WO2017149490A1 - A hair dye composition with plant-derived and synthetic pigments, the method of preparation thereof and method of applying the hair dye - Google Patents

A hair dye composition with plant-derived and synthetic pigments, the method of preparation thereof and method of applying the hair dye Download PDF

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Publication number
WO2017149490A1
WO2017149490A1 PCT/IB2017/051222 IB2017051222W WO2017149490A1 WO 2017149490 A1 WO2017149490 A1 WO 2017149490A1 IB 2017051222 W IB2017051222 W IB 2017051222W WO 2017149490 A1 WO2017149490 A1 WO 2017149490A1
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Prior art keywords
hair
krtap
protein
pigment
solution
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PCT/IB2017/051222
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French (fr)
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Shefali SABHARANJAK
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Sabharanjak Shefali
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Publication of WO2017149490A1 publication Critical patent/WO2017149490A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/10—Preparations for permanently dyeing the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/42—Colour properties
    • A61K2800/43—Pigments; Dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances

Definitions

  • a hair dye composition with plant-derived and synthetic pigments the method of preparation thereof and method of applying the hair dye
  • the present invention relates to a hair dye composition
  • a hair dye composition comprising the Keratin Associated Protein (KRTAP-2) family protein conjugated with a natural hair dye such as lawsone or indigo.
  • a hair dye such as lawsone or indigo.
  • the present invention also relates to a method of conjugation of protein with a pigment and method of applying the hair dye to human hair.
  • Hair dye or hair coloring is one of the oldest known cosmetic preparations being used by many ancient cultures in different parts of the world. Initially, wide range of hair coloring agents derived from plant based sources was used. In 1909, French Chemist Eugene Schuller created first safe synthetic coloring agent based on paraphenylenediamine.
  • hair dyes include dyes, modifiers, antioxidants, alkalizers, soaps, ammonia, wetting agents, fragrance and variety of other chemicals used in small amounts that impart special qualities to hair such as softening the texture or give a desired action to the dye such as making it more or less permanent.
  • the dye chemicals are usually amino compounds such as 4-amino-2-hydroxytoluene and m-aminophenol.
  • the composition further contains metal oxides such as titanium dioxide and iron oxide as pigments as well.
  • the Food and Drug Administration regulates the safety of cosmetic products.
  • the chemical products contained in the hair dyes include aromatic amines such as 4-methoxy-m-phenylenediamine 4-chloro-m- phenylenediamine, 2, 4-toluenediamine, 2-nitro-p-phenylenediamine, and 4- amino-2-nitrophenol. These compounds penetrate into the human skin and are known to be mutagenic and carcinogenic.
  • the human hair follicle is an intriguing structure wherein, the hair follicle is divided into three regions namely, the lower segment termed as bulb and suprabulb, the middle segment termed as isthmus, and the upper segment termed as infundibulum.
  • the lower segment extends from the base of the follicle to the insertion of the erector pili muscle, which is also known as the arrector pili muscle.
  • the middle segment is a short section that extends from the insertion of the erector pili muscle to the entrance of the sebaceous gland duct.
  • the upper segment extends from the entrance of the sebaceous gland duct to the follicular orifice.
  • Hair color is due to the presence of melanin pigment in the keratinocytes of the hair shaft cortex. Hair color modification is achieved by removal of cortical melanin pigment through bleaching or addition of artificial pigment through application of a hair dye.
  • hair dyes are associated with many adverse effects, such as, temporary skin irritation, allergy, skin discoloration, hair breakage, hair color differences. Further, long-term use is also associated with carcinogenicity, mutagenicity etc. [0011] In order to overcome these problems, many hair dye compositions are formulated with natural coloring agents wherein the natural coloring agents are derived from different species of plants.
  • keratin filaments comprise the hair shaft in human hair.
  • KRTAP are family of proteins, which binds to keratins and the cuticle of the hair shaft. Further, these Keratin-associated proteins are grouped into several families. Approximately 100 KRTAP genes have been identified in humans till date with each of the gene classified into a number of families based on their sequence homology and the nature of the repeat structures. KRTAP are useful in the composition of hair dyes.
  • the Patent Application "DE4410162C2" titled “Process for dyeing of human hair” discloses a novel, improved process for dyeing of human hair with vegetable dyes.
  • the invention relates to a method of dyeing human hair using pflanzli chen dyes.
  • the method comprises the step of applying a first composition comprising substantive plant dye on the hair and a second composition comprising a vegetable dye is applied after approximately five to fifteen minutes. After twenty to sixty minutes exposure, the dyed hair is washed and dried.
  • the invention does not disclose the use of plant-based dyes.
  • the Patent Application "US 6656229B1" titled “Hair dye and hair-dyeing methods using the same” discloses a composition of hair dye and hair dying methods.
  • the hair dye comprises a formulation composed of a dye component including indicant derived from indigo plant.
  • the hair dye further comprises a plant-based component derived from a plant selected from the group consisting of a tea leaf, comfrey, kamille, chlorella, rosemary, seaweed, and a mixture thereof.
  • the invention also discloses the method of applying the hair dye to the human hair. However, the invention is silent with respect to the conjugation of dye with KRTAP proteins.
  • the Patent Application "CN102198058A” titled "Herbal hair dye” discloses a natural herbal hair dye.
  • the dye components consist of henna, tannic acid and ferrite.
  • the invention further discloses a method of using the hair dye.
  • the hair dye dyes the hair into various colors including pure black and the dyeing time is shorter, the color of the dyed hair lasts longer, without inducing any serious side effects on human body.
  • the present invention discloses the use of henna flowers, leaves and other plant parts. The relevant parts are subjected to grinding and the resulting fine powder, henna flowers, leaves or plant milled powder is extracted with water or alcohol using 30-95% ethanol extraction.
  • the present invention discloses a composition of a hair dye comprising the KRTAP-2 family protein conjugated with a natural hair dye such as lawsone or indigo.
  • a natural hair dye such as lawsone or indigo.
  • the present invention discloses a method of conjugation of KRTAP-2 protein with lawsone or indigo.
  • the present invention further discloses a method of applying the hair dye to the human hair.
  • the hair dye composition comprises the protein-pigment conjugate.
  • the protein is KRTAP-2 family protein and the pigment is selected from a natural source. Lawsone or indigo are used as natural pigments.
  • the composition also comprises ethanolamine, antimicrobial agent and hair modifying agent.
  • the KRTAP-2 family protein is conjugated with a hair dye.
  • the hair dye used in the present invention is a natural hair dye derived from the plant source. Lawsone is a naphthaquinone derived from Lawsonia inermis. KRTAP-2 family protein binds with the lawsone through the attachment of side groups via a covalent bond.
  • indigo derived from Indigofera tinctorea, indican or indigotein are also conjugated with KRTAP- 2 proteins.
  • the conjugation is enhanced by the addition of linker molecule during conjugation.
  • the conjugate product is also created by the use of synthetic hair dyes used in the regular hair dyes.
  • the present invention also discloses a method of applying the hair dye to the human hair.
  • the method comprises the steps of cleaning and drying the hair to remove the presence of shampoos, conditioners or any other substances.
  • the next step involves the spraying of ethanolamine to disintegrate the cuticle of the hair.
  • This step is followed by spraying the KRTAP -pigment conjugated solution.
  • the spray is allowed for a suitable time to allow the penetration of the KRTAP-pigment conjugate into the hair shaft.
  • the hair is rinsed off from all the reagents.
  • a hair protective substance such as oil may be applied on the hair to retain the color.
  • conjugated product of KRTAP-2 family protein with a natural or synthetic dye reduces the adverse effects induced by the hair dyes.
  • the use of natural dyes in conjugation is non-carcinogenic and non-mutagenic.
  • the conjugated protein-pigment complex results in high rate of penetration of the dye into the hair shaft.
  • FIG 1 illustrates the chemical structure of the pigment lawsone produced by Lawsonia inermis.
  • FIG 2 illustrates the chemical structure of the pigment indigo produced by Indigofera tinctorea.
  • FIG 3 illustrates the flow chart for a method of preparing the conjugated product of KRTAP-2 protein with lawsone.
  • FIG 4 illustrates the flow chart for the method of preparing the conjugated product of KRTAP-2 protein with indigo.
  • FIG 5 illustrates the flow chart for the method of spraying the hair dye to the human hair.
  • Diae refers to a colored substance that has an affinity to the substrate to which it is being applied.
  • Carcinogen refers to any substance or an agent directly involved in causing cancer.
  • Mutas refers to a physical or chemical agent that changes the genetic material, usually DNA of an organism and thus increasing the frequency of mutations.
  • the present invention relates to a composition of the hair dye comprising a complex of a KRTAP-2 family protein conjugated with a plant-derived dye.
  • the present invention further discloses the method of conjugation of the pigment with KRTAP-2 protein and also the method of applying the hair dye comprising the conjugated product to the human hair.
  • hair dye is associated with many adverse effects due to the presence of carcinogenic, allergic or mutagenic activity of the ingredients present in the composition of the hair dye. This is overcome by the present invention which uses natural dyes derived from the plant sources. Further, the use of dyes is associated with low rate of penetration into the hair shaft. The use of conjugated complex of protein and the pigment in the present invention provides high rate of penetration of the dye.
  • the present invention discloses the composition of the hair dye.
  • the hair dye comprises a natural pigment lawsone at the concentration of 1-20 mgml "1 or indigo at a concentration of 1-5 mgml "1 .
  • Lawsone is dissolved in acetonitrile.
  • Indigo is dissolved in a solution comprising 50 mM Tris-HCl buffer and 100 mM sodium chloride with a pH range 4 to 7.5.
  • KRTAP-2 protein at a concentration of 1-5 mgml "1 is dissolved in a solution comprising 50 mM Tris-hydrochloride buffer and 100 mM sodium chloride solution with a pH range of 4 to 7.5.
  • the composition further comprises ethanolamine solution, anti-bacterial agent, anti-fungal agent, hair-modifying agent, hair-bleaching agent, hair relaxer chemical such as thioglycolate.
  • the hair dye composition is provided in the form of a kit, which further comprises hair crimping agent, shampoo, conditioner, hair pre-treatment, hair post-treatment reagents, hair moisturizer, hair straightener oil, cream/any other protective products, keratin protein of natural/synthetic origin and formaldehyde/any other chemicals, which modify the structure of the human hair.
  • the KRTAP-2 family protein is conjugated with a plant-derived dye.
  • the recombinant human KRTAP-2 family protein is expressed in bacterial, yeast or any other mammalian protein production systems using the methods known in the art with the help of the suitable promoter and expression system.
  • the recombinant KRTAP-2 family proteins are also produced by processing of pre-cut human hair.
  • the recombinant protein thus obtained is used in the preparation of conjugation complex.
  • FIG 1 illustrates the chemical structure of the pigment lawsone produced by Lawsonia inermis.
  • the conjugation of KRTAP-2 family protein with lawsone is achieved by a chemical reaction.
  • the dye lawsone is a naphthaquinone derived from Lawsonia inermis.
  • Lawsone is also isolated from different species of Lawsonia. Lawsone is a 2-hydroxy-l,4-naphthoquinone with a molecular formula (CioH 6 N3) and forms a covalent bond with KRTAP-2 family proteins through side chains. Lawsone forms a sulfhydryl adducts to proteins during conjugation with mammalian protein.
  • FIG 2 illustrates the chemical structure of the pigment indigo produced by Indigofera tinctorea.
  • KRTAP-2 family protein is conjugated with indican, indigotein or any other plant-derived or synthetic pigments to form a protein- pigment complex, which is used in the composition of the hair dye.
  • Indigo may be derived from Indigofera tinctorea and other species of Indigofera.
  • lawsone at the concentration of 1-20 mgml "1 is dissolved in acetonitrile solution.
  • KRTAP-2 protein at a concentration of 1-5 mgml "1 is dissolved in a solution comprising 50 mM Tris-hydrochloride buffer and 100 mM sodium chloride solution at the pH in the range of 4 to 7.5.
  • the composition is also prepared by using indigo instead of lawsone. Indigo at a concentration of 1-5 mgml "1 is dissolved in 50 mM Tris-HCl buffer, 100 mM sodium chloride solution at the pH in the range of 4 to 7.5.
  • FIG 3 illustrates the flow chart for a method of preparing the conjugated product of KRTAP-2 protein with lawsone.
  • the method (100) of preparing protein-pigment conjugated product of the KRTAP-2 protein and lawsone starts at step (101) of adding the lawsone solution in acetonitrile to the KRTAP-2 protein solution.
  • the solution is stirred continuously in the presence of argon at room temperature for a period of 2-6 hours in dark condition i.e. in the absence of light.
  • the solution is re-suspended in a solution comprising 50mM Tris-HCl buffer, lOOmM sodium chloride at pH 7.
  • the protein-pigment conjugate is ascertained by High Performance Liquid Chromatography (HPLC).
  • HPLC High Performance Liquid Chromatography
  • FIG 4 illustrates the flow chart for the method of preparing the conjugated product of KRTAP-2 protein with indigo.
  • the method (200) of preparing protein- pigment conjugated product of the KRTAP-2 protein and indigo starts at step (201) of dissolving indigo at a concentration of 1-5 mgml "1 in a solution comprising 50 mM Tris-HCl buffer and 100 mM sodium chloride solution at the pH in the range of 4 to 7.5.
  • step (202) beta-glucosidase at the concentration of 80 units per gram is diluted to 2-5 units per ml and the diluted solution of beta- glucosidase is added to the indigo solution.
  • KRTAP-2 protein at the concentration of 1-5 mgml "1 dissolved in a solution of 50 mM Tris-HCl buffer and lOOmM sodium chloride at pH range 4-7.5 is added to the reaction mixture.
  • indigo is converted to indoxyl by the action of beta-glucosidase.
  • indoxyl reacts with free amino groups on KRTAP-2 proteins to form a peptide bond. The formation of the peptide bond restores the blue color of the indigo to the indoxyl group.
  • the protein-pigment conjugate is ascertained by High Performance Liquid Chromatography (HPLC).
  • HPLC High Performance Liquid Chromatography
  • a linker chemical group is added to lawsone or indigo during conjugation, which facilitates cross-linkage of lawsone or indigo to derivatives of KRTAP-2 proteins.
  • the present invention also discloses the use of chemical cross linker during crosslinking of lawsone, indigo, indican and indigotein and subsequent crosslinking of this cross linked conjugate to the KRTAP-2 protein subfamily.
  • the KRTAP-2 family protein with lawsone or indigo results in improved penetration of the dye into the hair shaft.
  • the protein-pigment complex is used in different forms such as solution, cream, dry powder etc.
  • the use of antibacterial or antifungal agents prevents the degradation of other agents and retains long-term stability of the components.
  • the hair modifying agents dissolve the hair cuticle, bleach or modify the texture of the hair in order to make it straighter or curlier than the original texture.
  • the present invention further discloses the method of spraying the hair dye to the human hair.
  • FIG 5 illustrates the flow chart for the method of spraying the hair dye to the human hair.
  • the method (300) of spraying the hair dye to the human hair starts with a step (301) of cleaning and drying of hair to remove extraneous coatings of shampoo, conditioner or any other compounds.
  • the ethanolamine is sprayed to partially disintegrate the cuticle of hair. The disintegration of the cuticle results in the enhanced penetration of the hair dye.
  • the KRTAP-2 family protein-pigment conjugate complex is sprayed to the hair. KRTAP-2 family protein conjugate with a lawsone or indigo or any other natural or synthetic dye is used in this step.
  • the sprayed dye is allowed for a suitable time to allow penetration of the KRTAP-2 family protein- pigment conjugate complex into the hair shaft.
  • the applied reagents are rinsed with water.
  • hair protecting substances such as oil or cream of the sealing conditioners are applied to maintain and retain the hair color.
  • the present invention is not restricted to the use of lawsone, indigo, indican or indigotein.
  • other classes of natural and synthetic dyes such as alizarin, munjistin, purpurin, red dye from Caesalpinia sappan and Caesalpinia echinata plants, bark and roots of Morinda citrofolia, juglone from Juglans regia, carthamin from safflower, yellow dyes from turmeric (Curcuma) species, saffron (Crocus sativus), annatto and barberry (Berberis aristata), rind of pomegranate fruits (Punica granatum), fruits of the myrobalan tree (Terminalia chebula), Marigold plants (Tagetus species.), Butea species (Flame of the Forest plants), Mallotus philipensis, Onion peels (Allium cepa), European
  • the present invention is not restricted to the KRTAP-2 protein family.
  • KRTAP-2 protein family proteins isolated from human and sheep source such as KAP1, KAP2, KAP3, KAP4, KAP5, KAP6, KAP7, KAP8, KAP9, KAP10, KAP11, KAP12, KAP13, KAP14, KAP15, KAP16, KAP17, KAP18, KAP19, KAP20, KAP21, KAP22, KAP23, KAP24, KAP25, KAP26 and KAP27 are also useful for conjugation with natural or synthetic dye.
  • the present invention with a kit of hair dye composition comprising the conjugated protein-pigment complex allows usage of small quantities of coloring reagents such that the cost of manufacturing the kits as well as the adverse effects on human is reduced.
  • the method of application of the hair dye reduces human exposure of allergenic and potentially carcinogenic ingredients because of the high specificity of the principal ingredients.
  • the pigments used as dye are isolated from plant origin and from other natural sources. Hence are associated with low risk of carcinogenicity, mutagenicity and are less allergic.
  • the usage of human proteins to attach the pigments to human hair also allows inclusion of safe pigments to color hair.
  • the composition allows for inclusion of new plant-derived and synthetic hair pigments to produce novel color combinations and novel coloring strategies.

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Abstract

he present invention discloses a composition of hair dye comprising the KRTAP-2 family protein conjugated with a pigment. The present invention discloses the use of lawsone or indigo as natural pigment in the hair dye. The composition also comprises ethanolamine, antimicrobial agent and hair modifying agent. In order to achieve the conjugation of pigment and the protein, lawsone at the concentration of 1-20 mgml-1 is dissolved in acetonitrile solution, indigo at a concentration of 1-5 mgml-1 is dissolved in Tris-HCl buffer. KRTAP-2 protein at a concentration of 1-5 mgml-1 is dissolved in Tris-HCl buffer. The invention discloses the method of conjugation of KRTAP-2 family protein with lawsone or indigo and the method of spraying the hair dye composition to human hair. The hair dye composition is associated with low risk of carcinogenicity, mutagenicity and allergy and increases the penetration of pigment to hair shaft.

Description

TITLE OF THE INVENTION
A hair dye composition with plant-derived and synthetic pigments, the method of preparation thereof and method of applying the hair dye
[001] Technical field of the invention
[002] The present invention relates to a hair dye composition comprising the Keratin Associated Protein (KRTAP-2) family protein conjugated with a natural hair dye such as lawsone or indigo. Further, the present invention also relates to a method of conjugation of protein with a pigment and method of applying the hair dye to human hair.
[003] Background of the invention
[004] Hair dye or hair coloring is one of the oldest known cosmetic preparations being used by many ancient cultures in different parts of the world. Initially, wide range of hair coloring agents derived from plant based sources was used. In 1909, French Chemist Eugene Schuller created first safe synthetic coloring agent based on paraphenylenediamine.
[005] Most of the commercial hair dye formulations are complex with number of ingredients wherein the formulas differ considerably from one manufacturer to another. In general, hair dyes include dyes, modifiers, antioxidants, alkalizers, soaps, ammonia, wetting agents, fragrance and variety of other chemicals used in small amounts that impart special qualities to hair such as softening the texture or give a desired action to the dye such as making it more or less permanent. The dye chemicals are usually amino compounds such as 4-amino-2-hydroxytoluene and m-aminophenol. The composition further contains metal oxides such as titanium dioxide and iron oxide as pigments as well.
[006] The Food and Drug Administration (FDA) regulates the safety of cosmetic products. FDA checks for the safety of the raw materials used in different commercial products. The chemical products contained in the hair dyes include aromatic amines such as 4-methoxy-m-phenylenediamine 4-chloro-m- phenylenediamine, 2, 4-toluenediamine, 2-nitro-p-phenylenediamine, and 4- amino-2-nitrophenol. These compounds penetrate into the human skin and are known to be mutagenic and carcinogenic.
[007] The human hair follicle is an intriguing structure wherein, the hair follicle is divided into three regions namely, the lower segment termed as bulb and suprabulb, the middle segment termed as isthmus, and the upper segment termed as infundibulum. The lower segment extends from the base of the follicle to the insertion of the erector pili muscle, which is also known as the arrector pili muscle. The middle segment is a short section that extends from the insertion of the erector pili muscle to the entrance of the sebaceous gland duct. The upper segment extends from the entrance of the sebaceous gland duct to the follicular orifice. [008] Hair color is due to the presence of melanin pigment in the keratinocytes of the hair shaft cortex. Hair color modification is achieved by removal of cortical melanin pigment through bleaching or addition of artificial pigment through application of a hair dye.
[009] Usually, commercial hair dyes are classified into the following three main subtypes according to color permanence, such as, temporary, semi -permanent, and permanent. Temporary dyes are retained for very short period. The dyes adhere to the hair shaft cuticle via van der Waal force without penetrating into the cortex. Semi-permanent dyes coat the hair shaft cuticle in a similar fashion to temporary dyes while also partially infiltrating the cortex. In contrast, permanent dyes penetrate deeper into the hair shaft, passing through the hair cuticle and are deposited completely into the cortex.
[0010] The use of hair dyes is associated with many adverse effects, such as, temporary skin irritation, allergy, skin discoloration, hair breakage, hair color differences. Further, long-term use is also associated with carcinogenicity, mutagenicity etc. [0011] In order to overcome these problems, many hair dye compositions are formulated with natural coloring agents wherein the natural coloring agents are derived from different species of plants.
[0012] Usually, keratin filaments comprise the hair shaft in human hair. It is known that KRTAP are family of proteins, which binds to keratins and the cuticle of the hair shaft. Further, these Keratin-associated proteins are grouped into several families. Approximately 100 KRTAP genes have been identified in humans till date with each of the gene classified into a number of families based on their sequence homology and the nature of the repeat structures. KRTAP are useful in the composition of hair dyes.
[0013] The Patent Application "DE4410162C2" titled "Process for dyeing of human hair" discloses a novel, improved process for dyeing of human hair with vegetable dyes. The invention relates to a method of dyeing human hair using pflanzli chen dyes. The method comprises the step of applying a first composition comprising substantive plant dye on the hair and a second composition comprising a vegetable dye is applied after approximately five to fifteen minutes. After twenty to sixty minutes exposure, the dyed hair is washed and dried. However, the invention does not disclose the use of plant-based dyes.
[0014] The Patent Application "US 6656229B1" titled "Hair dye and hair-dyeing methods using the same" discloses a composition of hair dye and hair dying methods. The hair dye comprises a formulation composed of a dye component including indicant derived from indigo plant. The hair dye further comprises a plant-based component derived from a plant selected from the group consisting of a tea leaf, comfrey, kamille, chlorella, rosemary, seaweed, and a mixture thereof. The invention also discloses the method of applying the hair dye to the human hair. However, the invention is silent with respect to the conjugation of dye with KRTAP proteins.
[0015] The Patent Application "CN102198058A" titled "Herbal hair dye" discloses a natural herbal hair dye. The dye components consist of henna, tannic acid and ferrite. The invention further discloses a method of using the hair dye. The hair dye dyes the hair into various colors including pure black and the dyeing time is shorter, the color of the dyed hair lasts longer, without inducing any serious side effects on human body. The present invention discloses the use of henna flowers, leaves and other plant parts. The relevant parts are subjected to grinding and the resulting fine powder, henna flowers, leaves or plant milled powder is extracted with water or alcohol using 30-95% ethanol extraction. In case of henna powder, henna is grinded and the resulting powder exhibited the particle size between 0.01 to 1000 mm. However, the invention discloses only the use of henna and is silent with respect to the conjugation of dye with KRTAP proteins. [0016] Hence, there is a need for a composition of a hair dye with natural dyes. There is also a need for a method of delivery of hair dyes into the hair. The use of natural dye or synthetic dye is known in the art. However, the carcinogenicity or mutagenicity or other adverse effects of these dyes is overcome by the present invention. [0017] Summary of the invention
[0018] The present invention discloses a composition of a hair dye comprising the KRTAP-2 family protein conjugated with a natural hair dye such as lawsone or indigo. The present invention discloses a method of conjugation of KRTAP-2 protein with lawsone or indigo. The present invention further discloses a method of applying the hair dye to the human hair.
[0019] The hair dye composition comprises the protein-pigment conjugate. The protein is KRTAP-2 family protein and the pigment is selected from a natural source. Lawsone or indigo are used as natural pigments. The composition also comprises ethanolamine, antimicrobial agent and hair modifying agent. [0020] The KRTAP-2 family protein is conjugated with a hair dye. The hair dye used in the present invention is a natural hair dye derived from the plant source. Lawsone is a naphthaquinone derived from Lawsonia inermis. KRTAP-2 family protein binds with the lawsone through the attachment of side groups via a covalent bond. In addition to lawsone, other pigments such as indigo derived from Indigofera tinctorea, indican or indigotein are also conjugated with KRTAP- 2 proteins. The conjugation is enhanced by the addition of linker molecule during conjugation. The conjugate product is also created by the use of synthetic hair dyes used in the regular hair dyes.
[0021] Recombinant KRTAP-2 family proteins are produced in the bacterial, yeast or other mammalian systems. [0022] The present invention also discloses a method of applying the hair dye to the human hair. The method comprises the steps of cleaning and drying the hair to remove the presence of shampoos, conditioners or any other substances. The next step involves the spraying of ethanolamine to disintegrate the cuticle of the hair. This step is followed by spraying the KRTAP -pigment conjugated solution. After spraying, the spray is allowed for a suitable time to allow the penetration of the KRTAP-pigment conjugate into the hair shaft. The hair is rinsed off from all the reagents. Finally, a hair protective substance such as oil may be applied on the hair to retain the color.
[0023] The use of conjugated product of KRTAP-2 family protein with a natural or synthetic dye reduces the adverse effects induced by the hair dyes. The use of natural dyes in conjugation is non-carcinogenic and non-mutagenic. The conjugated protein-pigment complex results in high rate of penetration of the dye into the hair shaft.
[0024] Brief description of drawings [0025] FIG 1 illustrates the chemical structure of the pigment lawsone produced by Lawsonia inermis.
[0026] FIG 2 illustrates the chemical structure of the pigment indigo produced by Indigofera tinctorea.
[0027] FIG 3 illustrates the flow chart for a method of preparing the conjugated product of KRTAP-2 protein with lawsone.
[0028] FIG 4 illustrates the flow chart for the method of preparing the conjugated product of KRTAP-2 protein with indigo. [0029] FIG 5 illustrates the flow chart for the method of spraying the hair dye to the human hair.
[0030] Detailed description of the invention
[0031] In order to make the matter of the invention clear and concise, the following definitions are provided for specific terms used in the following description.
[0032] The term "Dye" refers to a colored substance that has an affinity to the substrate to which it is being applied.
[0033] The term "Carcinogen" refers to any substance or an agent directly involved in causing cancer.
[0034] The term "Mutagen" refers to a physical or chemical agent that changes the genetic material, usually DNA of an organism and thus increasing the frequency of mutations.
[0035] The present invention relates to a composition of the hair dye comprising a complex of a KRTAP-2 family protein conjugated with a plant-derived dye. The present invention further discloses the method of conjugation of the pigment with KRTAP-2 protein and also the method of applying the hair dye comprising the conjugated product to the human hair.
[0036] The use of hair dye is associated with many adverse effects due to the presence of carcinogenic, allergic or mutagenic activity of the ingredients present in the composition of the hair dye. This is overcome by the present invention which uses natural dyes derived from the plant sources. Further, the use of dyes is associated with low rate of penetration into the hair shaft. The use of conjugated complex of protein and the pigment in the present invention provides high rate of penetration of the dye.
[0037] The present invention discloses the composition of the hair dye. The hair dye comprises a natural pigment lawsone at the concentration of 1-20 mgml"1 or indigo at a concentration of 1-5 mgml"1. Lawsone is dissolved in acetonitrile. Indigo is dissolved in a solution comprising 50 mM Tris-HCl buffer and 100 mM sodium chloride with a pH range 4 to 7.5. KRTAP-2 protein at a concentration of 1-5 mgml"1 is dissolved in a solution comprising 50 mM Tris-hydrochloride buffer and 100 mM sodium chloride solution with a pH range of 4 to 7.5. In addition to the conjugated KRTAP-2 family protein, the composition further comprises ethanolamine solution, anti-bacterial agent, anti-fungal agent, hair-modifying agent, hair-bleaching agent, hair relaxer chemical such as thioglycolate. The hair dye composition is provided in the form of a kit, which further comprises hair crimping agent, shampoo, conditioner, hair pre-treatment, hair post-treatment reagents, hair moisturizer, hair straightener oil, cream/any other protective products, keratin protein of natural/synthetic origin and formaldehyde/any other chemicals, which modify the structure of the human hair.
[0038] The KRTAP-2 family protein is conjugated with a plant-derived dye. The recombinant human KRTAP-2 family protein is expressed in bacterial, yeast or any other mammalian protein production systems using the methods known in the art with the help of the suitable promoter and expression system. In addition to expression systems, the recombinant KRTAP-2 family proteins are also produced by processing of pre-cut human hair. The recombinant protein thus obtained is used in the preparation of conjugation complex. [0039] FIG 1 illustrates the chemical structure of the pigment lawsone produced by Lawsonia inermis. The conjugation of KRTAP-2 family protein with lawsone is achieved by a chemical reaction. The dye lawsone is a naphthaquinone derived from Lawsonia inermis. Lawsone is also isolated from different species of Lawsonia. Lawsone is a 2-hydroxy-l,4-naphthoquinone with a molecular formula (CioH6N3) and forms a covalent bond with KRTAP-2 family proteins through side chains. Lawsone forms a sulfhydryl adducts to proteins during conjugation with mammalian protein.
[0040] FIG 2 illustrates the chemical structure of the pigment indigo produced by Indigofera tinctorea. KRTAP-2 family protein is conjugated with indican, indigotein or any other plant-derived or synthetic pigments to form a protein- pigment complex, which is used in the composition of the hair dye. Indigo may be derived from Indigofera tinctorea and other species of Indigofera. [0041] In order to achieve the conjugation of pigment and the protein, lawsone at the concentration of 1-20 mgml"1 is dissolved in acetonitrile solution. KRTAP-2 protein at a concentration of 1-5 mgml"1 is dissolved in a solution comprising 50 mM Tris-hydrochloride buffer and 100 mM sodium chloride solution at the pH in the range of 4 to 7.5. The composition is also prepared by using indigo instead of lawsone. Indigo at a concentration of 1-5 mgml"1 is dissolved in 50 mM Tris-HCl buffer, 100 mM sodium chloride solution at the pH in the range of 4 to 7.5.
[0042] FIG 3 illustrates the flow chart for a method of preparing the conjugated product of KRTAP-2 protein with lawsone. The method (100) of preparing protein-pigment conjugated product of the KRTAP-2 protein and lawsone starts at step (101) of adding the lawsone solution in acetonitrile to the KRTAP-2 protein solution. At step (102), the solution is stirred continuously in the presence of argon at room temperature for a period of 2-6 hours in dark condition i.e. in the absence of light. At step (103), the solution is re-suspended in a solution comprising 50mM Tris-HCl buffer, lOOmM sodium chloride at pH 7. At step (104), the protein-pigment conjugate is ascertained by High Performance Liquid Chromatography (HPLC). At step (105), protein-pigment conjugate is separated from the by-products using HPLC.
[0043] The conjugation of pigment and the protein is also achieved by using a natural dye indigo.
[0044] FIG 4 illustrates the flow chart for the method of preparing the conjugated product of KRTAP-2 protein with indigo. The method (200) of preparing protein- pigment conjugated product of the KRTAP-2 protein and indigo starts at step (201) of dissolving indigo at a concentration of 1-5 mgml"1 in a solution comprising 50 mM Tris-HCl buffer and 100 mM sodium chloride solution at the pH in the range of 4 to 7.5. At step (202), beta-glucosidase at the concentration of 80 units per gram is diluted to 2-5 units per ml and the diluted solution of beta- glucosidase is added to the indigo solution. At step (203), KRTAP-2 protein at the concentration of 1-5 mgml"1 dissolved in a solution of 50 mM Tris-HCl buffer and lOOmM sodium chloride at pH range 4-7.5 is added to the reaction mixture. At step (204), indigo is converted to indoxyl by the action of beta-glucosidase. At step (205), indoxyl reacts with free amino groups on KRTAP-2 proteins to form a peptide bond. The formation of the peptide bond restores the blue color of the indigo to the indoxyl group. At step (206), the protein-pigment conjugate is ascertained by High Performance Liquid Chromatography (HPLC). At step (207), protein-pigment conjugate is separated from the by-products using HPLC.
[0045] A linker chemical group is added to lawsone or indigo during conjugation, which facilitates cross-linkage of lawsone or indigo to derivatives of KRTAP-2 proteins. The present invention also discloses the use of chemical cross linker during crosslinking of lawsone, indigo, indican and indigotein and subsequent crosslinking of this cross linked conjugate to the KRTAP-2 protein subfamily.
[0046] The crosslinking of lawsone, indigo, indican or indigotein or synthetic hair pigments exhibits optimum physical proximity to bind to natural or recombinant KRTAP-2 family proteins .
[0047] The KRTAP-2 family protein with lawsone or indigo results in improved penetration of the dye into the hair shaft. The protein-pigment complex is used in different forms such as solution, cream, dry powder etc. The use of antibacterial or antifungal agents prevents the degradation of other agents and retains long-term stability of the components. The hair modifying agents dissolve the hair cuticle, bleach or modify the texture of the hair in order to make it straighter or curlier than the original texture. [0048] The present invention further discloses the method of spraying the hair dye to the human hair.
[0049] FIG 5 illustrates the flow chart for the method of spraying the hair dye to the human hair. The method (300) of spraying the hair dye to the human hair starts with a step (301) of cleaning and drying of hair to remove extraneous coatings of shampoo, conditioner or any other compounds. At step (302), the ethanolamine is sprayed to partially disintegrate the cuticle of hair. The disintegration of the cuticle results in the enhanced penetration of the hair dye. At step (303), the KRTAP-2 family protein-pigment conjugate complex is sprayed to the hair. KRTAP-2 family protein conjugate with a lawsone or indigo or any other natural or synthetic dye is used in this step. At step (304), the sprayed dye is allowed for a suitable time to allow penetration of the KRTAP-2 family protein- pigment conjugate complex into the hair shaft. At step (305), the applied reagents are rinsed with water. At step (306), hair protecting substances such as oil or cream of the sealing conditioners are applied to maintain and retain the hair color.
[0050] The present invention is not restricted to the use of lawsone, indigo, indican or indigotein. Based on the requirement of the novel color and combination of colors, other classes of natural and synthetic dyes such as alizarin, munjistin, purpurin, red dye from Caesalpinia sappan and Caesalpinia echinata plants, bark and roots of Morinda citrofolia, juglone from Juglans regia, carthamin from safflower, yellow dyes from turmeric (Curcuma) species, saffron (Crocus sativus), annatto and barberry (Berberis aristata), rind of pomegranate fruits (Punica granatum), fruits of the myrobalan tree (Terminalia chebula), Marigold plants (Tagetus species.), Butea species (Flame of the Forest plants), Mallotus philipensis, Onion peels (Allium cepa), European Weld (Reseda luteola), Indian Dolu (Rheum emodi), brown and black dyes from oak species, Acacia catechu species, black dye obtained by iron mordanting of extracts from Haematoxylon campechianum, whole plant of Eupatorium odoratum, Ageratum conyzoides, bark of Atro carpus heterophyllus, Eucalyptus camaldulensis, flowers of Delonix regia, Dahlia variabilis, leaves of Tectona grandis, Acacia nilotica, Ziziphus mauritiana, Moringa pterygosperma, Spathodea companulata, Tamarindus indica , rhizomes of Nymphaea alba, fruits of Emblica officinalis, petals of Kigelia pinnata, seeds of Rumex maritmus, wood of Pterocarpus santalinus are also useful in the hair dye composition.
[0051] The present invention is not restricted to the KRTAP-2 protein family. The other members of KRTAP family proteins isolated from human and sheep source such as KAP1, KAP2, KAP3, KAP4, KAP5, KAP6, KAP7, KAP8, KAP9, KAP10, KAP11, KAP12, KAP13, KAP14, KAP15, KAP16, KAP17, KAP18, KAP19, KAP20, KAP21, KAP22, KAP23, KAP24, KAP25, KAP26 and KAP27 are also useful for conjugation with natural or synthetic dye. [0052] The present invention with a kit of hair dye composition comprising the conjugated protein-pigment complex allows usage of small quantities of coloring reagents such that the cost of manufacturing the kits as well as the adverse effects on human is reduced. The method of application of the hair dye reduces human exposure of allergenic and potentially carcinogenic ingredients because of the high specificity of the principal ingredients. The pigments used as dye are isolated from plant origin and from other natural sources. Hence are associated with low risk of carcinogenicity, mutagenicity and are less allergic. The usage of human proteins to attach the pigments to human hair also allows inclusion of safe pigments to color hair. The composition allows for inclusion of new plant-derived and synthetic hair pigments to produce novel color combinations and novel coloring strategies.

Claims

[001] Claims
I claim:
A composition of a hair dye, wherein the hair dye comprises:
a. a KRTAP-2 family protein conjugated with a pigment; b. ethanolamine solution;
c. an anti-microbial agent; and
d. a hair-modifying agent.
2. The composition as claimed in claim 1, wherein KRTAP-2 family protein at a concentration of 1-5 mgml"1 is dissolved in a solution of 50 mM Tris- hydrochloride buffer, 100 mM sodium chloride solution at the pH in the range of 4 to 7.5.
3. The composition as claimed in claim 1, wherein the pigment is selected from a group of natural or synthetic pigments.
4. The composition as claimed in claim 1, wherein the natural pigment is selected from a group of lawsone, indigo, indican or indigotein.
5. The composition as claimed in claim 1, wherein the hair dye composition reduces the risk of carcinogenicity, allergy or mutagenicity.
6. The composition as claimed in claim 1, wherein the hair dye composition increases the penetration of pigment into the hair shaft due to the presence of KRTAP-2 family protein-pigment conjugate.
7. A method of preparing the conjugated product of KRTAP-2 protein with lawsone, the method (100) comprising the steps of:
a. adding a solution of lawsone dissolved in acetonitrile to the KRTAP-2 protein solution (101);
b. stirring the solution continuously in the presence of argon at room temperature for a period of 2-6 hours in dark condition (102); c. suspending the stirred solution in a solution comprising 50mM Tris-HCl buffer, lOOmM sodium chloride at pH 7 (103); d. ascertaining the KRTAP-2 family protein-law sone conjugate by High Performance Liquid Chromatography (HPLC) (104); and e. separating the KRTAP-2 family protein-law sone conjugate from the by-products using HPLC (105).
A method of preparing the conjugated product of KRTAP-2 protein with indigo, the method (200) comprising the steps of:
a. dissolving indigo at a concentration of 1-5 mgml"1 in 50 mM Tris- HC1 buffer, 100 mM sodium chloride solution at the pH in the range of 4 to 7.5 (201);
b. adding the beta-glucosidase solution diluted to 2-5 units per ml to the indigo solution (202);
c. adding the KRTAP-2 protein solution at the concentration of 1- 5mg/ml in a solution of 50 mM Tris-HCl buffer, lOOmM sodium chloride at the pH range 4-7.5 to the reaction mixture (203);
d. allowing the reaction to convert indigo to indoxyl by the action of beta-glucosidase (204);
e. allowing the reaction of indoxyl with free amino groups on KRTAP-2 proteins to form a peptide bond (205);
f. ascertaining the KRTAP-2 family protein-indigo conjugate by High Performance Liquid Chromatography (HPLC) (206); and g. separating the KRTAP-2 family protein-indigo conjugate from the by-products using HPLC (207).
A method of applying the hair dye to the human hair, the method (300) comprising the steps of:
a. cleaning and drying of hair to remove extraneous coatings of shampoo, conditioner or any such one or more compounds (301); b. spraying ethanolamine to partially disintegrate a cuticle of hair (302);
c. spraying the KRTAP-2 family protein-pigment conjugate complex to the hair (303); d. allowing the sprayed dye for a suitable time to promote the penetration of the KRTAP-2 family protein-pigment conjugate complex into the hair shaft (304);
e. rinsing the applied reagents with water (305); and
f. applying one or more hair protecting substances such as oil or cream of the sealing conditioners to maintain and retain the hair dye (306).
10. The method as claimed in claim 9, wherein the method of spraying increases the rate of penetration of the dye into the hair shaft due to the presence of KRTAP-2 family protein-pigment conjugate.
PCT/IB2017/051222 2016-03-02 2017-03-02 A hair dye composition with plant-derived and synthetic pigments, the method of preparation thereof and method of applying the hair dye WO2017149490A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11357809B2 (en) 2019-08-14 2022-06-14 Ge Nutrients, Inc. Herbal preparation for stimulation of hair growth, control of hair fall, dandruff and infections thereof using Ageratum spp
WO2023238152A1 (en) * 2022-06-07 2023-12-14 Sabharanjak Shefali Protein conjugate for hair dye, a construct, composition and method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050170366A1 (en) * 2001-11-13 2005-08-04 Jun Kudo Novel hair keratin-associated proteins
US20090098074A1 (en) * 2005-12-01 2009-04-16 Basf Se Keratin-Binding Effector Molecules Containing Reactive Dyes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050170366A1 (en) * 2001-11-13 2005-08-04 Jun Kudo Novel hair keratin-associated proteins
US20090098074A1 (en) * 2005-12-01 2009-04-16 Basf Se Keratin-Binding Effector Molecules Containing Reactive Dyes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11357809B2 (en) 2019-08-14 2022-06-14 Ge Nutrients, Inc. Herbal preparation for stimulation of hair growth, control of hair fall, dandruff and infections thereof using Ageratum spp
WO2023238152A1 (en) * 2022-06-07 2023-12-14 Sabharanjak Shefali Protein conjugate for hair dye, a construct, composition and method thereof

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