hair or preventing the progression of hair loss, or a salt thereof, or a chemically modified product thereof]

[0030] As described above, the hair growth agent contains one or more amino acids or peptides selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly, salts thereof, or chemically modified products thereof. In this specification, unless otherwise specified, "amino acid" is represented by a three-character abbreviation. Further, unless otherwise specified, "amino acid" means an L-amino acid. For the "peptide" in this specification, for example, "Pro-Hyp" means a peptide (dipeptide) in which proline and hydroxyproline are arranged in the order from the N-terminal side to the C-terminal side, and "Glu-Hyp-Gly" means 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 dipeptides and tripeptides other than "Pro-Hyp" and "Glu-Hyp-Gly".

[0031] Preferably, the hair growth agent contains one or more amino acids or peptides selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Pro-Ala, Pro-Gly, Pro-Pro, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, and Pro-Ala-Gly, salts thereof, or chemically modified products thereof. More preferably, the hair growth agent contains at least one of the peptide Pro-Hyp or Hyp-Gly, salts thereof, or chemically modified products thereof. Further, the hair growth agent may contain a combination of Hyp and Pro, or a combination of Hyp and Gly. In this case, the hair growth agent can more significantly exhibit a promoting effect on the growth of dermal papilla cells.

[0032] The "salts" of the above-mentioned amino acids and peptides are formed, for example, as inorganic acid salts of the above-mentioned amino acids or peptides such as hydrochloride, sulfate, and phosphate; organic acid salts such as methanesulfonate, benzenesulfonate, succinate, and oxalate; inorganic base salts such as sodium salt, potassium salt, and calcium salt; and organic base salts such as triethylammonium salt.

[0033] The "chemically modified product" of each amino acid and peptide refers to a compound in which the free functional groups of the amino acid residues as constituent units are chemically modified. The chemical modification can be carried out, for example, on the hydroxyl group of hydroxyproline, on the amino group on the N-terminal (amino-terminal) side of the amino acid, and on the carboxyl group on the C-terminal (carboxyl-terminal) side of the amino acid. For the specific manner and treatment conditions of the chemical modification, known conventional chemical modification techniques for amino acids and peptides are applied. The chemically modified products of each amino acid and peptide obtained by such chemical modification can enhance the solubility under weakly acidic to neutral conditions, enhance the compatibility with other active ingredients, etc.

[0034] For example, as the chemical modification of the hydroxyl group in hydroxyproline, O-acetylation can be carried out on the tripeptide Glu-Hyp-Gly. The O-acetylation can be carried out by applying acetic anhydride to the peptide in an aqueous solvent or a non-aqueous solvent. As the chemical modification of the carboxyl group in glycine, esterification, amidation, etc. can be carried out. The esterification can be carried out by suspending the peptide in methanol and then passing dry hydrogen chloride gas through the resulting suspension. The amidation can be carried out by applying carbodiimide, etc. to the peptide.

[0035] As the chemical modification of the free amino group in the peptide, methylation can be carried out. As the chemical modification of the free hydroxyl group in the peptide, at least one of phosphorylation and sulfation can be carried out.

[0036] Preferably, the amino acids and peptides are derived from collagen. Herein, the collagen as a raw material can be obtained by carrying out known conventional degreasing or decalcifying treatment, extraction treatment, etc. on, for example, the skin, dermis, bone, cartilage, tendon, etc. of animals (usually cows, pigs, sheep, chickens or ostriches) or on the bone, skin, scales, etc. of fish. In addition, gelatin can be used as the raw material for the peptide. The gelatin can be obtained by treating the collagen thus obtained by known conventional methods (such as extraction with hot water). For the collagen and gelatin, commercially available products can be used as raw materials.

[0037] The amino acids and peptides can be obtained by hydrolyzing the collagen and / or the gelatin with a combination of two or more endopeptidases and exopeptidases. The amino acids and peptides can be obtained as a collagen peptide mixture, which coexists with other collagen peptides due to hydrolysis. However, any of the collagen peptide mixtures themselves and the mixtures obtained by partially purifying the collagen peptide mixture can be used as the hair growth agent according to the present invention. That is, it is also preferred that the hair growth agent is a collagen peptide mixture containing at least one of the above amino acids or peptides. In addition, by further purifying the collagen peptide mixture, a purified product containing one of the above amino acids and peptides with high purity can be obtained. When the amino acids and peptides are derived from collagen, it is preferred to obtain the amino acids and peptides by using a method in which collagen or gelatin is enzymatically treated in two stages as described below.

[0038] In addition, preferably, the weight average molecular weight of the collagen peptide mixture is 100 Da or more and 8000 Da or less. The weight average molecular weight of the collagen peptide mixture is more preferably 100 Da or more and 6000 Da or less, and still more preferably 100 Da or more and 4000 Da or less. When the weight average molecular weight of the collagen peptide mixture is within the above range, the hair growth agent more significantly exhibits the promoting effect on the growth of dermal papilla cells, and thus at least one of the promoting effects on hair development and hair growth and the preventing effect on the progression of hair loss can be sufficiently obtained. If the weight average molecular weight is greater than 8000 Da, the above effects of the hair growth agent may be insufficient.

[0039] The weight average molecular weight of the collagen peptide mixture can be determined by performing size exclusion chromatography (SEC) under the following measurement conditions.

[0040] Instrument: High performance liquid chromatography (HPLC) (manufactured by TOSOH CORPORATION)

[0041] Column: TSKGel (registered trademark) G2000SW XL

[0042] Column temperature: 40 °C

[0043] Column size: 7.8 mm (I.D.) × 30 cm, 5 μm

[0044] Eluent: 45 mass% acetonitrile (containing 0.1 mass% trifluoroacetic acid)

[0045] Flow rate: 1.0 mL / min

[0046] Injection volume: 10 μL

[0047] Detection: UV 214 nm

[0048] Molecular weight markers: The following five types are used

[0049]

[0050] 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 through a 0.2 μm filter to prepare a sample (measurement sample) for measuring the weight-average molecular weight. By using size exclusion chromatography on the said measurement sample, the weight-average molecular weight of the collagen peptide mixture can be determined.

[0051] [Method for producing hair growth agent]

[0052] The amino acids or peptides contained in the hair growth agent can be obtained by known conventional methods. For example, the amino acid (Hyp) can be obtained by purchasing commercially available amino acids. Amino acids can also be obtained by using methods including hydrolysis of collagen or gelatin.

[0053] The said peptides (Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly) can be obtained respectively by using known conventional liquid-phase or solid-phase peptide synthesis methods, or methods including hydrolysis of collagen or gelatin. From the viewpoint of efficiency, it is preferred to prepare the said peptides by using chemical synthesis methods using amino acids as described below, or methods including enzymatic treatment of collagen or gelatin in two stages as described below. In addition, the said peptides can be produced by using methods including enzymatic treatment using only secondary enzymes and omitting primary enzymes, or methods including enzymatic treatment using both primary enzymes and secondary enzymes, instead of using methods including enzymatic treatment of collagen or gelatin in two stages. Hereinafter, as an example of the method for manufacturing the peptides contained in the hair growth agent, the method for manufacturing "Glu-Hyp-Gly" in particular among the peptides contained in the hair growth agent will be described.

[0054] [Chemical synthesis method]

[0055] The said peptides can be obtained by using conventional peptide synthesis methods. As peptide synthesis methods, solid-phase synthesis methods and liquid-phase synthesis methods are known. As solid-phase synthesis methods, the Fmoc method and the Boc method are known. The said peptides can be obtained by using either the Fmoc method or the Boc method. As a solid-phase peptide synthesis method, the method for synthesizing tripeptides represented by Glu-Hyp-Gly can be carried out as follows.

[0056] First, beads of a polystyrene polymer gel with a diameter of about 0.1 mm and having a surface modified with an amino group are provided as a solid phase. In addition, diisopropylcarbodiimide is provided as a condensing agent. Next, the amino group of glycine, which is the amino group on the C-terminal (carboxyl-terminal) side of the amino acid sequence, is protected with Fmoc (fluorenyl-methoxy-carbonyl), and the carboxyl group of glycine is peptide-bonded to the amino group of the solid phase by a dehydration reaction using the condensing agent. Further, the solid phase is washed with a solvent to remove the residual condensing agent and amino acid, and then the protecting group (deprotection) of the amino group of glycine peptide-bonded to the solid phase is removed.

[0057] Subsequently, hydroxyproline in which the amino group is protected with an Fmoc group is provided, and the carboxyl group of the hydroxyproline is peptide-bonded to the deprotected amino group of glycine by using a condensing agent. Thereafter, in the same manner as described above, the amino group of the hydroxyproline is deprotected, glutamic acid protected with an Fmoc group is provided, and a reaction for peptide-bonding glutamic acid to hydroxyproline is carried out to synthesize a tripeptide represented by Glu-Hyp-Gly as a solid phase. 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.

[0058] <Method for producing collagen and gelatin>

[0059] In addition, a method for producing a tripeptide represented by Glu-Hyp-Gly by enzyme-treating collagen or gelatin in two stages can be carried out as follows.

[0060] The term "enzyme-treating (collagen or gelatin) in two stages" means the following. That is, primary enzyme treatment is carried out by a conventional method known for breaking 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 a combination of 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. A method for enzyme-treating collagen or gelatin in two stages will be described in more detail below.

[0061] (Primary enzyme treatment)

[0062] The enzyme used in the primary enzyme treatment may be any enzyme that can break the peptide bonds of collagen or gelatin, without particular limitation, and any proteolytic enzyme can be used. Specifically, examples thereof include collagenase, thiol protease, serine protease, acidic protease, alkaline protease, and metalloprotease. One selected from the group consisting of these enzymes can be used alone, or two or more of them can be used in combination. As the thiol protease, papain, papain, bromelain, and ficin derived from plants, cathepsin and calcium-dependent protease derived from animals, etc. can be used. As the serine protease, trypsin, cathepsin D, etc. can be used. As the acidic protease, pepsin, chymotrypsin, etc. can be used. Considering that the hair growth agent of the present invention is used for drugs, etc., preferably, as the enzyme used in the primary enzyme treatment, an enzyme other than the enzyme derived from pathogenic microorganisms is used.

[0063] Based on 100 parts by mass of collagen or gelatin, the amount of the enzyme in the primary enzyme treatment is preferably, for example, 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 20,000 Da, more preferably 500 to 10,000 Da, and still more preferably 500 to 8,000 Da. It can be said that when the weight average molecular weight is within the above range, peptides with appropriate molecular weights can be appropriately produced. If necessary, the enzyme can be inactivated after the primary enzyme treatment. In this case, the inactivation temperature is preferably 70 to 100 °C, for example. The weight average molecular weight of the collagen peptide mixture precursor can be determined by using the SEC method.

[0064] (Secondary enzyme treatment)

[0065] Examples of the enzyme used in the secondary enzyme treatment include an enzyme having aminopeptidase N activity, an enzyme having both aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity, and a combination 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, and when an amino acid other than proline or hydroxyproline is present at the second position on the N-terminal side, the enzyme acts. 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, and the peptide has proline or hydroxyproline at the third position on the N-terminal side. Considering that the hair growth agent of the present invention is used for drugs, etc., preferably, as the enzyme used in the secondary enzyme treatment, an enzyme other than the enzyme derived from pathogenic microorganisms is used.

[0066] 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)).

[0067] By performing secondary enzyme treatment, a collagen peptide mixture containing peptides not included in the precursor of the collagen peptide mixture can be obtained. Specifically, a collagen peptide mixture containing Glu-Hyp-Gly can be obtained.

[0068] Based on 100 parts by mass of the precursor of the collagen peptide mixture, the amount of the enzyme in the secondary enzyme treatment is preferably, for example, 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 10,000 Da, more preferably 100 to 8,000 Da, still more preferably 100 to 4,000 Da. The weight-average molecular weight of the collagen peptide mixture can be determined by a method using SEC.

[0069] The secondary enzyme treatment is mainly carried out to produce Glu-Hyp-Gly tripeptide. Therefore, it is preferable to adjust the amount of the enzyme, treatment temperature, treatment time, and pH in the secondary enzyme treatment so that the peptides contained in the precursor of the collagen peptide mixture are not overly 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 preferable 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.

[0070] In the secondary enzyme treatment, not only enzymes having aminopeptidase N activity and enzymes having prolyl tripeptidyl aminopeptidase activity can be used, but also enzymes having different activities can be used, and two or more enzymes having different activities can be used in combination. Therefore, by-products can be digested and removed. Preferably, the enzymes used in this case are appropriately selected according to the type of collagen used as a raw material and the type of enzyme used in the primary enzyme treatment. Examples of different activities include dipeptidase activity, such as prolinase activity and hydroxyprolinase activity. Therefore, by-products (such as dipeptides) can be digested and removed.

[0071] In addition, the aminopeptidase N activity is essentially the activity that causes the sequential release of amino acids on the N-terminal side. Therefore, in the case where the collagen peptide mixture precursor obtained by the primary enzyme treatment contains peptides with very large molecular weights, when only the enzyme with aminopeptidase N activity is used for the secondary enzyme treatment, the duration of the secondary enzyme treatment significantly increases. To address this situation, for example, prolyl oligopeptidase can be used in the secondary enzyme treatment. The proline oligopeptidase is an endopeptidase having the activity of causing the hydrolysis of proline on the carboxyl side (proline enzyme activity). Therefore, the secondary enzyme treatment can be effectively carried out.

[0072] In a method for enzymatic treatment of collagen or gelatin in two stages, the primary enzyme treatment can produce peptides with relatively large molecular weights. 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, the enzyme with aminopeptidase N activity acts on the peptide represented by [X1-Gly-X2-Glu-Hyp-Gly], thereby releasing X1 at the N-terminus to obtain a peptide having an amino acid sequence represented by [Gly-X2-Glu-Hyp-Gly]. Next, the enzyme with aminopeptidase N activity acts on the peptide represented by [Gly-X2-Glu-Hyp-Gly] twice, thereby releasing glycine and X2 to obtain a peptide represented by [Glu-Hyp-Gly].

[0073] (Purification of collagen peptide mixture)

[0074] By carrying out the enzymatic 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, it is preferable to purify the collagen peptide mixture. As a purification method in this case, known conventional methods can be used, and examples thereof include ultrafiltration and various types of liquid chromatography, such as size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and affinity chromatography.

[0075] Specifically, the collagen peptide mixture can be purified according to the following steps. That is, a collagen peptide mixture of about 2 g / 10 ml is loaded onto an ion exchange column (for example, "TOYOPEARL" (registered trademark) DEAE-650 (trade name) manufactured by TOSOH CORPORATION), and then the first void volume fraction eluted with distilled water is collected. Subsequently, the first void volume fraction is loaded onto a column having an ion exchange group opposite to that of the above ion exchange column (for example, "TOYOPEARL" (registered trademark) SP-650 manufactured by TOSOH CORPORATION), and then the second void volume fraction eluted with distilled water is collected.

[0076] Next, the second void volume fraction is loaded onto a gel filtration column (for example, "SEPHADEX LH-20" (trade name) produced by GE Healthcare Japan Corporation), and eluted with a 30% by mass aqueous methanol solution to collect the fraction containing the Glu-Hyp-Gly tripeptide. Finally, using high performance liquid chromatography (HPLC) with a reversed-phase column (for example, "μBondasphere 5μ C18 Column" (trade name)), the fraction is fractionated according to a linear concentration gradient of an aqueous acetonitrile solution of 32% or less containing 0.1% by mass of trifluoroacetic acid. In this way, Glu-Hyp-Gly can be obtained with high purity.

[0077] [Cell growth promoter for dermal papilla cells, hair growth promoter for hair or hair growth promoter, and agent for preventing progression of hair loss]

[0078] The hair growth agent of the present invention is preferably a cell growth promoter for dermal papilla cells. As described above, the hair growth agent contains one or more amino acids or peptides selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly, salts thereof, or chemically modified products thereof, and thus can exert a cell growth promoting effect on dermal papilla cells. This enables the hair growth agent to exhibit at least one of the effects of promoting hair development or hair growth and preventing the progression of hair loss. Therefore, the hair growth agent can be used as a cell growth promoter for dermal papilla cells to promote the cell growth of dermal papilla cells.

[0079] Preferably, the hair growth agent is a promoter of hair follicle development or hair growth or an agent for preventing hair loss progression, because the hair growth agent contains any one of the above-mentioned amino acids or peptides, their salts, or chemically modified products. As described above, the hair growth agent has a promoting effect on the growth of dermal papilla cells, and thus can be used as a promoter of hair follicle development or hair growth for treating to promote hair follicle development or hair growth by promoting the growth of dermal papilla cells. In addition, the hair growth agent can be used as an agent for preventing hair loss progression for the purpose of growing dermal papilla cells to inhibit and prevent the progression of hair loss due to the reduction of dermal papilla cells.

[0080] The hair growth agent can be administered orally or parenterally in various forms. For these forms, when the hair growth agent is administered orally, it can take the following dosage forms, such as tablets, granules, capsules, powders, liquids, suspensions, and emulsions. The hair growth agent contains at least one of, for example, the above-mentioned amino acids, combinations of amino acids or peptides, which are rapidly absorbed in the intestine and thus can be administered orally.

[0081] When administered parenterally, the hair growth agent can be in the form of dosage forms such as external preparations like ointments, creams, and lotions, as well as transdermal preparations. In addition, the hair growth agent can be applied to the scalp skin in the form of a solution or a coating.

[0082] The dosage of the hair growth agent varies according to differences in the age, sex, weight, and sensitivity of the subject, the administration method, the administration interval, the type of preparation, etc. When the hair growth agent 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 hair growth agent is, for example, a tablet, the amount of the hair growth agent contained in the tablet can be 0.001 to 80% by mass per tablet, and when the dosage form of the hair growth agent is, for example, a powder, the amount of the hair growth agent contained in the powder can be 0.001 to 100% by mass. When the hair growth agent is administered parenterally or by another form of preparation, the dosage can be appropriately determined with reference to the dosage for oral administration. The hair growth agent can be administered once a day or in several divided doses, or once a day or once every few days.

[0083] The hair growth agent may appropriately contain other active ingredients, pharmaceutical carriers, etc., as long as it does not have an adverse effect on the effects of the present invention. Examples of other active ingredients include inulin, caffeic acid, quinic acid, its derivatives, marjoram extract, crude drugs such as Kinfukan, motherwort (polygala tenuifolia), Hakubiso, and Desmos chinensis Lour, royal jelly, echinacea extract, acai extract, Cupuacu extract, etc. In addition, examples of pharmaceutically acceptable carriers for formulating into pharmaceutical preparations include diluents, binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone), excipients (lactose, sucrose, corn starch, potassium phosphate, sorbitol, and glycine), lubricants (magnesium stearate, talc, polyethylene glycol, and silica), disintegrants (potato starch), and wetting agents (sodium lauryl sulfate).

[0084] [Use of the Invention]

[0085] As described above, the hair growth agent according to the present invention contains one or more amino acids or peptides selected from the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly, salts thereof, or chemically modified products thereof. The hair growth agent has a promoting effect on the growth of dermal papilla cells, which is an unknown property of the above amino acids and peptides, and thus can have at least one of a promoting effect on hair follicle development or hair growth and a preventing effect on the progression of hair loss. In other words, the present invention is any one of amino acids or peptides, salts thereof, or chemically modified products thereof for promoting hair follicle development or hair growth or preventing the progression of hair loss.

[0086] Examples

[0087] Hereinafter, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.

[0088] [Example 1: Cell Biology Test (In Vitro Test)]

[0089] [Preparation of Samples]

[0090] [Preparation of Amino Acid, Peptide, and Collagen Peptide Mixture]

[0091] As samples for evaluating the promoting effect on the growth of dermal papilla cells, the amino acids, combinations of amino acids, dipeptides, tripeptides, and collagen peptide mixtures shown in Tables 1 and 2 below were produced using the above methods or purchased from the manufacturers described later and provided.

[0092] Here, for the amino acids, combinations of amino acids, and peptides shown in Table 1, abbreviations in which each amino acid is represented by one character are used. In Table 1, "PO" represents a dipeptide of proline - hydroxyproline (trade name: "G - 3025", manufactured by BACHEM), and "OG" represents a dipeptide of hydroxyproline - glycine (trade name: "G - 2365", manufactured by BACHEM). "GPO" represents a tripeptide of glycine - proline - hydroxyproline (manufactured by PH Japan Co., Ltd.). "PO" refers to a peptide in which proline and hydroxyproline are arranged in sequence from the N - terminal side to the C - terminal side. The same applies to the description of peptides other than "PO".

[0093] In addition, in Table 1, "AOG" represents a tripeptide of alanine - hydroxyproline - glycine (manufactured by PH Japan Co., Ltd.), and "EOG" represents a tripeptide of glutamic acid - hydroxyproline - glycine (manufactured by PH Japan Co., Ltd.). "SOG" represents a tripeptide of serine - hydroxyproline - glycine (manufactured by PH Japan Co., Ltd.), and "GP" represents a dipeptide of glycine - proline (trade name: "G - 3015", manufactured by BACHEM). "LO" represents a dipeptide of leucine - hydroxyproline (manufactured by PH JapanCo., Ltd.), "FO" represents a dipeptide of phenylalanine - hydroxyproline (manufactured by PH Japan Co., Ltd.), and "EO" represents a dipeptide of glutamic acid - hydroxyproline (manufactured by PH Japan Co., Ltd.).

[0094] "PA" represents proline-alanine dipeptide (manufactured by PH Japan Co., Ltd.), and "PAG" represents proline-alanine-glycine tripeptide (manufactured by PH Japan Co., Ltd.). "PG" represents proline-glycine dipeptide (manufactured by PH Japan Co., Ltd.). "PP" represents proline-proline dipeptide (manufactured by PH Japan Co., Ltd.). "O" represents hydroxyproline (trade name: "080-01642", manufactured by FUJIFILM Wako Pure Chemical Corporation), "G" represents glycine (trade name: "073-00732", manufactured by FUJIFILM Wako Pure Chemical Corporation), "P" represents proline (trade name: "161-04602", manufactured by FUJIFILM Wako Pure Chemical Corporation), "P+O" represents a combination of proline and hydroxyproline, and "O+G" represents a combination of hydroxyproline and glycine.

[0095] In addition, in the quantitative analysis by LC-MS / MS under the conditions described below, it was found that the collagen peptide mixture A (trade name: "TYPE-S", manufactured by Nitta Gelatin Inc., weight-average molecular weight (Mw): approximately 750 Da) shown in Table 2 has the following composition.

[0096] Pro-Hyp: 8 ppm, Hyp-Gly: 7,389 ppm, Gly-Pro-Hyp: 8 ppm, Ala-Hyp-Gly: 199 ppm, Glu-Hyp-Gly: 9 ppm, Ser-Hyp-Gly: 176 ppm, Gly-Pro: 1,159 ppm, Pro-Ala-Gly: 2,229 ppm, total: 11,177 ppm.

[0097] In addition, in the quantitative analysis by LC-MS / MS under the conditions described below, it was found that the collagen peptide mixture B (trade name: "COLLAPEP PU", manufactured by Nitta Gelatin Inc., weight-average molecular weight (Mw): approximately 630 Da) shown in Table 2 has the following composition.

[0098] Pro-Hyp: 8 ppm, Hyp-Gly: 3,447 ppm, Gly-Pro-Hyp: 36 ppm, Ala-Hyp-Gly: 436 ppm, Glu-Hyp-Gly: 4 ppm, Ser-Hyp-Gly: 120 ppm, Gly-Pro: 2,379 ppm, Pro-Ala-Gly: 2,645 ppm, Total: 9,074 ppm.

[0099] Quantitative analysis by LC-MS / MS was performed under the following conditions.

[0100] HPLC instrument: "ACQUITY UPLC H-Class Bio" (manufactured by Waters Corporation)

[0101] Column: "Hypersil GOLD PFP 2.1×150 mm, 5 μm" (manufactured by Thermo Fisher Scientific.Inc.)

[0102] 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% methanol

[0105] (Gradient setting)

[0106]

[0107]

[0108] Injection volume: 0.5 μl

[0109] MS / MS instrument: "Xevo TQ-XS", manufactured by Waters Corporation

[0110] Ionization method: positive ESI

[0111] Capillary voltage (kV): 1

[0112] Desolvation temperature (°C): 500

[0113] Source temperature (°C): 150

[0114] MRM conditions:

[0115]

[0116] <Preparation of dermal papilla cells>

[0117] First, human normal dermal papilla cells HFDPC-C (manufactured by Takara Bio Inc.) were obtained, and then the dermal papilla cells were inoculated at 0.2×10 4 cells / well into each well of a 96-well plate for cell culture (manufactured by Corning Inc.). In addition, 200 μL of a basal medium (trade name: "Hair Follicle Dermal Papilla Cell Basal Medium", manufactured by Takara Bio Inc.) containing growth factors accompanying the obtained dermal papilla cells was supplied to each well, and the dermal papilla cells were pre-cultured in each well at 37 °C for 24 hours.

[0118] Next, it was confirmed that the dermal papilla cells were sub-confluent, and then the basal medium in each well was replaced with 200 μL of another basal medium (trade name: "Hair Follicle Dermal Papilla Cell Basal Medium", manufactured by Takara Bio Inc.) without the above-mentioned growth factors. In this way, dermal papilla cells were prepared for determining whether the addition of the above sample promoted cell growth.

[0119] [Cell Growth Test]

[0120] Amino acids, combinations of amino acids, peptides, and collagen peptide mixtures as the above sample were added to the dermal papilla cells prepared as described above at the final concentrations shown in Tables 1 and 2, and the dermal papilla cells were cultured in each well at 37 °C for 72 hours. Here, 20 μL of purified water was added to one of the wells containing the dermal papilla cells prepared as described above, and like the other dermal papilla cells, it was cultured at 37 °C for 72 hours to prepare a control test sample (control). Thereafter, for each dermal papilla cell in the wells containing purified water or the sample, the number of live cells (number of live cells) was counted by the neutral red method. Here, the "neutral red method" is the method described below: Neutral red was added to the wells of the cultured cells at a final concentration of 150 μg / mL, the cells were cultured for 20 minutes, washed with PBS (phosphate-buffered saline), 200 μL of a 50% ethanol solution containing 1% acetic acid was added to the wells as an extract, the mixture was stirred, and the absorbance of the wells containing neutral red was measured at a wavelength of 540 nm to measure the number of live cells in the wells.

[0121] The viable cell count of dermal papilla cells in the wells containing the sample was determined relative to the viable cell count of dermal papilla cells (control sample) in the wells containing purified water as the cell growth rate (%) to evaluate the promoting effect on the growth of dermal papilla cells in the sample. In addition, statistical processing was performed on the cell growth rate (%) to evaluate the significance of the promoting effect on the growth of dermal papilla cells in the sample. For significance assessment, statistical processing was performed using software (“Excel (Ver 2016)” (trade name), manufactured by Social Survey Research Information Co., Ltd.), Smirnov-Grubbs (two-sided test) was performed, and the significance level (P value) was set at 0.05 as the threshold. Thereafter, a student t-test (t-test) was performed to evaluate the significance. Tables 1 and 2 show the results. In Tables 1 and 2, the samples with “++” were determined to have a significant promoting effect on the growth of dermal papilla cells. In the samples with “+”, the cell growth rate (%) exceeded 100.

[0122] [Table 1]

[0123] Table 1

[0124]

[0125]

[0126] [Table 2]

[0127] Table 2

[0128]

[0129] <Discussion>

[0130] As can be seen from Tables 1 and 2, an amino acid or peptide, a salt thereof, or a chemically modified product thereof selected from one or more of the group consisting of Hyp, Pro-Hyp, Hyp-Gly, Gly-Pro, Leu-Hyp, Phe-Hyp, Pro-Ala, Pro-Gly, Pro-Pro, Glu-Hyp, Gly-Pro-Hyp, Ala-Hyp-Gly, Glu-Hyp-Gly, Pro-Ala-Gly, and Ser-Hyp-Gly has a promoting effect on the growth of dermal papilla cells. A collagen peptide mixture containing an amino acid or peptide has a promoting effect on the growth of dermal papilla cells. In addition, the combination of the amino acids Pro and Hyp and the combination of the amino acids Hyp and Gly have a promoting effect on the growth of dermal papilla cells. This indicates that the above-mentioned amino acids and peptides and the collagen peptide mixture containing the amino acids and peptides are effective as hair growth agents, particularly as cell growth promoters for dermal papilla cells, hair development or hair growth promoters, or agents for preventing the progression of hair loss.

[0131] [Example 2: Experiment to confirm hair development effect and hair growth effect using hairless mice (in vivo experiment)]

[0132] Thirty 8-week-old male and female hairless mice were purchased from Hoshino Laboratory Animals, Inc. The hairless mice were divided into five groups, each group consisting of six mice, regardless of gender. Specifically, the multiple groups consisted of: a normal group fed with normal feed (trade name: "Labo MR Stock", manufactured by Nosan Corporation), a control group fed with a magnesium-deficient special feed (trade name: "HR-AD Feed", manufactured by Nosan Corporation), a first group fed with a mixed feed obtained by adding 0.3% by mass of Pro-Hyp to the magnesium-deficient special feed, a second group fed with a mixed feed obtained by adding 5% by mass of collagen peptide mixture A to the magnesium-deficient special feed, and a third group fed with a mixed feed obtained by adding 2.5% by mass of collagen peptide mixture C having the following composition to the magnesium-deficient special feed.

[0133] Quantitative analysis by LC-MS / MS under the above conditions found that collagen peptide mixture C (a collagen peptide mixture developed by Nitta Gelatin Inc.) had the following composition.

[0134] Pro-Hyp: 12,772 ppm, Hyp-Gly: 6,353 ppm, Gly-Pro-Hyp: 32,010 ppm, Ala-Hyp-Gly: 454 ppm, Glu-Hyp-Gly: 24 ppm, Ser-Hyp-Gly: 239 ppm, Gly-Pro: 26,387 ppm, Pro-Ala-Gly: 2,183 ppm, total: 80,422 ppm.

[0135] After the above groups of hairless mice were reared from 3 weeks to 11 weeks of age, their heads were immediately observed under the following conditions: temperature was 23 ± 2 °C, relative humidity was 55 ± 10%, light cycle was 12 hours, the light cycle started at 7:00 and ended at 19:00, and the mice were allowed to eat freely. Here, it is known that hairless mice start to lose hair after 2 weeks of age and become hairless at about 4 weeks of age. When obtaining hairless mice, they have no hair on their heads. Figure 1 The head of an 11-week-old hairless mouse in the control group fed with the magnesium-deficient special feed is shown. Figure 2 The head of an 11-week-old hairless mouse in the first group fed with the magnesium-deficient special feed containing Pro-Hyp is shown.

[0136] As a result, as can be seen from Figure 1and Figure 2 Hair development occurred in the hairless mice fed the mixed feed containing Pro-Hyp, as understood from the comparison between

[0137] In addition, hairless mice at 2 weeks of age before hair loss began were obtained from Hoshino Laboratory Animals, Inc. The amount of head hair of each 11-week-old hairless mouse in each group was visually measured relative to the amount of head hair of this hairless mouse defined as 10. Table 3 shows the results. Each value in the table represents the average value of the hair amount of 6 hairless mice in each group.

[0138] [Table 3]

[0139] Table 3

[0140] group score normal group 0 control group 0 the first group 8 the second group 3 the third group 7

[0141] [Discussion]

[0142] The above results indicate that Pro-Hyp, collagen peptide mixture A, and collagen peptide mixture C have a promoting effect on the growth of dermal papilla cells and are therefore effective as hair growth agents, particularly as cell growth promoters for dermal papilla cells, hair development or hair growth promoters, or agents for preventing the progression of hair loss.

[0143] [Example 3: Control test (Cell biology test (in vitro test)]

[0144] [Preparation of samples]

[0145] [Preparation of amino acids, peptides, and collagen peptide mixtures]

[0146] As samples for evaluating the promoting effect on the growth of dermal papilla cells, the following were prepared: alanine (trade name: "L-alanine", manufactured by Kanto Kagaku Co., Inc., product number: 01101-30), arginine (trade name: "L-arginine", manufactured by FUJIFILM Wako Pure Chemical Corporation, product number: 015-04613), glutamine (trade name: "L-glutamine", manufactured by FUJIFILM Wako Pure Chemical Corporation, product number: 074-00522), and proline (trade name: "L-proline", manufactured by FUJIFILM Wako Pure Chemical Corporation, product number: 161-04602).

[0147] [Preparation of dermal papilla cells]

[0148] The dermal papilla cells were prepared by the same method as described in the <Preparation of dermal papilla cells> section of Example 1 above.

[0149] [Cell growth test]

[0150] The promoting effect on dermal papilla cell growth and the significance in each sample (amino acid) were evaluated in the same manner as described in the [Cell growth test] section of Example 1 above. Table 4 shows the results. No significant promoting effect on dermal papilla cell growth was exhibited in any sample.

[0151] [Table 4]

[0152] Table 4

[0153]

[0154] [Discussion]

[0155] As can be seen from Table 4, the promoting effect on dermal papilla cell growth of alanine, arginine, glutamine, and proline is poor, and only specific amino acids such as Hyp have a promoting effect on dermal papilla cell growth.

[0156] Although the embodiments and examples of the present invention have been described above, the configurations of the above embodiments and examples can be appropriately combined as originally envisioned.

[0157] The embodiments and examples disclosed herein should be considered illustrative and not restrictive in any way. The scope of the present invention is given by the appended claims rather than the foregoing description, and all changes falling within the scope of the appended claims and their equivalents are included therein.

Claims

1. A hair growth agent comprising the following peptides 8 ppm–12772 ppm of Pro-Hyp, 3447 ppm–7389 ppm of Hyp-Gly, 8 ppm–32010 ppm of Gly-Pro-Hyp, 199 ppm–454 ppm of Ala-Hyp-Gly, 4 ppm–24 ppm of Glu-Hyp-Gly, 120 ppm–239 ppm of Ser-Hyp-Gly, 1159 ppm–26387 ppm of Gly-Pro and 2183 ppm–2645 ppm of Pro-Ala-Gly, or a salt thereof.

2. The hair growth agent according to claim 1, wherein the peptide is derived from collagen.

3. The hair growth agent according to claim 1 or 2, wherein the hair growth agent is a collagen peptide mixture containing at least the peptide according to claim 1 or 2.

4. The hair growth agent according to claim 3, wherein the weight-average molecular weight of the collagen peptide mixture is 100 Da or more and 8000 Da or less.

5. The hair growth agent according to claim 1 or 2, wherein the hair growth agent is a cell growth promoter for dermal papilla cells.

6. The hair growth agent according to claim 1 or 2, wherein the hair growth agent is a hair development or hair growth promoter for hair, or an agent for preventing hair loss progression.

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