JP7758723B2 - Hair carbonyl protein reducer - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies. Posting address: https://ifscc.org/news/welcome-to-the-ifscc-2020-virtual-yokohama-congress/ Posting date: October 14, 2020 Meeting name: IFSCC Congress 2020 Date held: October 21-30, 2020 Location: Online

The present invention relates to agents, compositions and methods for reducing carbonylated proteins in hair, which increase with age.

Like skin, hair changes with age, such as an increase in gray hair and a decrease in hair volume. For women over 40, the increase in gray hair is a particular concern. Until now, the only way to address gray hair has been to correct the color by dyeing, and there is currently no known method for reversing gray hair. In addition to gray hair, other concerns include hair becoming irregular in shape, such as becoming wavy or frizzy, with age. It has also been reported that aging can cause a decrease in hair luster, density, and lipid content, as well as changes in the morphology of the cuticle layer.

As a means of preventing or improving gray hair, Patent Document 1 discloses an agent for preventing or improving gray hair or age-related alopecia, an agent for inhibiting or improving DNA damage in hair follicle constituent cells, and an agent for inhibiting the decrease of melanocyte stem cells, each of which contains at least one active ingredient selected from the group consisting of berberrubine, its derivatives, and salts thereof.

Furthermore, as a novel material that can improve hair appearance and a composition containing the same, Patent Document 2 discloses a composition for improving hair appearance, which contains as an active ingredient a peptide having the amino acid sequence of WY or a pharmaceutically acceptable salt or solvate thereof.

Patent Publication No. 2021-004235 Japanese Patent Publication No. 2020-158413 Patent No. 6802458

I. Iwai, T. Hirao, Skin Pharmacol Physiol., 2008, Vol.21, p.269-273 Y. Kobayashi, I. Iwai, N. Akutsu, T. Hirao, Int. J. Cosmet. Sci., 2008, Vol. 30, pp. 35-40 T. Toda, Biomed. Gerontol., 2011, Vol.35, p.17-22 S. Sato, T. Sakamoto, E. Miyazawa, Y. Kikugawa, Tetrahedron60, 2004, pp.7899-7906 S. Nagase, Y. Kajiura, A. Mamada, H. Abe, S. Shibuichi, N. Satoh, T. Itou, Y. Shinohara, Y. Amemiya, J. Cosmet. Sci., 2009, Vol.60, p.637-648

Aging-related changes in hair quality are a particular concern for women in their 40s and older, and effectively improving hair quality can further improve their quality of life (QOL).

Therefore, the object of the present invention is to provide new methods and agents for improving the qualitative changes in hair that occur with aging.

To solve the above problem, the inventors analyzed age-related changes in hair quality, focusing on black and white hair in people aged 40 and over.

First, we performed external observations of hair from people in their 40s, 50s, and 60s and found that irregular hair shapes increased with age under both high and low humidity conditions. In particular, wavy and most irregular hair shapes were observed more frequently in gray hair than in black hair.

The irregular shapes caused by changes in humidity differed between gray and black hair, suggesting that the two hair types differ in their reactivity to water. We then applied the water penetration measurement method described in Patent Document 3 to evaluate the water behavior of gray and black hair, and found that gray hair has higher water permeability and lower water retention than black hair. Since the contribution of the cortex, which accounts for more than 80% of the cross-sectional area of the hair shaft, is greater than that of the cuticle, the inventors believe that denaturation of keratin proteins in the cortex is one of the factors behind the difference in water behavior.

Therefore, the inventors evaluated the degree of carbonylation, an indicator of aging, using gray and black hair from women of various age groups. The results showed that the content of carbonylated proteins in hair increased with age, and significant differences were observed in the carbonylation of gray and black hair in women in their 50s and 60s compared to women in their 40s.

It has been reported that skin carbonylation increases with age and leads to various cosmetic defects (Non-Patent Documents 1 and 2). These documents relate to the correlation between carbonylated proteins in the stratum corneum and physiological parameters of the skin. These documents show that protein carbonylation is inversely proportional to the water content of the stratum corneum, and that carbonylation of keratin proteins in each layer reduces the water-retaining capacity. The inventors hypothesized that hair keratin proteins may also undergo a similar phenomenon by undergoing carbonylation in the same way as skin keratin proteins.

Until now, it was thought that carbonylated proteins could not be restored to their previous state (Non-Patent Document 3). However, the present inventors have demonstrated that reductive amination treatment using 5-ethyl-2-methylpyridineborane (PEMB) or Hantzsch ester can reduce the amount of carbonylated proteins in hair that increase with age. They also demonstrated that reductive amination treatment improves hair's water-retaining capacity, reduces the mean curvature of hair, and improves irregular hair shape.

The one-pot reductive amination of aldehydes and ketones is an important transformation in organic synthesis, and it is known that carbonyl compounds can be directly converted to amines through a simple procedure (Non-Patent Document 4). However, it was not known that reductive amination was possible for organic materials. The present inventors have demonstrated for the first time that reductive amination is also useful for organic materials. The present invention was made based on these findings.

The present invention provides a hair carbonyl protein reducer containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source.

The present invention also provides the agent for reducing carbonylated protein in hair, wherein the reducing agent is an organoborane complex or a compound represented by the following general formula (I):
(In general formula (I), ^R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a carbohydrate having 1 to 16 carbon atoms, adenine dinucleotide, or adenine dinucleotide phosphate; ^R2 and ^R3 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; ^R4 and ^R5 each independently represent a hydrogen atom, -COO- ^R6 , or -CO- ^{NR6R7 ;} and ^R6 and ^R7 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.)

The present invention also provides the hair carbonyl protein reducer, wherein the reducing agent is at least one selected from the group consisting of 5-ethyl-2-methylpyridine borane (PEMB), 5-ethyl-2-methylpyridine borane complex, picoline borane complex, sodium cyanoborohydride (NaBH3CN), and sodium triacetoxyborohydride.

The present invention also provides the agent for reducing hair carbonyl protein, wherein the reducing agent is a compound represented by the general formula (I) above, ^R1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, at least one of ^R4 and ^R5 represents —COO— ^R6 , and ^R6 represents an alkyl group having 1 to 3 carbon atoms.

The present invention also provides the agent for reducing hair carbonylated proteins, wherein the reducing agent is a compound represented by the general formula (I) above, ^R1 represents a hydrogen atom, a carbohydrate having 1 to 16 carbon atoms, adenine dinucleotide, or adenine dinucleotide phosphate, ^R4 represents a hydrogen ^atom , ^R5 represents -CO- ^NR6R7 , and ^R6 and ^R7 represent hydrogen atoms.

The present invention also provides the hair carbonylated protein reducer, wherein the reducing agent is at least one selected from the group consisting of reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide, and Hantzsch ester.

The present invention also provides the hair carbonyl protein reducer, wherein the acid catalyst is at least one selected from the group consisting of acetic acid, citric acid, formic acid, propionic acid, phosphoric acid, and oxalic acid.

The present invention also provides the hair carbonyl protein reducer, wherein the nitrogen source is ammonia or an ammonium salt.

The present invention also provides a hair modifying composition containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source, and a solvent.

The present invention also provides the above hair modifying composition, in which the above reducing agent is an organoborane complex or a compound represented by the above general formula (I).

The present invention also provides the above hair modifying composition, wherein the reducing agent is at least one selected from the group consisting of 5-ethyl-2-methylpyridine borane (PEMB), 5-ethyl-2-methylpyridine borane complex, picoline borane complex, sodium cyanoborohydride (NaBH3CN), and sodium triacetoxyborohydride.

The present invention also provides the hair modifying composition, wherein the reducing agent is a compound represented by general formula (I) above, ^R1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, at least one of ^R4 and ^R5 represents —COO— ^R6 , and ^R6 represents an alkyl group having 1 to 3 carbon atoms.

The present invention also provides the hair modifying composition, wherein the reducing agent is a compound represented by general formula (I), ^R1 represents a carbohydrate having 1 to 16 carbon ^atoms , ^R4 represents a hydrogen atom, ^R5 represents —CO— ^NR6R7 , and ^R6 and ^R7 represent hydrogen atoms.

The present invention also provides the above-mentioned hair modifying composition, in which the reducing agent is at least one selected from the group consisting of reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide, and Hantzsch ester.

The present invention also provides the hair modifying composition, wherein the acid catalyst is at least one selected from the group consisting of acetic acid, citric acid, formic acid, propionic acid, phosphoric acid, and oxalic acid.

The present invention also provides the hair modifying composition, wherein the nitrogen source is ammonia or an ammonium salt.

The present invention also provides the above hair modifying composition, wherein the solvent is water or ethanol.

The present invention also provides a hair moisture retention improving agent containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source.

The present invention also provides a hair curvature reducing agent containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source.

The present invention also provides a method for reducing carbonylated proteins in hair, which comprises subjecting hair to reductive amination treatment with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source.

The present invention also provides a method for improving the water retention capacity of hair, which comprises treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source by reductive amination.

The present invention also provides a method for reducing hair curvature, which comprises treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source by reductive amination.

The present invention can reduce carbonylated proteins in hair, which increase with age. Therefore, the present invention can improve hair's water retention, which decreases with age. Furthermore, the present invention can reduce hair curvature, which increases with age, and improve irregular hair shapes. Therefore, the present invention can contribute to further improving quality of life (QOL).

Diagram showing hair bundles in the forties (40s), fifties (50s) and sixties (60s) under relative humidity (RH) conditions of 20% RH (a), 50% RH (b) or 90% RH (c). A diagram showing the average curvature of hair for each age group. FIG. 1 shows an example of irregular shapes observed in white hair and dyed white hair. FIG. 10 shows measurement points for line mapping using an optical microscope image of untreated hair. A diagram showing the behavior of water in the cortex of black hair (diamond shape) and white hair (triangle shape) of a Japanese woman in her 50s. A diagram showing the localization of carbonylated proteins in cross-sections of hair from each age group. Graph showing the ratio of mean fluorescence intensity in cross sections of white and black hair for each age group. A diagram showing the localization of carbonylated proteins in a cross-section of hair from a Japanese woman in her 50s. A diagram showing the behavior of water in the cortex of black hair (diamond shape), white hair (triangle), and white hair treated with reductive amination (square) from a Japanese woman in her 50s. FIG. 10 shows the mean curvature of hair from the same subjects as in FIGS. 2 and 9. FIG. 1 is a graph showing the level of amino groups introduced into hair treated with reductive amination. FIG. 1 is a graph showing the effect of reducing carbonylated proteins when Hantzsch ester is used as a reducing agent. FIG. 1 is a diagram showing a comparison of the effects of different solvents on reducing carbonylated proteins.

The present invention provides a hair carbonyl protein reducer containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source.

As used herein, "reducing carbonylated proteins in hair" means reducing the amount of carbonylated proteins contained in hair. As used herein, "hair" includes both the hair shaft extending from the scalp and the hair root within the scalp. "Reducing carbonylated proteins in hair" includes reducing carbonylated proteins in at least one of the hair cuticle, cortex, and medulla. Additionally, as used herein, "reducing carbonylated proteins" includes inhibiting an increase in carbonylated proteins.

Carbonylated proteins are proteins in which amino acids such as proline, arginine, lysine, or threonine have been oxidized by reactive oxygen species (ROS) to form carbonyl derivatives. Carbonylated proteins are known to be produced in vivo by aging and oxidative stress.

The hair carbonyl protein reducing agent of the present invention can modify hair by reducing hair carbonyl proteins. That is, the hair carbonyl protein reducing agent of the present invention can be used as a hair modifying agent. As used herein, "modifying hair" includes improving hair's water retention, reducing the mean curvature of hair, correcting irregular hair shapes, and improving hair appearance.

As used herein, the term "reductive aminating agent" refers to an agent capable of converting a carbonyl group of a carbonylated protein to an amino group through a reductive amination reaction. Reductive amination is a chemical reaction that converts an aldehyde or ketone to an amine. In the present invention, the reductive aminating agent is a mixture containing a reducing agent, an acid catalyst, and a nitrogen source.

In the present invention, a reducing agent is used to reduce the reaction intermediate imine to an amine. For example, an organoborane complex can be used as the reducing agent. Examples of organoborane complexes include 5-ethyl-2-methylpyridine borane (PEMB), 5-ethyl-2-methylpyridine borane complex, picoline borane complex, sodium cyanoborohydride (NaBH3CN), and sodium triacetoxyborohydride. From the standpoint of toxicity and safety, 5-ethyl-2-methylpyridine borane complex is preferred.

Furthermore, as the reducing agent in the present invention, a compound having a 1,4-dihydropyridine structure represented by the following general formula (I) can be used.

In general formula (I), ^R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a carbohydrate having 1 to 16 carbon atoms. The alkyl group represented by ^R1 can be, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, or a cyclopropyl group.

In general formula (I), ^R2 and ^R3 each independently represent a hydrogen atom or an alkyl group having 1 to ³ carbon atoms. ^R2 and ^R3 may be the same or different. The alkyl group represented by ^R2 and R3 may be, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, or a cyclopropyl group.

In general formula (I), ^R4 and ^{R5 each} independently represent a hydrogen atom, —COO— ^R6 , or —CO— ^NR6R7 . ^R4 and ^R5 may be the same or different.

In general formula (I), ^R6 and ^R7 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. ^R6 and ^R7 may be the same or different. The alkyl group represented by ^R6 and ^R7 may be, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, or a cyclopropyl group.

As used herein, the term "alkyl group" includes straight-chain and branched alkyl groups. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, and hexyl groups.

As used herein, "carbohydrate" includes monosaccharides, disaccharides, oligosaccharides, amino sugars, and alditols. Monosaccharides can be pyranosides or furanosides. Examples of carbohydrates include D-glucose, D-mannose, D-galactose, D-allose, D-altrose, D-idose, D-talose, D-xylose, D-ribose, and D-arabinose. Carbohydrates can also be structures in which two or more monosaccharides are linked by one to three phosphate groups. The monosaccharides constituting carbohydrates can be, for example, nucleosides or nucleotides in which ribose is linked to a nucleic acid base. Carbohydrates can be, for example, adenine dinucleotide or adenine dinucleotide phosphate.

In the present invention, alkyl groups and carbohydrates may have a substituent. Examples of the substituent include halogen atoms, nitrogen atoms, sulfur atoms, hydroxyl groups, nitro groups, cyano groups, methoxy groups, ethoxy groups, carboxyl groups, carboxymethyl groups, carboxyethyl groups, and methylenedioxy groups. Halogen atoms include fluorine, chlorine, bromine, and iodine atoms.

In general formula (I), preferably, ^R1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, at least one of ^R4 and ^R5 represents -COO- ^R6 , and ^R6 represents an alkyl group having 1 to 3 carbon atoms. More preferably, ^R1 represents a hydrogen atom, ^R2 and ^R3 each represent a methyl group, ^R4 and ^R5 each represent -COO- ^R6 , and ^R6 represents an ethyl group. The reducing agent in the present invention is preferably a Hantzsch ester represented by the following formula (II):

In general formula (I), preferably, ^R1 represents a hydrogen atom or a carbohydrate having 1 to 16 carbon atoms, ^R4 represents a hydrogen atom, ^R5 represents -CO- ^NR6R7 , and ^R6 and ^R7 represent hydrogen atoms. More preferably, ^R1 represents a hydrogen atom, adenine ^dinucleotide , or adenine dinucleotide phosphate, ^R2 , ^R3, and ^R4 represent hydrogen atoms, ^R5 represents -CO- ^NR6R7 , and ^R6 and ^R7 each represent a hydrogen atom. The reducing agent in the present invention is, for example, reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced nicotinamide adenine dinucleotide (NADH), or reduced nicotinamide. NADPH/ ^NADH is a compound having the following structure. Reduced niacinamide is a compound obtained by isolating only the active site of NADH/NADPH, and both are biological molecules.

Hantsch ester is preferably used as the reducing agent in the present invention from the standpoint of economy and stability. Hantsch ester can be purchased in large quantities at low cost and is easy to store.

The compound represented by general formula (I) is used as a reducing agent in its reduced form. This compound functions as a reducing agent by releasing two electrons to become its oxidized form.

By using the compound represented by the above general formula (I) as the reducing agent of the present invention, it is possible to reduce the burden on the environment and increase safety for the human body. Furthermore, because water or ethanol, etc., can be used as the solvent, safety for the human body can be further increased.

Compounds of general formula (I) can be synthesized by any synthetic procedure known to those skilled in the art. Commercially available compounds can be used, including 5-ethyl-2-methylpyridine borane (PEMB), 5-ethyl-2-methylpyridine borane complex, picoline borane complex, sodium cyanoborohydride (NaBH3CN), and sodium triacetoxyborohydride, as well as reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide, and Hantzsch ester. Furthermore, any of the reduced compounds can be obtained by reducing the oxidized form using standard procedures.

In the present invention, the acid catalyst is an acid used to activate the carbonyl group by adding a proton. The acid catalyst may be any Brønsted acid, and examples include acetic acid, citric acid, formic acid, propionic acid, phosphoric acid, and oxalic acid. From the standpoint of safety and simplicity, the acid catalyst is preferably acetic acid or phosphoric acid, although it is not particularly limited.

In the present invention, a nitrogen source is used to convert a carbonyl group to an imine. The nitrogen source is not particularly limited, but ammonia or an ammonium salt can be used, for example. Ammonia and ammonium salts can restore the carbonylated amino acid to its original amino acid structure. Specific examples of nitrogen sources that can be used include aqueous ammonia, ammonium formate, ammonium acetate, ammonium carbonate, and ammonium bicarbonate. From the perspective of ease of operation, the nitrogen source is not particularly limited, but ammonia water or ammonium acetate is preferably used.

The present invention also provides a hair modifying composition containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source, and a solvent.

The solvents used in the present invention include, but are not limited to, protic solvents, aprotic polar solvents, and aprotic solvents. Protic solvents include, for example, water, methanol, ethanol, 1-propanol, 2-propanol, propylene glycol, glycerin, butylene glycol, and 1,3-BG. Aprotic polar solvents include, for example, dimethyl sulfoxide, dimethylformamide, acetonitrile, and hexamethylphosphoramide. Aprotic solvents include, for example, diethyl ether, chloroform, toluene, 1,4-dioxane, and liquid hydrocarbons. Of these, water, methanol, or ethanol are preferred due to their reactivity and ease of handling. Furthermore, water or ethanol is preferred due to its safety for the human body.

The present invention also provides a method for reducing carbonylated proteins in hair, which comprises subjecting hair to reductive amination treatment with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source.

In the method of the present invention, the reducing agent, acid catalyst, nitrogen source, and solvent can be those described in detail above. That is, in the method of the present invention, hair can be treated using the agent or composition of the present invention described above.

In the method of the present invention, the method of reductively aminating hair with a reducing agent can be, for example, a method of preparing a composition containing a nitrogen source such as an aqueous ammonia solution and a reducing agent in a solvent containing an acid catalyst, and treating hair with this composition.

The present invention also provides an agent for improving hair water retention, which contains a reductive amination agent including a reducing agent, an acid catalyst, and a nitrogen source. The present invention also provides a method for improving hair water retention, which comprises treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source through reductive amination.

In this specification, "improving hair's water retention ability" means improving hair's water retention ability that has decreased with age, and includes improving hair's water retention ability that has decreased with age and inhibiting the decrease in water retention ability that occurs with age.

The present invention also provides an agent for reducing hair curvature, which contains a reductive amination agent comprising a reducing agent, an acid catalyst, and a nitrogen source.The present invention also provides a method for reducing hair curvature, which comprises treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source by reductive amination.

As used herein, "reducing hair curvature" includes reducing hair curvature that has increased with age and inhibiting the increase in hair curvature due to age. "Hair curvature" refers to the degree of disorder of hair, with higher curvature meaning greater disorder of hair.

In addition to the above-mentioned components, the agent and composition of the present invention may contain any other components depending on the intended use, as long as the effects of the present invention are not impaired. For example, the agent and composition of the present invention may contain anionic surfactants such as lauryl sulfate, polyoxyethylene lauryl ether sulfate, lauryl benzene sulfonate, and sodium lauroylmethyl-β-alanine; amphoteric surfactants such as 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazonium betaine, coconut oil fatty acid amidopropyl betaine, and coconut oil alkyl N-carboxyethyl-N-hydroxyethylimidazolinium betaine sodium; polymers such as cationized cellulose, polyacrylic acid, and poly(diallyldimethylammonium chloride); sorbitol, inositol, glycerin, propylene glycol, butylene glycol, etc. It may also contain humectants such as ethylene glycol, dipropylene glycol, and isoprene glycol; anti-dandruff ingredients such as zinc pyrithione and benzalkonium chloride; lower alcohols such as ethanol, methanol, propyl alcohol, and isopropyl alcohol; higher alcohols such as cetyl alcohol, stearyl alcohol, and behenyl alcohol; quaternary cations such as behentrimonium chloride and stearyltrimonium chloride; amino acids such as DL-alanine, L-arginine, glycine, L-cysteine, and L-threonine; as well as ultraviolet absorbers, preservatives, sugars, fragrances, colorants, sequestering agents, antioxidants, and various other drugs.

The agents and compositions of the present invention can be in any form, such as liquid, emulsion, cream, gel, paste, and foam, depending on their intended use.

The agents and compositions of the present invention can be in the form of, for example, hair cosmetics. Hair cosmetics include, for example, hair rinses, hair conditioners, hair treatments, leave-in treatments, hair packs, hair foams, hair waxes, pomades, hair gels, hair creams, hair sprays, hair mists, hair water, hair liquids, hair oils, hair tonics, hair lotions, hair colors, perm agents, and hair care products. These hair cosmetics can be produced by conventionally known methods.

(Samples and Materials)
The subjects were healthy Japanese women aged 40-60 who had not dyed or permed their hair for one week prior to the test. They only used shampoo the day before the test, and did not use any hair styling products or hair treatments on the day of the test. The hair samples were immersed in ion-exchange water for 10 minutes to remove temporary hair shapes, and the intrinsic shape of the hair was evaluated.

Deuterium oxide (99.8% D) was obtained from Kanto Chemical Co., Ltd. (Tokyo, Japan). 2-(N-morpholino)ethanesulfonic acid (MES) and phosphate-buffered saline (PBS) were obtained from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fluorescein-5-thiosemicarbazide (5-FTSC) and 5-ethyl-2-methylpyridine borane complex (PEMB) were obtained from Sigma (St. Louis, Missouri, USA). The chemical structure of PEMB is shown below.

(statistical analysis)
Statistical analysis was performed using Dunnett's multiple comparison test or Student's t-test, with significance defined as p<0.05.

[Test 1: External observation of hair]
Hair samples were prepared from 20 strands each from individuals in their 40s, 50s, and 60s. The hair was stored at 25°C and the relative humidity (RH) was varied from 50% to 90% or from 50% to 20%. The appearance of the hair was observed. Figure 1 shows hair tresses from individuals in their 40s (40s), 50s (50s), and 60s (60s) under 20% (a), 50% (b), or 90% (c) RH conditions. External observations indicated that irregular hair shape increased with age under both high and low humidity conditions.

To quantify this irregular shape, the mean curvature of the hair was measured using the method described in Non-Patent Document 5. Specifically, the hair fibers were immersed in water at 25°C for 10 minutes to remove temporary water set formed during drying of the hair fibers and resulting from the exchange of hydrogen bonds. Next, the hair fibers were placed in an image scanner (GT-X830, Seiko Epson Corporation) to capture two-dimensional images of the hair shape, and the curvature of each curve from the root to the tip was analyzed. Because hair color has little effect on hair shape, shape measurements were performed without distinguishing between color-treated and uncolored hair.

Figure 2 shows the average hair curvature for each age group (mean ± SD (n = 20)). As shown in Figure 2, the average curvature was found to increase with age.

Furthermore, careful observation of these hair strands revealed that the most irregular shapes were observed in white hair or dyed white hair. Figure 3 shows an example of the irregular shapes observed in white hair and dyed white hair.

[Test 2: Water behavior evaluation]
The irregular shape caused by changes in humidity was different between white and black hair, suggesting that the reactivity to water differs between the two hair types. Therefore, the behavior of water inside the hair was measured.

Water behavior was evaluated using a method adapted from the water penetration measurement method described in Patent Document 3. Specifically, smooth hair sections were sandwiched between diamond cells developed in-house using a rotary microtome, and heavy water was injected into the cell gap. The hair sections were continuously evaluated using a Fourier transform infrared microscope (Thermo Fisher Scientific, Nicolet iN10). Water behavior was visualized and quantified in real time by evaluating the intensity of the OD stretching peak at approximately 2500 cm ^-1 , which indicates heavy water, every 10 μm from the medulla to the cuticle using line mapping measurements. Figure 4 shows the line mapping measurement points using an optical microscope image of untreated hair. The measurements were performed with a resolution of 8 cm ^-1 , 256 integrations, and a wavenumber range of 4000 to 650 cm ^-1 .

Figure 5 shows the behavior of water in the cortex of black hair (diamond shape) and white hair (triangle shape) from the same Japanese woman in her 50s (OD stretching peak intensity at approximately 2500 cm ^-1 ; first 30 minutes under heavy water penetration conditions, followed by a 4-hour drying process at 25°C and 50% RH). This result indicates that the rate of water penetration from the outside is faster in white hair than in black hair. During the drying process, white hair lost moisture one hour earlier than black hair. These results indicate that the water retention capacity of white hair is reduced compared to black hair.

Test 3: Evaluation of local carbonylation of hair
Changes in hair proteins were thought to be responsible for the different water behavior observed between white and black hair, so carbonylation was assessed in white and black hair at different age groups.

After adjusting the hair samples to a uniform thickness using a rotary microtome (Leica RM2265), they were immersed in 20 μM 5-FTSC/0.1 M MES-Na (pH 5.5) and incubated in the dark for 1 hour. The hairs were then washed four times with PBS and observed under a fluorescence microscope. The resulting images were analyzed using ImageJ, and the fluorescence intensity per unit area of the hair sample was defined as the degree of carbonylation.

Figure 6 shows the localization of carbonylated proteins in cross-sections of hair from each age group ((a) black hair from a person in their 40s; (b) black hair from a person in their 50s; (c) black hair from a person in their 60s; (d) white hair from a person in their 40s; (e) white hair from a person in their 50s; (f) white hair from a person in their 60s). As shown in Figure 6, an increase in carbonylated proteins was observed with age. It was also shown that white hair contained more carbonylated proteins than black hair.

These measurements were quantified using fluorescence intensity. Figure 7 shows the ratio of mean fluorescence intensity in cross-sections of white and black hair for each age group (n = 10; **p < 0.01 vs. hair in women in their 40s (Dunnett's multiple comparison test)). Compared to women in their 40s, women in their 50s and 60s showed significantly higher carbonylation in white hair compared to black hair.

[Test 4: Effect of reductive amination reaction]
Aged hair was subjected to the following reductive amination reaction treatment. A dispersion solution containing 28% aqueous ammonia and 201 mg of PEMB was prepared in methanol:acetic acid (10:1; 5.5 mL). Then, thinly cut hair samples (100 mg) were placed in the solution at room temperature for 4 days. After the reaction, the hair samples were washed four times with methanol.

Local carbonylation was then assessed. Figure 8 shows the localization of carbonylated proteins in hair sections from a Japanese woman in her 50s (Reagent: reductive amination treatment). Reductive amination treatment was shown to reduce the amount of carbonylated proteins, regardless of whether the hair was black or white.

We also evaluated the water behavior of hair that had undergone reductive amination. Figure 9 shows the water behavior in the cortex of black hair (diamond), white hair (triangle), and reductively aminated white hair (square) from the same Japanese woman in her 50s (OD stretching peak intensity at approximately 2500 cm ^-1 ; first 30 minutes under heavy water penetration conditions, followed by a 4-hour drying process at 25°C and 50% RH). The reductively aminated white hair showed improved water retention and water behavior equivalent to that of black hair.

Figure 10 shows the mean curvature of hair from the same subjects as in Figures 2 and 9 (mean ± SD (n = 20); * p < 0.05 vs. hair from people in their 50s at 90% RH (Student's t-test)). At 90% RH, the mean curvature of hair from people in their 50s that underwent reductive amination treatment was significantly reduced compared to hair that did not undergo reductive amination treatment. This result indicates that reductive amination treatment also improves hair shape.

[Test 5: Level of amino group introduction into hair treated with reductive amination]
We confirmed whether externally applied amino groups were introduced into hair through reductive amination. Ammonia was replaced with a fluorescent reagent containing amino groups (DBD-ED), and reductive amination was performed. Specifically, a dispersion solution containing 21 mg of DBD-ED (solid) and 10.26 mg of PEMB was prepared in methanol:acetic acid (10:1; 1 mL). Hair (mixed black and white) from a person in their 60s was cut into 1 cm pieces and used. Ten hair samples were placed in the solution at room temperature for four days. After the reaction, the hair samples were washed four times with methanol. As a control, a sample without PEMB was also tested in the same manner.

The fluorescence intensity in the cross section of the treated hair was measured, and the level of amino group introduction was quantified. Figure 11 shows the level of amino group introduction in hair treated with reductive amination. As shown in Figure 11, the level of amino group introduction was higher in hair treated with PEMB compared to the control without PEMB, indicating that a large amount of DBD-ED was introduced into the hair. These results demonstrate that PEMB treatment introduces amino groups into hair.

Test 6: Reductive amination with Hantzsch ester
The reduction of carbonylated proteins was investigated using Hantsch ester as a reducing agent. Aged hair was subjected to the following reductive amination reaction. A dispersion solution containing 28% aqueous ammonia, 201 mg of Hantsch ester (obtained from Tokyo Chemical Industry Co., Ltd.), and 30 mg of Hantsch ester was prepared in methanol:acetic acid (10:1; 5.5 mL). Next, a 1 cm section of hair from the roots was used as a sample and stirred in the solution at room temperature for 4 days. After the reaction, the hair sample was washed four times with methanol. A control test was also performed using a sample without Hantsch ester.

Then, sections of the sample hair were prepared and the carbonyl levels were evaluated using fluorescent staining. Figure 12 shows the effect of reducing carbonylated proteins when Hantsch ester was used as a reducing agent. Hair treated with Hantsch ester for reduction showed a reduced amount of carbonylated proteins.

[Test 7: Confirmation of solvent effect]
The effect of using water or ethanol as a solvent other than methanol on reducing carbonylated proteins was investigated. Aged hair was subjected to the following reductive amination reaction. A dispersion solution containing 28% aqueous ammonia and 30 mg of Hantsch ester (201 mg) was prepared in various solvents: acetic acid (10:1; 5.5 mL). Next, a 1 cm section of hair from the roots was used as a sample and stirred in the solution at room temperature for 4 days. After the reaction, the hair sample was washed four times with methanol. The solvents used were water, methanol, and ethanol. A control test was also performed using a sample without Hantsch ester but using methanol as the solvent.

Then, sections of the sample hair were prepared and the carbonyl levels were evaluated using fluorescent staining. Figure 13 shows a comparison of the effect of different solvents on reducing carbonylated proteins. The results showed that the amount of carbonylated proteins also decreased in hair that had been reduced using water or ethanol as the solvent.

(Conclusion)
These results indicate that gray hair, which increases with age, has a higher concentration of carbonylated proteins than black hair from the same individual, leading to reduced water-retaining capacity and an irregular water-related shape. Furthermore, while it was previously thought that carbonylated proteins could not return to their original shape, these test results demonstrated that carbonylated proteins were reduced by reductive amination using PEMB and Hantzsch ester. Furthermore, reductive amination also improved hair water-retaining capacity and curvature. These results suggest that modifying hair shape through reductive amination may be useful for improving quality of life.

The present invention can be suitably used in cosmetics and pharmaceuticals for modifying hair quality.

Claims (22)

A hair carbonyl protein reducer containing a reductive aminating agent including a reducing agent, an acid catalyst and a nitrogen source. 2. The hair carbonyl protein reducer according to claim 1, wherein the reducing agent is an organoborane complex or a compound represented by the following general formula (I):
(In general formula (I), ^R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a carbohydrate having 1 to 16 carbon atoms, adenine dinucleotide, or adenine dinucleotide phosphate; ^R2 and ^R3 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; ^R4 and ^R5 each independently represent a hydrogen atom, -COO- ^R6 , or -CO- ^{NR6R7 ;} and ^R6 and ^R7 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.) The hair carbonyl protein reducer according to claim 2, wherein the reducing agent is at least one selected from the group consisting of 5-ethyl-2-methylpyridine borane (PEMB), 5-ethyl-2-methylpyridine borane complex, picoline borane complex, sodium cyanoborohydride (NaBH3CN), and sodium triacetoxyborohydride. The agent for reducing hair carbonyl protein according to claim ² , wherein the reducing agent is a compound represented by general formula (I), ^R1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, at least one of ^R4 and ^R5 represents -COO-R6, and ^R6 represents an alkyl group having 1 to 3 carbon atoms. The agent for reducing hair carbonylated proteins according to claim ² , wherein the reducing agent is a compound represented by general formula (I), ^R1 represents a hydrogen atom, a carbohydrate having 1 to 16 carbon atoms, adenine dinucleotide, or adenine dinucleotide phosphate, R4 represents a hydrogen ^atom , ^R5 represents -CO- ^NR6R7 , and ^R6 and ^R7 represent hydrogen atoms. A hair carbonylated protein reducer according to claim 2, wherein the reducing agent is at least one selected from the group consisting of reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide, and Hantzsch ester. A hair carbonyl protein reducer according to any one of claims 1 to 6, wherein the acid catalyst is at least one selected from the group consisting of acetic acid, citric acid, formic acid, propionic acid, phosphoric acid, and oxalic acid. A hair carbonyl protein reducer according to any one of claims 1 to 7, wherein the nitrogen source is ammonia or an ammonium salt. A hair modifying composition containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source, and a solvent. 10. The hair modifying composition according to claim 9, wherein the reducing agent is an organoborane complex or a compound represented by the following general formula (I):
(In general formula (I), ^R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a carbohydrate having 1 to 16 carbon atoms, adenine dinucleotide, or adenine dinucleotide phosphate; ^R2 and ^R3 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; ^R4 and ^R5 each independently represent a hydrogen atom, -COO- ^R6 , or -CO- ^{NR6R7 ;} and ^R6 and ^R7 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.) The hair modifying composition according to claim 10, wherein the reducing agent is at least one selected from the group consisting of 5-ethyl-2-methylpyridine borane (PEMB), 5-ethyl-2-methylpyridine borane complex, picoline borane complex, sodium cyanoborohydride (NaBH3CN), and sodium triacetoxyborohydride. The hair modifying composition according to claim 10, wherein the reducing agent is a compound represented by general formula (I), ^R1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, at least one of ^R4 and ^R5 represents -COO- ^R6 , and ^R6 represents an alkyl group having 1 to 3 carbon atoms. The hair modifying composition according to claim 10, wherein the reducing agent is a compound represented by general formula (I), ^R1 represents a hydrogen atom, a carbohydrate having 1 to 16 carbon atoms, adenine dinucleotide, or adenine dinucleotide phosphate, ^R4 represents a hydrogen ^atom , ^R5 represents -CO- ^NR6R7 , and ^R6 and ^R7 represent hydrogen atoms. The hair modifying composition according to claim 10, wherein the reducing agent is at least one selected from the group consisting of reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide, and Hantzsch ester. A hair modifying composition according to any one of claims 9 to 14, wherein the acid catalyst is at least one selected from the group consisting of acetic acid, citric acid, formic acid, propionic acid, phosphoric acid, and oxalic acid. A hair modifying composition according to any one of claims 9 to 15, wherein the nitrogen source is ammonia or an ammonium salt. A hair modifying composition according to any one of claims 9 to 16, wherein the solvent is water or ethanol. A hair moisture retention improver containing a reductive aminating agent including a reducing agent, an acid catalyst, and a nitrogen source. A hair curvature reducer containing a reductive aminating agent including a reducing agent, an acid catalyst and a nitrogen source. A method for reducing carbonylated proteins in hair, comprising treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source through reductive amination. A method for improving the water retention capacity of hair, comprising treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source through reductive amination. A method for reducing hair curvature, comprising treating hair with a reducing agent in a solvent in the presence of an acid catalyst and a nitrogen source by reductive amination.