JPH08104614A - Hair pretreating agent, hair pretreating cosmetic and method for dyeing hair - Google Patents

Abstract

PURPOSE: To obtain a hair pretreating agent capable of providing high hair dyeing effects and improving the hair even in the organoleptic aspects without damaging the hair and causing the skin irritancy. CONSTITUTION: This hair pretreating agent comprises a soluble keratinous protein or a keratinous protein prepared by carrying out the reducing treatment of keratinous proteinic fibers having a cross-linked structure in a liquid, then removing insoluble substances, adding a cationic surfactant to the resultant solution and subsequently removing the reducing agent. Hair is treated with a hair pretreating cosmetic containing the hair pretreating agent and then dyed with a hair dye containing an antibody, having immunological activities against the keratinous protein and modified with a pigment, a coloring matter or an enzyme.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hair pretreatment agent used in a hair dyeing method using an anti-hair keratin antibody that does not adhere to the skin and specifically binds to the hair, and a hair pretreatment cosmetic composition containing the same. And a hair dyeing method.

[0002]

2. Description of the Related Art In recent years, research on cosmetics using antibodies has been conducted (Japanese Patent Laid-Open No. 5-163123). Cosmetics using an antibody have advantages such as not damaging hair and less irritation to the skin because the antibody is a protein.

However, when this is used as a hair dye, it is not possible to bind a sufficient amount of dye to the antibody, and the binding power of the antibody to hair becomes insufficient, so that it is practically used as a hair dye. I couldn't. To solve this problem, Japanese Patent Application No. 4-351973 and Japanese Patent Application No. 4-3
Japanese Patent Laid-Open No. 51971 and Japanese Patent Application Laid-Open No. 2-134012 disclose a method in which an antibody modified with a pigment or a dye or modified with an enzyme is used as a hair dye. However, in order to actually fully exhibit the hair dyeing effect, it was necessary to further efficiently bind the antibody itself to the hair.

[0004]

Therefore, an object of the present invention is to use an anti-hair antibody, which has good coloring properties, is resistant to fading, does not stain the skin, does not cause skin irritation, and does not cause hair damage. It is an object of the present invention to provide a hair pretreatment agent that enhances the hair dyeing effect of a hair dye or a hair cosmetic composition that improves the feel of the hair, and a hair cosmetic composition that contains the hair pretreatment agent.

[0005]

Therefore, as a result of intensive studies on the method for achieving the above object, the present inventors have conducted a reduction treatment of keratin protein fibers having a crosslinked structure in a liquid and then removing the insoluble matter. To the hair pretreatment agent comprising soluble keratin protein or keratin protein obtained by removing the reducing agent after adding a cationic surfactant to the hair, and after treating the hair with a hair treatment agent containing the hair pretreatment agent, The present invention has been completed by discovering that coloring properties can be dramatically improved by dyeing hair with a hair dye containing an antibody immunologically active against keratin protein modified with a pigment or a dye or an enzyme.

That is, according to claim 1 of the present invention, a keratin protein fiber having a crosslinked structure is reduced in a liquid,
Then, after adding the cationic surfactant to the solution in which the insoluble matter has been removed, the soluble keratin protein obtained by removing the reducing agent or the keratin protein fiber having a crosslinked structure is added to the liquid containing the reducing agent and the cationic surfactant. A pretreatment agent for hair comprising soluble keratin protein, which is obtained by contacting with, and then removing the reducing agent from the solution from which insoluble matter has been removed.

A second aspect of the present invention is a hair pretreatment cosmetic containing the hair pretreatment agent according to the first aspect.

Further, claim 3 is directed to a keratin protein modified with a pigment or a dye after treating the hair with the hair pretreatment agent according to claim 1 or the hair pretreatment cosmetic composition according to claim 2. A hair dyeing method characterized by dyeing hair with an antibody having immunological activity.

[0009] A fourth aspect of the present invention is that the hair is treated with the hair pretreatment agent according to the first aspect or the hair pretreatment cosmetic agent according to the second aspect, and then has an immunological activity against a keratin protein modified with an enzyme. It is a hair dyeing method characterized by dyeing hair with an antibody.

The present invention will be described in detail below. As the keratin-containing substance used in the present invention, hair and nails of humans, sheep, goats, horses, pigs, cows, rabbits and the like and feathers of various birds are preferably used, but wool is preferable from the economical viewpoint. Further, from the viewpoint of the effect, it is desirable to use a species close to the species of the animal in which the antigen was prepared when producing the antibody.

As the reducing agent, a general reducing agent may be used, and thioglycolic acid, mercaptoethanol or sodium hydrogen sulfite is used. The concentration of these reducing agents is 0.05 to 0.5 mol based on 10 g of the keratin-containing substance. As the solvent used for the reduction treatment of the keratin-containing substance, water or a buffer solution is used because it is easy to use. The amount is
The ratio of the keratin-containing substance to the solvent is preferably about 0.5 to 10% by weight.

Hair, nails, animal hair, feathers, horns, hoofs, etc. sometimes have insufficient solubility in a liquid medium due to hydrogen bonds even if the disulfide bonds are cleaved. In such a case, the solubility of the reduced product was imparted to the liquid medium by adding a protein denaturing agent such as urea or thiourea, an alkali such as sodium hydroxide or ammonia, or an inorganic salt such as sodium chloride as a dissolution aid. It is better to use a solution. Such a solubilizing agent is more effective as the dose is larger, but an appropriate amount is determined in consideration of the solubility in a liquid medium and the efficiency of the subsequent removal operation of the reducing agent and the like.

The reductive solubilization reaction can give good results even if it is neutral, but it is preferably alkaline, more preferably
It is preferable to carry out at pH 10-11. Further, the reaction temperature and the reaction time are appropriately combined so that the reduction reaction is completed. For example, it is sufficient to carry out the reaction at room temperature for 3 to 6 hours, at 5 ° C for 24 to 48 hours, and at 40 to 60 ° C for 30 to 120 minutes.

Before removing the reducing agent, the solubilizing agent and the like in the obtained keratin solution, insoluble matters existing in the solution are removed in advance by centrifugation or filtration.

As the cationic surfactant added to the keratin solution after removing the insoluble matter, quaternary ammonium salts such as stearyl trimethyl ammonium chloride, lauryl trimethyl ammonium chloride, lauryl dimethyl benzyl ammonium chloride, and N-hydroxyethyl propyl alkyl amide are used. Nitrate or the like is used.

The amount of the cationic surfactant added to the solution is 0.1 to 10% by weight, preferably 0.3 to 5% by weight, more preferably 0.5 to 2% by weight. The addition amount varies depending on the concentration of the keratin solution and the kind of the raw material keratin, but if it exceeds 10% by weight, it tends to be economically unfavorable.

Further, the cationic surfactant may be added in advance when the reducing agent is first added.

The reducing agent is removed by means such as dialysis, gel filtration, membrane filtration and electrodialysis. By this removal, the keratin solution can be obtained as a liquid having no turbidity or insoluble matter.

The obtained keratin solution can be concentrated by a film or dried, and by further drying, a dry powder can be obtained. The keratin thus obtained has a very high water solubility. Further, it is a high molecular weight keratin containing 0.5 to 5 cysteines and 0.5 to 5 cystines per 100 amino acid residues in the keratin protein and having a molecular weight of 40,000 to 70,000. Moreover, this storage stability was very high, and no insoluble matter was generated in the solution state for at least half a year.

The hair pretreatment agent comprising the soluble keratin protein is in the form of a solution, shampoo, rinse, styling foam, hair conditioner,
It is incorporated into hair packs, hair creams, hair liquids, hair nicks, permanent wave shampoos, rinses, setting agents and perms, and applied to hair.

The blending amount is 0.0 at the final keratin concentration.
1 to 10% by weight is preferable, and 0.1 to 10% is more preferable.
It is 2.0% by weight.

In the present invention, physical immobilization, chemical bonding or the like is used to fix the hair pretreatment agent or hair cosmetic and the hair.

Physical adsorption can be carried out by applying a hair pretreatment agent or a hair cosmetic containing the hair pretreatment agent to the hair.

In the case of chemical bonding, the functional group of hair (amino group, carboxyl group, sulfide group, etc.) is used.

These functional groups can be combined with the hair pretreatment agent or the hair cosmetic composition containing the hair pretreatment agent by a known method. When the functional group is an amino group, it is glutaraldehyde. When it is a carboxyl group, it is a water-soluble carbodiimide, 1-ethyl-3.
It can be linked by-(3-dimethylaminopropyl) carbodiimide. In the case of SH group, a crosslinking reagent such as N-succinimidyl-3- (2-pyridyldithio) -propionate (hereinafter abbreviated as SPDP) can be used.

Antibodies having an immunological activity against the hair protein used in the present invention are disclosed in Japanese Patent Application No. 4-351973, Japanese Patent Application No. 4-351971, and Japanese Patent Application Laid-Open No. 2-1.
The method described in Japanese Patent No. 34012 can be used. That is, cows, horses, sheep, goats, rabbits, chickens and the like are immunized with the above hair protein, and their normal milk, colostrum, serum,
It can be obtained by extracting from egg yolk or the like, but it is preferable because a large amount of antibody obtained from bovine normal milk or colostrum or egg yolk can be obtained.

Purification of the antibody from the above-mentioned antibody raw material may be carried out in accordance with a known method. For example, the lipid is removed by an appropriate method and then purified by the ammonium sulfate fractionation method, the alcohol precipitation method or the membrane separation method. A crudely purified antibody can be obtained. If necessary, further purification can be performed by ion exchange chromatography, gel filtration chromatography or the like to obtain a purified antibody. Further, if necessary, high-purity purified antibody can be obtained by performing affinity chromatography using the antigen used for immunization as a ligand.

Further, an antibody can be obtained as a monoclonal antibody from a fused cell of an antibody-producing cell producing the desired antibody and a myeloma cell.

The antibody obtained as described above may be an antibody fragment obtained by treating with an enzyme such as papain or pepsin to remove the Fc portion of immunoglobulin. Also, 2
-H chain or L chain obtained by reducing the antibody with mercaptoethanol may be used. Further, it may be a product of a microorganism or a cultured cell into which an immunoglobulin gene fragment cloned from spleen cells or lymphocytes of an immunized animal has been introduced.

In the present invention, to immobilize the antibody on the pigment, there are a method of directly immobilizing and a method of interposing a polymer carrier, both of which have a binding force to hair as compared with a dye-bound antibody (direct binding). However, the method of directly fixing is preferable in that the binding force to hair is stronger, while the method of interposing a polymer carrier is preferable in that the functional improvement is remarkable. In order to immobilize the antibody on the dye,
A method using a polymer carrier may be mentioned.

The polymer carrier used in the present invention includes
Examples include insoluble polymer carriers and water-soluble polymer carriers.
The insoluble polymer carrier is preferable in that it significantly improves the firmness, elasticity, strain, slippage, cohesion, and texture of the hair, and the water-soluble polymer carrier is the moist feeling, smoothness, suppleness, smoothness, and texture of the hair. Is preferable in that the improvement of is remarkable.

Examples of the insoluble polymer carrier include synthetic polymer, insoluble protein, insoluble polysaccharide, liposome and the like.

Examples of synthetic high molecular polymers include polystyrene, poly (α-methylstyrene), polyvinyltoluene, polychloromethylstyrene, polychlorostyrene,
Polyvinyl chloride, polyvinyl bromide, polyacrylonitrile, polymethacrylonitrile, polyethyl acrylate,
Octyl polyacrylate, hydroxypropyl polyacrylate, butyl polyacrylate, methoxyethyl polyacrylate, hydroxyethyl polyacrylate, lauryl polyacrylate, ammonium polyacrylate, polymethyl methacrylate, polyethyl methacrylate, polymethacryl Propyl acid, butylaminoethyl polymethacrylate, polyhydroxymethacrylic acid, polyvinyl acetate, polyacrylic acid, polymethacrylic acid, polymaleic acid, polystyrenesulfonic acid, poly (2-acrylamido-2)
-Methyl propane sulfonic acid), polyacrylamide,
Polymethacrylamide, poly {N- (2-hydroxypropyl) methacrylamide}, poly (2-hydroxyethyl methacrylate), poly (glycerol monomethacrylate), poly (2-oxyethyl acrylate),
Examples thereof include polymers such as poly (2-oxyethyl methacrylate), polyethylene glycol methacrylate, polyethylene, polypropylene, polybutene, polyisobutene, copolymers thereof, polyurethane, copolymers of polyurethane and silicone, nylon beads and the like. Moreover, the thing which modified the surface of those beads can also be used. Among these synthetic polymers, polystyrene is preferable because the particle size can be easily controlled.

As the insoluble protein, a neutral insoluble protein or a water-soluble protein crosslinked to be insolubilized / polymerized may be used. Specific examples include fibroin, gelatin, collagen and the like.

Examples of insoluble polysaccharides include crosslinked agarose, dextran, chitin and the like.

Examples of lipids used as raw materials for liposomes include phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and lysolecithin.

The particle form of these insoluble polymer carriers is not particularly limited, and spherical or plate-like particles can be used, but spherical particles having a uniform particle size are preferred.
The size is not particularly limited, but is 0.00
The size is preferably 1 to 100 μm, and particularly 0.001 to
1 μm is preferable in that a high hair dyeing degree can be obtained.

The amount ratio of the insoluble polymer carrier to the antibody varies depending on the insoluble polymer carrier to be used and cannot be specified unconditionally, but 0.
It is preferable that 01 to 100 mg is immobilized.

The water-soluble polymer carrier may be a natural product or a synthetic product. Examples of such substances include polysaccharides and proteins derived from natural products. Examples of the polysaccharides include plant-derived starch, amylose, amylopectin, pectin, carrageenan, mannan, galactan, sodium alginate, gum tragacanth, gum arabic, dextran derived from microorganisms, pullulan, curdlan, levan, glucan, succinoglucan, Examples include xanthan gum and other animal-derived hyaluronic acid, chondroitin sulfate, and the like.

Examples of proteins include glue, gelatin, casein, collagen, fibroin and the like.

Examples of the semi-synthesized product include viscose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, solubilized starch, carboxymethyl starch, dialdehyde starch and the like.

As the synthetic material, polyvinyl alcohol, carboxyvinyl polymer, sodium polyacrylate,
Examples thereof include polyvinylpyrrolidone, polyethylene oxide, polylysine, polyglutamic acid and polyaspartic acid.

The particle form of these water-soluble polymer carriers is not particularly limited, and straight-chain type or branched chain type can be used, but those having a uniform molecular weight to some extent are preferable, for example, a molecular weight of 10,000. To 2 million.

The amount ratio of the water-soluble polymer to the antibody varies depending on the polymer used and cannot be specified unconditionally,
It is preferable that 0.01 mg to 1 g of the antibody be immobilized per 1 g of the water-soluble polymer.

The pigments and dyes used in the present invention include
Titanium oxide such as titanium black and white titanium, iron oxide,
Inorganic pigments such as magnetic particles, organic pigments, various synthetic pigments including tar-based pigments, reactive pigments, fluorescent pigments, synthetic melanin pigments, animals, plants, microorganism-derived natural pigments and the like, pigments and Tar-based dyes are preferred because they are less irritating. The pigment is also preferable in that it strengthens the binding of the antibody to the hair.

The size of the pigment cannot be specified unconditionally, but is preferably 0.01 to 6 μm.

The pigment, the dye and the amount ratio of the dye and the antibody differ depending on the kind and cannot be defined unconditionally, but when the antibody is directly immobilized, 5 to 100 g of the pigment is added to 1 g of the antibody.
(10 to 200 mg of antibody per 1 g of pigment) is preferable,
When immobilized via a polymer carrier, 1 mg to 10 g is preferable per 1 g of the polymer carrier.

In the present invention, physical adsorption or chemical bonding is used to directly immobilize the pigment and the antibody. Physical adsorption can be performed by mixing the pigment and the antibody. In the case of chemical bonding, the pigment may have an organic functional group, but when it does not have an organic functional group like an inorganic pigment, it is necessary to introduce an organic functional group on the surface of the pigment. There is. Examples of the method for introducing an organic functional group onto the surface of the pigment include a method using a coupling agent and a method using silicon treatment. Examples of the coupling agent include silane coupling agents, titanate coupling agents, aluminum coupling agents, zircoaluminate coupling agents, and the like. Examples of materials used for the silicon treatment include amino-modified silicon and carboxy-modified silicon. For example, by reacting with 3-aminopropyltriethoxysilane, an amino group can be introduced on the pigment surface. It is also possible to convert this amino group into a carboxyl group by reacting glutaric anhydride or succinic anhydride. Also,
The SH group can be introduced by reacting with 3-mercaptopropyltrimethoxysilane.

These organic functional groups can be bound to the antibody by a known method. When the functional group is an amino group, it is glutaraldehyde. When it is a carboxyl group, it is a water-soluble carbodiimide, 1-ethyl-3.
It can be linked by-(3-dimethylaminopropyl) carbodiimide. Further, in the case of SH group, a crosslinking reagent such as N-succinimidyl-3- (2-pyridyldithio) -propionate can be used.

In the present invention, the method of holding the pigment or dye on the insoluble polymer carrier or the water-soluble polymer carrier includes a physical method and a chemical method. As a physical method, a pigment, an adsorption method of adsorbing a dye on an insoluble polymer carrier, a pigment at the time of making an insoluble polymer carrier, an internal addition method of adding a dye, an encapsulation method of wrapping the pigment in the insoluble polymer carrier, There is a polymerization method in which a dye precursor is polymerized in an insoluble polymer carrier. When an insoluble polymer carrier or a water-soluble polymer carrier having a functional group is used, it can be immobilized by a chemical bond. As the functional group, an amino group, a carboxyl group, an aldehyde group, a hydroxyl group, a thiol group or the like is used. The polysaccharide may be used as it is, but may be chemically modified if necessary. For example, an aldehyde group can be generated by oxidizing a polysaccharide with metaperiodic acid or the like. It is also possible to introduce an amino group by reacting this aldehyde group with a diamine and a carboxyl group by reacting with ε-aminocaproic acid or the like. Also,
It is also possible to make the polysaccharide reactive by reacting the polysaccharide with an activating reagent such as cyanuric chloride or bromine cyanide.

In the present invention, the method for immobilizing an antibody on an insoluble polymer carrier or a water-soluble polymer carrier includes
Physical adsorption or chemical bonding may be mentioned. The functional group of the antibody used for the chemical bond includes an amino group, a carboxyl group, a thiol group, a sugar chain portion, etc., and can be immobilized in the same manner as in the case of binding the dye and the carrier. For example, when the sugar chain portion is used for conjugation, the sugar chain portion can be immobilized by Schiff base formation with the amino group of the carrier by oxidizing the sugar chain portion with metaperiodic acid or the like to generate an aldehyde group. When a thiol group is used, it can be immobilized using an SPDP reagent or the like.

The antibody-immobilized pigment or dye obtained as described above is prepared as a solution or dispersion using an appropriate solvent such as an aqueous solution, or by an operation such as freeze-drying or spray-drying. It is provided as the obtained dried product.

The enzyme-linked antibody can be prepared by the method described below. These enzymes and antibodies can be bound by a known method. When the functional group is an amino group, it can be bound by glutaraldehyde, and when it is a carboxyl group, it can be bound by a water-soluble carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Further, in the case of SH group, a crosslinking reagent such as N-succinimidyl-3- (2-pyridyldithio) -propionate can be used. When this enzyme-linked antibody is used as a hair dye, the hair can be dyed by the action of the enzyme by using a coloring agent after binding the antibody to the hair.

The modified antibody thus obtained bears a sufficient amount of pigment or dye for coloring, can specifically react with hair and is colored, and
Sliding, cohesiveness, texture, moist feeling, smoothness,
It is also possible to improve the feel of hair such as suppleness.
In particular, when a synthetic polymer is used as the insoluble polymer carrier, the hair feels soft and elastic, when an insoluble protein is used, the suppleness is obtained, and when an insoluble polysaccharide is used, a moist feeling is obtained. When a liposome is used, gloss can be imparted, and when a water-soluble polymer carrier is used, a moist feeling can be imparted. In addition, a hair dye in which an antibody is directly immobilized on a pigment has a stronger binding force to a hair protein than a conventional dye (direct) -bonded antibody.

The content of the hair dye in the hair cosmetic composition of the present invention may be appropriately determined depending on the type of the cosmetic composition and the like, but is generally about 0.01 to 80% by weight based on the total cosmetic composition of 100.

In the present invention, the anti-hair protein antibody modified with a dye, a pigment, an enzyme or the like is combined with the hair pretreatment agent of the present invention or the hair pretreatment cosmetic composition containing the hair pretreatment agent, which has never been obtained before. A hair dyeing method having a high hair dyeing degree can be provided.

Further, by using the hair pretreatment agent of the present invention or the hair pretreatment cosmetic composition containing the hair pretreatment agent, the binding force of the hair dye containing the anti-hair protein antibody to the hair is increased, Furthermore, it is possible to obtain an excellent effect that the improvement is significantly improved in terms of sensory aspects.

[0058]

[Function] By the specific binding of the anti-hair keratin antibody having the hair dye to the keratin applied to the hair,
A high hair dyeing effect can be obtained.

[0059]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Preparation of Soluble Keratin) 20 g of wool (hybrid) was mixed with 3% β-mercaptoethanol.
It was immersed in an 8 M urea aqueous solution (600 ml) containing 1 mM EDTA, and the pH was adjusted to 10.5 with 10% potassium hydroxide. Then, deaeration operation was performed and then the container was sealed. After stirring at room temperature for 3 hours for reduction, the pH was returned to neutral with 6N HCl,
Centrifuge at 12,000 rpm for 30 minutes at 4 ℃ 7
40 ml of supernatant was obtained. Stearyl trimethyl ammonium chloride (cation
AB600 (63% product) to a final concentration of 2% and homogenize it completely, and then use a dialysis membrane to remove the reducing agent, and use 10 liters of ion-exchanged water for 3 hours each for 3 hours.
The dialysis operation was performed once. As a result, it was possible to obtain a transparent keratin solution containing no insoluble matter. The recovery of solubilized keratin protein from 20 g of wool was 76.5% by weight. The protein concentration was 30.0 mg / ml. In addition, the molecular weight was measured to be 40,0 in the absence of reducing agent.
It was 00 to 70,000. The molecular weight was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Comparative Example 1 A keratin solution was obtained in the same manner as in Example 1 except that sodium dodecyl sulfate (manufactured by Hanai Co., Ltd.) was used as the anionic surfactant instead of the cationic surfactant. As a result, the recovery rate of solubilized keratin protein was 70% by weight. The molecular weight was 40,000 to 70,000.

Example 2 (Preparation of anti-carboxymethylated keratin antibody) Preparation of antigen (carboxymethylated keratin) Mix 5 g of healthy male hair and 5 g of healthy female hair,
2% sodium polyoxyethylene lauryl sulfate (3E.
O.) Washed with aqueous solution. 2500 washed healthy hair
The mixture was stirred in 50 ml of 0.2 M Tris-HCl buffer (pH 9.2) containing 8 M urea and 0.2 M 2-mercaptoethanol for 1 hour under nitrogen bubbling at 50 ° C., and triturated with a Teflon homogenizer. The above extraction procedure was repeated, and the obtained extract was centrifuged at 1,000 × g for 30 minutes to remove insoluble matter, and a hair keratin antigen extract was obtained. Add 200 g of monoiodoacetic acid (40
(Dissolved in 760 ml of a solution containing 0 g of Tris) was added, and the mixture was stirred and reacted for 1 hour at room temperature in the dark. 7 ml of 2-
The reaction was stopped by adding mercaptoethanol, dialyzed against a sufficient amount of water, passed through a 5 μm filter to remove insolubles, and an aqueous hair keratin antigen solution was obtained (6 liters).
Further, to 4 parts by volume of this solution, 1 part by volume of 0.5 M sodium acetate buffer (pH 4.2) was added (prepared with acetic acid so as to obtain pH 4.2), and hair keratin was isoelectrically precipitated. 1
Centrifugation was carried out at 10,000 × g for 10 minutes, the supernatant was removed, and the precipitate was collected. Dissolve the precipitate with physiological saline,
The cells were sterilized through a 0.2 μm filter, and further concentrated with an ultrafiltration membrane to obtain carboxymethylated keratin (2.6 g as protein).

B. Immunization of cattle The protein concentration of the carboxymethylated keratin antigen solution prepared above was adjusted to 20 mg / ml with physiological saline, and the solution and Freund's complete adjuvant were mixed at a volume ratio of 1: 1 in oil. A water-based emulsion was prepared. 5.0 ml of the above emulsion was subcutaneously administered to the necks of two pregnant Holstein cows two months before delivery. Then one
At 0 day intervals, an emulsion containing the same amount of antigen as the initial immunization prepared with Freund's incomplete adjuvant was administered subcutaneously or intramuscularly to immunize (1st-3rd times; subcutaneous administration, 4-5th times). ; Muscle note).

C. Collection and purification of antibody Colostrum of cattle immunized with the above antigen was collected for 3 days immediately after birth. The fat layer was removed from the colostrum using a cream separator to obtain skim milk. The skim milk thus obtained was subjected to fractional purification of the antibody by the following method. That is, 0.1N hydrochloric acid was added to skim milk to obtain a pH of 4.
It was adjusted to 5, and casein was precipitated. After the precipitate was roughly removed with a filter cloth, the supernatant was obtained by continuous centrifugation at 2,500 xg. After the obtained supernatant was neutralized, ammonium sulfate was added so as to achieve 33% saturation, and the antibody was salted out.
Precipitated portions were collected by continuous centrifugation at 2,500 × g and dissolved in physiological phosphate buffer (hereinafter abbreviated as PBS). This ammonium sulfate salting out operation was repeated. The obtained solution was dialyzed against 10 mM phosphate buffer (pH 7.5) and equilibrated with the same buffer to a 2-liter diethylaminocellulose column (DEAE-cellulose column, DE-52, manufactured by Whatman). It was applied in batches. After washing off unadsorbed protein with the same buffer, the antibody was eluted with the same buffer containing 50 mM sodium chloride, and this fraction was collected (200 g as antibody). The antibody purity of this fraction is 90% by weight
That was all. This fraction was applied to 400 ml of an affinity carrier (Affigel 15, Bio-Rad), to which purified hair keratin was bound by a conventional method, in 5 batches. Anti-hair keratin antibody bound to affinity carrier
Elution with a glycine hydrochloric acid buffer (pH 2.5) and immediately adjusting the pH to around 8 with 3M Tris solution, an anti-hair keratin antibody (affinity purification) that specifically binds to the hair keratin antigen was obtained. . Hereinafter, it is simply referred to as an anti-keratin antibody.

D. Preparation of control antibody An antibody was purified from colostrum of non-immunized cow in the same manner as this method, and this was used as a control antibody. The control antibody used in the comparative example had a purity of 90% by weight or more, which was purified by a DEAE-cellulose column.

Example 3 (Immobilization of anti-carboxymethylated keratin antibody on titanium black) A. Surface treatment of titanium black 1 g of titanium black 10S (particle diameter 0.025 μm, manufactured by Mitsubishi Metals) was ultrasonically dispersed in 10 ml of distilled water. To this dispersion was added 75 μl of a 1% 3-aminopropyltriethoxysilane aqueous solution, and the mixture was stirred at room temperature for 2 hours. 40 ° C
After dehydration under reduced pressure with an evaporator while heating to 11
An amino group was introduced on the surface of titanium black by drying at 0 ° C. for 10 minutes.

B. Reaction of silane-treated titanium black with glutaraldehyde Titanium black 80 with amino groups introduced by silane treatment
mg was ultrasonically dispersed in 4 ml of distilled water. 4 ml of a 0.25% glutaraldehyde aqueous solution was added to this dispersion, and the mixture was stirred at room temperature for 2 hours. 20 ° C, 10,000 rpm, 20
After centrifuging for minutes, it was redispersed in 8 ml of distilled water. This washing operation was repeated five times.

C. Binding of Titanium Black and Anti-Keratin Antibody 0.5 ml of anti-keratin antibody solution adjusted to 10 mg / ml with PBS was added to 0.5 ml of 1% dispersion of titanium black treated with glutaraldehyde, and the mixture was stirred overnight at 4 ° C. 20
PBS containing 0.1% bovine serum albumin (hereinafter abbreviated as BSA) after centrifugation for 20 minutes at 12,000 rpm at 20 ° C
Redispersed to 0.5 ml. Once again at 20 ℃, 12:00
Centrifuge for 20 minutes at 0 rpm, PBS containing 0.1% BSA
After redispersion to 0.5 ml, the mixture was left at room temperature for 1 hour. Next, 10 μl of 0.005 M sodium borohydride aqueous solution was added, and the mixture was left at room temperature for 1 hour. 20 ° C, 1
Centrifuge at 2,000 rpm for 20 minutes and 0.1% Twee
PBS containing n20 (polyoxyethylene sorbitan monolaurate, 20 E.O.) and 0.1% BSA
Redispersed to 0.5 ml. In the obtained hair dye, 144 mg of antibody was immobilized per 1 g of titanium black.

Comparative Example 2 In place of the anti-keratin antibody in Example 3, Example 2 is used.
Manufacture titanium black to which the control antibody of -D was immobilized,
This was designated as Comparative Example 2. The obtained hair dye had 243 mg of antibody immobilized per 1 g of titanium black.

Example 4 (Hair Treatment with Solubilized Keratin) The solubilized keratin prepared in Example 1 was adjusted to a protein concentration of 10 mg / ml with PBS. A human hair bundle of about 4 cm was immersed in this solubilized keratin solution. After 5 minutes, the gray hair bundle was taken out and dried. By this operation, human gray hair bundles in which solubilized keratin was immobilized on the surface or inside the hair could be prepared.

Test Example 1 (Hair Dyeing Test) The hair dyes of Example 3 and Comparative Example 2 were mixed with 0.1% Tween.
Dilute with PBS containing 20 and 0.1% BSA,
The pigment and latex concentrations were adjusted to 0.1%. The human hair bundle prepared in Example 4 or the untreated human hair bundle not treated with solubilized keratin prepared in Example 4 was dipped in 1 ml of each hair dye and rotated at room temperature for 1 hour. Then, this hair bundle was shaken in physiological saline containing 0.02% Tween 20 and air-dried, and the degree of hair dyeing was visually evaluated. In addition, ◯ means "dyeing" and x means "not dyed". Also, the larger the number of circles is, the better the hair is dyed, and the maximum number is 5. The results are summarized in Table 1.

[0072]

[Table 1]

As can be seen from Table 1, untreated human white hair was dyed with the hair dye of Example 3 using an anti-keratin antibody, but Example 4 treated with solubilized keratin.
The hair was dyed more than that, and the degree of hair dyeing was dramatically improved by pretreatment with solubilized keratin. On the other hand, in Comparative Example 2 in which the control antibody was used, hair was hardly dyed regardless of whether it was untreated or treated with solubilized keratin.

Comparative Example 3 (Hair Treatment with Anionic Surfactant Solubilized Keratin) The anionic surfactant solubilized keratin prepared in Comparative Example 1 was adjusted to a protein concentration of 10 mg / ml with PBS.
A human hair bundle of about 4 cm was immersed in this solubilized keratin solution. After 5 minutes, the gray hair bundle was taken out and dried. By this operation, human gray hair bundles in which solubilized keratin was immobilized on the surface or inside the hair could be prepared.

Comparative Example 4 The hair dye of Example 3 was mixed with 0.1% Tween 20 and 0.
It was diluted with PBS containing 1% BSA, and the concentration of pigment and latex was adjusted to 0.1%. The human hair bundle prepared in Example 4 and Comparative Example 3 was dipped in 1 ml of each hair dye and rotated at room temperature for 1 hour. Then, this hair bundle was shaken in physiological saline containing 0.02% Tween 20 and air-dried, and the degree of hair dyeing was visually evaluated. The evaluation method is the same as in Test Example 1. The results are summarized in Table 2.

[0076]

[Table 2]

As is clear from Table 2, when the hair dye of Example 3 using an anti-keratin antibody was used for hair dyeing, the human white hair prepared in Example 4 was better than the human white hair prepared in Comparative Example 3. It was dyed. That is, the solubilized keratin of the present invention prepared by solubilizing with a cationic surfactant,
It was revealed that the solubilized keratin prepared by solubilizing with an anionic surfactant is very excellent as a pretreatment agent for a hair dye.

Example 5 (Shampoo) The solubilized keratin of Example 1 was blended in a shampoo with the composition shown in Table 3 and produced according to a conventional method.

[0079]

[Table 3]

Example 6 (Rinse) The hair dye of Example 3 was blended in the composition shown in Table 4 to prepare a rinse having a coloring effect according to a conventional method.

[0081]

[Table 4]

The rinse of Example 6 was continuously used for one month. Then, the degree of dyeing of gray hair, the degree of dyeing of face and hands, discoloration due to shampoo, presence or absence of itching and rash on scalp, and sensory evaluation were performed. As a result, the rinse of Example 6 showed good dyeing of white hair, and no dyeing of the face or hands was observed. In addition, there was no discoloration due to shampoo, no itchiness or rash on the scalp, and improvement in sensuality.

Test Example 2 [Hair dyeing degree improvement test by pretreatment (practical test)] With the help of three volunteers with noticeable white hair, half-head, one shampoo of Example 4 and the other Example A shampoo having the same composition as in Example 5 was used except that the solubilized keratin of Example 4 was removed, and then the rinse containing the hair dye of Example 6 was used. As a result of continuous use for 1 week, the use of the shampoo of Example 5 gives better dyeing of gray hair,
It quickly became inconspicuous.

[0084]

According to the present invention, a high hair dyeing effect can be obtained without damaging the hair and causing no skin irritation.
Moreover, the hair can be improved in terms of sensory aspects.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeru Igarashi 5-328, Kotobuki-cho, Odawara-shi, Kanagawa Inside the Biochemical Research Center, Kanebo Co., Ltd. (72) Toshihiro Usui 5-chome, 3-cho, Odawara, Kanagawa No. 28 Kanebo Co., Ltd. Biochemistry Research Institute

Claims (4)

[Claims] 1. A soluble keratin protein obtained by subjecting a keratin protein fiber having a crosslinked structure to a reduction treatment in a liquid, adding a cationic surfactant to a solution from which insoluble matter has been removed, and then removing the reducing agent. Alternatively, a hair pretreatment agent composed of soluble keratin protein obtained by contacting a keratin protein fiber having a crosslinked structure with a liquid containing a reducing agent and a cationic surfactant, and then removing the reducing agent from the solution from which insoluble matter has been removed . 2. A hair pretreatment cosmetic, comprising the hair pretreatment agent according to claim 1. 3. An antibody having immunological activity against a keratin protein modified with a pigment or a dye, after treating the hair with the hair pretreatment agent according to claim 1 or the hair pretreatment cosmetic composition according to claim 2. A hair dyeing method characterized by dyeing hair with. 4. The hair is treated with the hair pretreatment agent according to claim 1 or the hair pretreatment cosmetic composition according to claim 2, and then dyed with an antibody having an immunological activity against a keratin protein modified with an enzyme. A hair dyeing method characterized by hair-giving.