WO2024188833A1 - Indigo-based dyeing agent with improved dyeing properties - Google Patents

Abstract

The invention relates to an agent, a kit and a method for the non-oxidative dyeing of keratin-containing fibers, more particularly human hair, using an indicane-containing component that was pretreated at a temperature in the range of 60°C to 200°C, and an enzyme mixture having cellulase activity.

Description

Indigo-based dye with improved dyeing properties

The invention relates to agents, kits and methods for coloring keratin-containing fibers, in particular human hair, using an indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C and an enzyme mixture with cellulase activity.

The desire to change one's hair color is a major need of many consumers. To satisfy this need, the cosmetics industry offers a diverse range of products. Hair dyes that achieve particularly long-lasting coloring with high coverage are usually oxidation dyes. These use oxidizing agents that can damage the hair structure. Certain cationic direct azo dyes are also able to enable hair color changes with excellent fastness properties. However, the azo dyes mentioned are synthetic dyes.

A growing number of consumers are demanding hair dyes and hair coloring processes based on natural dyes, even though these products and processes are often inferior to the aforementioned products and processes in terms of authenticity, coverage and color variety.

In addition to dyeing hair with henna, which is obtained from the plant Lawsonia inermis, dyeing hair with plants from which indigo can be produced has also been known for a long time.

State of the art

The production of storage-stable cosmetic products containing natural plant material is often difficult. The plant material contains enzymes that can react with other ingredients of the plant material or the cosmetic during storage. There has therefore been no lack of efforts in the state of the art to increase the storage stability of cosmetics containing plant material. When dyeing with indigo-producing plant materials, it is often observed that the color changes constantly over a period of up to 2 weeks after application of the dye and that the final color tone only remains after this period. From a concentration of 2% by weight of indigo-producing plant powder in water, the color tone on the hair changes in the first few days after dyeing from an initial turquoise-blue tone to a redder and darker color tone. so that a purple color impression is created. In the prior art, there has therefore been no lack of efforts to improve the coloring of keratin fibers, especially human hair, with indigo. According to the invention, "improvement" is to be understood as meaning that the formation of a red tone and a purple tone is suppressed as completely as possible or at least to a high degree. Furthermore, according to the invention, "improvement" is to be understood as meaning that the final blue color tone is achieved as quickly as possible after the end of the coloring process.

US20040055096A1 discloses a dye for the production of which powdered leaves of an indigo-producing plant, such as Polygonum tinctorium, Strobilanthes cusia, Indigofera tinctoria or Isatis indigotica, whose natural enzyme content has been deactivated by heat treatment, are mixed shortly before use with a beta-glucosidase-containing component (e.g. fresh leaves of an indigo-producing plant which have not been heat-treated, or the pressed juice from these non-heat-treated leaves, optionally in freeze-dried form, or extracts of various mushrooms, such as enokitake (Flammulina velutipes) and shiitake (Lentinus Edodes), or extracts of corn leaves, apricot kernels or almonds). By separating the indigo precursor indican, which is present in the plants in the form of 3-indoxyl-ß-D-glucopyranoside, from the plant's own enzyme beta-glucosidase, the natural dye's storage stability is improved. US20040055096A1 also deals with suppressing the red tone and achieves this by adding saponins or saponin-containing plants, particularly in powder form. The addition of saponins in the form of saponin-containing plant parts is particularly disadvantageous for the commercial production of indigo-based dyes, as these are natural products with fluctuating saponin content, which makes it difficult to manufacture products of consistent, standardized quality. Saponins also cause the dye to foam, which impairs the dye's ability to absorb color and is therefore undesirable.

In PCT application W02014104301A1, a similar approach to improved storage stability is disclosed as in US20040055096A1, namely the combination of two different indigo powders, whereby the natural enzyme content in the first leaf powder component was deactivated by heat treatment, while the second leaf powder component was dried at temperatures below 50 °C and thus contained active enzyme.

WO2015082482A1 discloses a method for dyeing hair with powdered plant parts from plants that produce indigo, in which the hair is treated with a strongly alkaline composition after indigo dyeing, which can be done in the presence of hydrogen peroxide. This is intended to accelerate the development of the final color tone and prevent the color from changing after the actual dyeing process has been completed. However, the hair is damaged by the alkaline pH and the oxidizing agent.

Task

An object of the present invention was to provide agents, kits and methods for non-oxidative To provide dyeing of keratin-containing fibers, in particular human hair, using plants that contain indican and produce indigo, with which the final color result of the indigo dyeing is obtained as soon as possible after completion of the actual dyeing process.

A further object of the present invention was to provide agents, kits and methods for the non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants that contain indican and produce indigo, with which the formation of a red tone and a purple tone is suppressed as completely as possible or at least to a high degree.

A further object of the present invention was to provide saponin-free or low-saponin agents, kits and methods for the non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants that contain indican and produce indigo, with which the formation of a red tone and a purple tone is suppressed as completely as possible or at least to a high degree.

A further object of the present invention was to provide agents, kits and methods for non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants containing indican and producing indigo, with improved storage stability.

Surprisingly, it was found that the dyes, dyeing processes and dyeing kits described in the patent claims can be used to achieve dyeings using parts or extracts of plants that contain indican and produce indigo, with which the final color result of the indigo dyeing is obtained as quickly as possible after completion of the actual dyeing process. The final color result of the indigo dyeing achieved according to the invention has only a small proportion of red, even without the addition of saponins or saponin-containing plant parts. The dyes and dyeing kits according to the invention also have a high storage stability.

A first subject of the present invention is therefore an agent for the non-oxidative coloring of keratin fibers, in particular human hair, comprising i) at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, furthermore ii) an enzyme mixture comprising at least one endo-1,4-beta-glucanase (EC 3.2.1.4), at least one beta-glucosidase (EC 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (EC 3.2.1.91) and iii) water. The indigo precursor indican occurs naturally in plants. Indican is a glucoside of indoxyl and occurs in some plants known as indigo plants, for example Isatis tinctoria (woad) or Indigofera tinctoria. Synonyms for indican are 3-indoxyl-ß-D-glucopyranosid, indoxyl-ß-D-glucoside, 3-(ß-D-glucosido)indole or plant indican.

Before the indican is converted into the dye indigo, the indican must be split into indoxyl and the glucoside residue. This splitting preferably takes place enzymatically. The splitting should take place in situ, i.e. just before application to the keratin fibers to be dyed.

Accordingly, the indican-containing component, which has been pretreated at a temperature in the range of 60 °C to 200 °C, and the enzyme mixture ii) according to the invention should be physically separated from each other until use.

A further subject of the present invention is therefore a kit for the non-oxidative coloring of keratinic fibers, in particular human hair, with natural dyes, which comprises the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, and ii) an aqueous composition (E) which contains an enzyme mixture comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91).

According to the invention, "non-oxidative" means that no oxidizing agents other than atmospheric oxygen are used in the agents, kits and methods according to the invention. In particular, the oxidizing agents hydrogen peroxide, persulfates, perbromates, percarbonates, perborates and percarbamides are excluded. Atmospheric oxygen does not constitute an oxidizing agent in the context of the invention.

Agents and kits for non-oxidative coloring preferred according to the invention are characterized in that the indican-containing component i) is selected from comminuted parts of an indigo-producing plant and from indican-containing extracts of an indigo-producing plant and mixtures of these indican-containing components i).

Particularly preferred agents and kits for non-oxidative dyeing according to the invention are characterized in that the indican-containing component i) is selected from indican-containing extracts of an indigo-producing plant. Further agents and kits for non-oxidative coloring preferred according to the invention are characterized in that the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore Ci-C4-alkanols and C2-C4-polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerin and 1,3-butylene glycol, and mixtures of these extraction agents, preferably selected from water, ethanol, mixtures of water and at least one Ci-C4-alkanol, mixtures of water and at least one C2-C4-polyol and water/ethanol mixtures, particularly preferably selected from water, ethanol and water/ethanol mixtures.

Preferred agents and kits for non-oxidative dyeing according to the invention are characterized in that the indigo-producing plant(s) is/are selected from at least one species of the following genera:

- Indigofera, in particular Indigofera tinctoria, Indigo suffruticosa, Indigofera articulata, Indigofera arrecta, Indigofera heterantha “Gerardiana”, Indigofera argentea or Indigofera longiracemosa;

- Isatis, especially Isatis tinctoria (woad);

- Persicaria, especially Persicaria tinctoria (dyer's knotweed);

- Wrightia, especially Wrightia tinctoria;

- Calanthe, especially Calanthe veratrifolia; and

- Baphicacanthus cusia, synonym Strobilanthes cusia.

Particularly preferred plants producing indigo according to the invention are selected from the genus Indigofera and in particular from Indigofera tinctoria.

The plants of the genera and species mentioned above contain no or only small amounts of indigo. Instead, the plants contain glycosidically bound indican, which is a precursor of indigo after enzymatic cleavage of the glycoside residue. The parts of the plant genera and species mentioned above that are rich in indican and also contain indigo are the leaves of these plants. To obtain the indican-containing component i) according to the invention, the plant leaves of the plant genera and species mentioned above are dried and ground and used as plant powder.

Plant powders preferred according to the invention have a particle size of less than 500 pm, particularly preferably in the range of 120 pm - 200 pm, extremely preferably in the range of 150 pm - 180 pm. Other plant powders preferred according to the invention have a bulk density in the range of 0.20 - 0.60 g/cm ³ , particularly preferably in the range of 0.25 - 0.40 g/cm ³ . Other plant powders preferred according to the invention have, based on their weight, a moisture content of less than 10 wt.%, preferably 0.1 - 6.0 wt.%, particularly preferably 0.2 - 3.0 wt.%. The moisture content is determined after 3 hours of drying at 105°C.

It is preferred according to the invention that the powder of the indigo-producing plant, based on its weight, consists of at least 50 wt. %, preferably at least 80 wt. %, particularly preferably more than 80 - 100 wt. %, of the leaves of the plant. Further preferred embodiments according to the invention are characterized in that the powder of the indigo-producing plant has a particle size of less than 500 pm, particularly preferably in the range of 120 pm - 200 pm, extremely preferably in the range of 150 pm - 180 pm, and also a bulk density in the range of 0.20 - 0.60 g/cm ³ , particularly preferably in the range of 0.25 - 0.40 g/cm ³ and, based on its weight, a moisture content of less than 10 wt. %, preferably 0.1 - 6.0 wt. %, particularly preferably 0.2 - 3.0 wt. %.

Further agents and kits preferred according to the invention are characterized in that the powder of the indigo-producing plant contains indican in an amount of 2 - 5 wt.%, preferably 2.5 - 4.5 wt.%, particularly preferably 3 - 4.1 wt.%, based on the weight of the powder, wherein the indican content is converted to pure indican.

Deactivation of the natural enzyme content in the indican-containing component i)

Further agents and kits for non-oxidative coloring preferred according to the invention are characterized in that the pretreatment of the at least one indican-containing component i) is carried out at a temperature in the range from 60 °C to 200 °C by heating, in particular by heating in a solvent, furthermore by treatment with steam or by dry heating, preferably for a period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extremely preferably 5 to 10 minutes.

Option 1 : Indican-containing component i) is an extract

Particularly preferred agents and kits for non-oxidative coloring according to the invention are characterized in that the indican-containing component i) is selected from at least one indican-containing extract of an indigo-producing plant.

Agents and kits for non-oxidative coloring which are particularly preferred according to the invention are characterized in that the indican-containing component i) is selected from at least one indican-containing extract of an indigo-producing plant, wherein the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore Ci-C4-alkanols and C2-C4-polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerin and 1,3-butylene glycol, and mixtures of these extraction agents, preferably selected from water, ethanol, mixtures of water and at least one Ci-C4-alkanol, mixtures of water and at least one C2-C4-polyol and water/ethanol mixtures, particularly preferably selected from water, ethanol and water/ethanol mixtures.

The indican can be extracted using these polar solvents. Since the extraction can be carried out easily at a temperature in the range of 60 °C to 200 °C by heating the solvent, this preferred procedure not only isolates the indican, but also deactivates the plant's own enzymes at the same time. The deactivation of the plant's own enzymes is desirable according to the invention, because it was surprisingly found that the coloring achieved can be significantly improved if the plant's own enzymes, which as a natural product are subject to a large number of qualitative and quantitative fluctuations in composition, are replaced by a standardized enzyme mixture.

In a further preferred embodiment of the invention, the extracted indican is not separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, for example by filtration or decanting, but is used directly as indican-containing component i), optionally after concentration by evaporation of the solvent or after dilution, wherein optionally further cosmetic aids, such as preservatives, thickeners, perfumes, pH regulators or hair conditioners, can be added.

In another, likewise preferred embodiment of the invention, the extracted indican is also separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, for example by filtration or decanting, for example by freeze-drying or by distilling off the solvent, so that the indican-containing component i) is obtained in powder form. To produce the colorant preferred according to the invention, the powdered indican-containing component i) can be taken up in water and then mixed with the enzyme mixture and optionally further water. In a further preferred embodiment, to produce the colorant preferred according to the invention, the powdered indican-containing component i) can be mixed with the enzyme mixture and with water.

Option 2: dried and crushed plant parts of an Indiqo-producing plant as indican-containing component i)

According to the invention, dried and crushed plant parts of an indigo-producing plant which have been pretreated at a temperature in the range of 60 °C to 200 °C can also be used as indican-containing component i).

The heat pretreatment of this embodiment of the invention is characterized in that the pretreatment of the at least one indican-containing component i) is carried out at a temperature in the range from 60 °C to 200 °C by heating, in particular by heating without solvent, further by treatment with steam or by dry heating, preferably for a Period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extremely preferably 5 to 10 minutes.

Further colorants preferred according to the invention are characterized in that at least one indican-containing component i) contains dried and comminuted parts of at least one indigo-producing plant, which have been pretreated at 60 - 200°C, in a total amount of 1 - 80 wt.%, preferably 2 - 50 wt.%, particularly preferably 3 - 25 wt.%, extremely preferably 4 - 15 wt.%, further extremely preferably 5 - 10 wt.%, further extremely preferably 6 - 7 wt.%, based on the weight of the agent.

Further colorants preferred according to the invention are characterized in that they contain as at least one indican-containing component i) powdered leaves of Indigofera tinctoria in an amount of 1-80% by weight, preferably 2-50% by weight, particularly preferably 3-25% by weight, extremely preferably 4-15% by weight, further extremely preferably 5-10% by weight, further extremely preferably 6-7% by weight, based on the weight of the agent.

Preferred agents for non-oxidative coloring according to the invention are characterized in that the indican-containing component i) contains, based on its weight, 0.01 - 6 wt.% indican. Preferred indican-containing components i) contain, based on their weight, 0.02 - 5 wt.%, preferably 0.05 - 4.5 wt.%, particularly preferably 0.1 - 4 wt.%, extremely preferably 0.2 - 3.9 wt.% indican. Depending on the type of indican-containing component i), different subranges from the previously mentioned broad ranges of amounts of indican can be particularly preferred.

If the indican-containing component i) is dried and crushed plant parts of an indigo-producing plant, its indican content, in each case based on its weight, is preferably 1 - 6 wt.%, particularly preferably 2 - 5 wt.%, particularly preferably 3 - 4.5 wt.%, extremely preferably 3.5 - 4.1 wt.%.

If the indican-containing component i) is the solvent-containing extract from dried plant parts of an indigo-producing plant, its indican content, in each case based on its weight, is preferably 0.01 - 2 wt.%, particularly preferably 0.02 - 1 wt.%, particularly preferably 0.1 - 0.5 wt.%, extremely preferably 0.2 - 0.4 wt.%.

The dyes according to the invention also contain water. The water essentially serves as a solvent for the enzymes and the at least one indican. If dried and chopped plant parts of an indigo-producing plant that have previously been subjected to heat treatment are used as the indican-containing component i), these are preferably dispersed in water in order to dissolve out the indican and make it accessible for the reaction to form indigo. If the indican-containing component i) is the solvent-containing extract from dried plant parts of an indigo-producing plant, it preferably contains water as a solvent.

Water in the entire dye

Colorants preferred according to the invention are characterized in that they contain, in each case based on their weight, water in an amount of 19.9 - 95.0 wt.%, preferably 30.0 - 90.0 wt.%, particularly preferably 50.0 - 88.0 wt.%, extremely preferably 60.0 - 85.0 wt.%.

Water in the Indican-containing extract

If the indican-containing component i) is the solvent-containing extract from dried plant parts of an indigo-producing plant, it contains, in each case based on its weight, preferably water in an amount of 0.5 - 99.5 wt.%, more preferably 10.0 - 95.0 wt.%, particularly preferably 30.0 - 90.0 wt.%, extremely preferably 50.0 - 85.0 wt.%, more extremely preferably 65 - 80 wt.%.

Water in the indican-containing plant powder dispersion

If dried and crushed plant parts of an indigo-producing plant which have previously been subjected to heat treatment are used as indican-containing component i), these are preferably dispersed in water, wherein this dispersion, in each case based on its weight, preferably contains water in an amount of 15.0 - 99.5 wt.%, more preferably 30.0 - 95.0 wt.%, particularly preferably 50.0 - 90.0 wt.%, extremely preferably 60.0 - 85.0 wt.%, more extremely preferably 70 - 80 wt.%.

Water in the indican-containing dry extract dispersion

If the dried extract from the plant parts of an indigo-producing plant is used as the indican-containing component i), this is preferably dispersed or dissolved in water, wherein this dispersion or solution, in each case based on its weight, preferably contains water in an amount of 15.0 - 99.5 wt.%, more preferably 30.0 - 95.0 wt.%, particularly preferably 50.0 - 90.0 wt.%, extremely preferably 60.0 - 85.0 wt.%, more extremely preferably 70 - 80 wt.%.

Water in composition (E) containing the enzyme mixture ii)

The enzyme mixture ii) used according to the invention, comprising at least one endo-1,4-beta-glucanase (EC 3.2.1.4), at least one beta-glucosidase (EC 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (EC 3.2.1.91), can preferably be dispersed or dissolved in water before it is combined with the indican-containing component i) to form a colorant according to the invention. This enzyme dispersion preferably contains, based on its weight, water in an amount of 15.0 - 99.5% by weight, more preferably 30.0 - 95.0% by weight, particularly preferably 50.0 - 90.0% by weight, extremely preferably 60.0 - 80.0% by weight, even more extremely preferably 64 - 75% by weight.

Water as part of an optional third compartment

The water content of the colorant according to the invention can also be regulated via an optional third compartment which comprises neither an indican-containing component i) nor an enzyme from the enzyme mixture ii) used according to the invention.

The colorant according to the invention is further characterized in that it contains an enzyme mixture comprising

- at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4),

- at least one beta-glucosidase (E.C. 3.2.1 .21) and

- contains at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91).

Surprisingly, it was discovered that an enzyme mixture used according to the invention can produce dyes in which the final color result of the indigo dyeing is achieved shortly after the actual dyeing process has been completed and does not change over time. If, on the other hand, an indican-containing component with its natural enzyme content is used, a very unstable dyeing result is obtained, the color of which shifts significantly within two weeks of dyeing.

The enzymes contained in the enzyme mixture ii) used according to the invention are selected from a group of enzymes generally referred to as cellulases.

The term "cellulase" as used herein refers to enzymes that catalyze the hydrolysis of 1,4-beta-D-glucoside bonds present in cellulose (cellobiose) and/or lichenin and/or beta-D-glucans. Cellulases are often also able to hydrolyze the 1,4-bonds in beta-D-glucans, which have 1,3-bonds in addition to the 1,4-bonds. Cellulases are able to split cellulose into beta-glucose. Consequently, cellulases act in particular on cellulose-containing or cellulose-derivative-containing residues and catalyze their hydrolysis. The decisive factor as to whether an enzyme is a cellulase within the scope of the invention is its ability to hydrolyze 1,4-beta-D-glucoside bonds in cellulose.

The term "cellulase activity" is defined here as an enzyme that catalyzes the hydrolysis of 1,4-beta-D-glucoside bonds in beta-1,4-glucan (cellulose). Cellulose activity is measured using a standard method, e.g. as follows: Cellulases release glucose from CMC (carboxymethylcellulose). The samples are analyzed under defined reaction conditions (100 mM sodium phosphate). phat buffer pH 7.5, 40°C, 15 min) with a substrate (1.25 wt.% CMC). The reaction with p-hydroxybenzoic acid hydrazide (PAHBAH) in the presence of bismuth produces a yellow dye that can be determined photometrically at 410 nm. An alkaline pH value is required during the color reaction. The amount of sugar released corresponding to the color is a measure of the enzyme activity (Lever, Anal. Biochem., 1972, 47 & 1977, 81).

Cellulases can be divided into three categories:

1. Endoglucanase (E.C. 3.2.1.4), also called endo-1,4-beta-glucanase, beta-1,4-glucanase, avice-lase, beta-1,4-endoglucanhydrolase, endo-1,4-beta-D-glucanohydrolase, carboxymethylcellu- lase or celludextrinase;

2. Cellulose-1,4-beta-cellobiosidase (non-reducing end) (E.C. 3.2.1.91), also called exoglucanase, 1,4-beta-cellobiohydrolase, 4-beta-D-glucan cellobiohydrolase (non-reducing end), avicelase, exo-1,4-beta-D-glucanase or exocellobiohydrolase, releases cellobiose from the non-reducing ends of the ß-D-glucan chains by hydrolysis of the (1->4)-beta-D-glucosidic bonds in cellulose and cellotetraose;

3. beta-Glucosidase (EC 3.2.1.21), also called cellobiase, beta-D-glucoside glucohydrolase, amygdalase or gentobiase, hydrolyzes terminal, non-reducing beta-D-glucosyl groups to release beta-D-glucose.

Cellulases preferably used according to the invention do not originate from plants, but are of bacterial or fungal origin. To produce cellulases particularly preferably used according to the invention, fungi are used which are selected from Aspergillus spp., in particular Aspergillus niger, Aspergillus niger SOI017, Aspergillus niger AS 3.4309, Aspergillus niger HDF 05, Aspergillus oryzae, Aspergillus strain SA 58, Aspergillus terreus EMOO 6-4, and mixtures of Aspergillus niger and Aspergillus oryzae, and also selected from Trichoderma reesei. Chemically modified or protein-technically modified mutants are included.

Examples of cellulases with endo-1,4-glucanase activity (EC 3.2.1.4) are described in WO 2002/099091, e.g. those with a sequence of at least 97% identity to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 of WO 2002/099091.

A preferred embodiment of the invention is characterized in that the agent for non-oxidative coloring comprises an enzyme mixture comprising at least one endo-1,4-beta-glucanase (EC 3.2.1.4) in a total amount of 20 - 35 wt.%, at least one beta-glucosidase (EC 3.2.1.21) in a total amount of 15 - 25 wt.% and at least one exo-1,4- beta-D-glucanase (EC 3.2.1.91) in a total amount of 25 - 35 % by weight, the amounts referring to the total weight of the enzyme mixture ii).

In a further preferred embodiment of the invention, in addition to the at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), the at least one beta-glucosidase (E.C. 3.2.1.21) and the at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91), at least one further enzyme with cellulase activity is additionally contained, selected from exo-1,4-beta-glucosidase (E.C. 3.2.1.74), cellulose-1,4-beta-cellobiosidase (E.C. 3.2.1.176) and oligooxyloglucan-reducing end-specific cellobiohydrolase (E.C. 3.2.1.150) as well as mixtures of these enzymes.

A further particularly preferred embodiment of the invention is characterized in that the agent for non-oxidative coloring comprises an enzyme mixture comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4) in a total amount of 20 - 35 wt.%, at least one beta-glucosidase (E.C. 3.2.1.21) in a total amount of 15 - 25 wt.%, and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91) in a total amount of 25 - 35 wt.%, as well as at least one further enzyme with cellulase activity, selected from exo-1,4-beta-glucosidase (E.C. 3.2.1.74), cellulose-1,4-beta-cellobiosidase (E.C. 3.2.1 .176) and oligooxyloglucan-reducing end-specific cellobiohydrolase (E.C. 3.2.1.150) as well as mixtures of these enzymes in a total amount which, together with the amounts of the other enzymes, makes up 100% by weight, the amounts referring to the total weight of the enzyme mixture ii).

A further particularly preferred embodiment of the invention is characterized in that the agent for non-oxidative coloring contains, based on its weight, an enzyme mixture with cellulase activity which contains at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91) in a total amount of 0.0001 - 1 wt.%, preferably 0.001 - 0.1 wt.%, more preferably 0.002 - 0.05 wt.%, particularly preferably 0.003 - 0.04 wt.%, extremely preferably 0.01 - 0.03 wt.%, more extremely preferably 0.015 - 0.02 wt.%.

The precursor of the dye indigo is indican. Indican, which occurs naturally in plants, is a glycoside, or more precisely a glucoside, which is split into indoxyl and the glycoside residue before the indican is converted into the dye indigo. This splitting preferably takes place enzymatically. The splitting should take place in situ, i.e. just before application to the keratin fibers to be dyed.

The above-discussed agent for non-oxidative coloration of keratin fibers is therefore preferably sold commercially as a kit comprising at least two components (Ind) and (E), which are physically separate from each other. A further subject matter of the present invention is therefore a kit for the non-oxidative coloring of keratinic fibers, in particular human hair, with natural dyes, which comprises the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, and ii) an aqueous composition (E) which contains an enzyme mixture comprising at least one endo-1,4-beta-glucanase (EC 3.2.1.4), at least one beta-glucosidase (EC 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (EC 3.2.1.91).

The enzyme mixture ii) used according to the invention, comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91), can preferably be dispersed or dissolved in water before it is mixed with the indican-containing component i) to form a colorant according to the invention. This enzyme dispersion, which represents component (E) of the kit according to the invention, preferably contains, based on its weight, water in an amount of 15.0 - 99.5% by weight, more preferably 30.0 - 95.0% by weight, particularly preferably 50.0 - 90.0% by weight, extremely preferably 60.0 - 80.0% by weight, more extremely preferably 64 - 75% by weight.

The enzyme active substance content in the aqueous composition (E) is preferably, in each case based on its weight, 1-70 wt.%, particularly preferably 5-50 wt.%, further particularly preferably 10-35 wt.%, extremely preferably 15-25 wt.%, further extremely preferably 18-22 wt.%.

Further colorants preferred according to the invention are characterized in that saponins are contained in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001% by weight, in each case based on the weight of the ready-to-use colorant.

With regard to further preferred embodiments of the dyeing kits according to the invention, what has been said about the dyeing agents according to the invention applies mutatis mutandis.

Another object of the present invention is a method for the non-oxidative coloring of keratin fibers, in particular human hair, which comprises the following process steps in the order given: a) providing a composition (Ind) which contains at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, and providing an aqueous composition (E) which contains an enzyme mixture, comprising at least one endo-1,4-beta-glucanase (EC 3.2.1.4), at least one beta-glucosidase (EC 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (EC 3.2.1.91), b) mixing the compositions (Ind) and (E) with one another to produce a dye, c) applying the dye obtained in step b) to the keratin fibers, d) leaving to act for a time of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 15 to 30 minutes, e) rinsing the keratin fibers with water, f) optionally drying the keratin fibers.

The ready-to-use dye obtained in step b) is applied to the keratin fibers. The ready-to-use dye obtained in step b) is preferably applied to the keratin fibers within 1 second to 30 minutes, particularly preferably within 10 seconds to 15 minutes after completion of process step b), i.e. immediately after the production of the ready-to-use dye.

Surprisingly, it was found that the temporal development of the dyeing result on the keratin fibers can be further accelerated if the ready-to-use dye according to the invention is allowed to act under the influence of heat, for example by means of a heat lamp or a drying hood or a hair dryer.

A particularly preferred embodiment of the dyeing process according to the invention is therefore characterized in that the ready-to-use dye obtained in step b) is allowed to act under the influence of heat, preferably at a temperature of 25 - 60 °C, particularly preferably at a temperature of 30 - 50 °C, extremely preferably at a temperature of 35 - 40 °C.

In conventional dyes and dyeing processes with natural dyes, compounds, especially salts, of metals other than alkali metals are often used to improve the adhesion of the natural dye to the keratin fibers. Such compounds are also referred to as "mordants". For example, US20040055096A1 discloses a pretreatment or post-treatment of the keratin fibers with metals or metal salts selected from aluminum, iron, zinc, nickel, calcium and magnesium. Other mordants are the transition metals and lanthanides as well as their salts and complex compounds.

The content of metal ions other than alkali metal ions, which are usually contained in the water used, e.g. in tap water or city water, is not taken into account here. Tap water can contain an average of 2 mg of copper ions per litre, i.e. about 0.0002% by weight of copper ions. Tap water contains about 20 - 120 mg of calcium ions per litre, i.e. about 0.002 - 0.012% by weight calcium ions. Tap water contains about 2 - 25 mg magnesium ions per liter, i.e. about 0.0002 - 0.0025% by weight magnesium ions. In such low concentrations, no perceptible mordant effect occurs. For the purposes of the present invention, the agents according to the invention are "free of mordants" if the mordant(s) is/are contained in maximum concentrations that are naturally usual for the ingredients used, such as water, enzymes and plant parts.

The dyes, dye kits and dyeing processes according to the invention can dispense with the use of such mordants.

Preferred dyeing agents and dyeing processes according to the invention are therefore characterized in that they are free of mordants.

In order to preserve the hair-protecting potential of natural dyes, preferred methods according to the invention are limited to those methods in which the keratin fibers were not treated with a mordant in a period of up to 7 days before and up to 7 days after the application of a dye according to the invention.

Further preferred colorants, coloring kits and coloring processes according to the invention are characterized in that they are free from hydrogen peroxide. Further preferred colorants, coloring kits and coloring processes according to the invention are characterized in that they are free from mordants and hydrogen peroxide.

With regard to further preferred embodiments of the dyeing processes according to the invention, what has been said about the dyeing agents and dyeing kits according to the invention applies mutatis mutandis.

The keratin fibres to be coloured, to which the colouring agent prepared in step b) by mixing the compositions (Ind) and (E) is applied in process step c), are preferably dry.

In order to preserve the hair-protecting potential of natural dyes, the claimed method is preferably limited to those methods in which the keratin fibers have not been treated with an oxidizing agent within a period of up to 7 days prior to the application of the dye according to the invention.

The oxidizing agents that are usually used in hair cosmetics, but which according to the invention are not intended to be used for hair treatment, not even as a pre-treatment, include hydrogen peroxide, persulfates, perbromates, percarbonates, perborates and percarbamides. The oxygen contained in the ambient air does not constitute an oxidizing agent in the context of the invention. In order to preserve the hair-protecting potential of natural dyes, preferred methods according to the invention are limited to those methods in which the keratin fibers have not been treated with a keratin-reducing compound within a period of up to 7 days prior to the application of a dye according to the invention.

The keratin fibers are preferably dried after rinsing out the dye prepared from the compositions (Ind) and (E). Drying can take place without actively applying heat. However, drying can also take place with the application of heat at a temperature of 25 - 120 °C, particularly preferably at a temperature of 30 - 80 °C, extremely preferably at a temperature of 35 - 60 °C. The heat is preferably applied using a heat lamp, a drying rod, a drying hood, a straightening iron or a hair dryer.

A further feature of the dyeing process according to the invention is that the dye prepared from the compositions (Ind) and (E) is allowed to act on the keratin fibers for a period of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 20 to 35 minutes, extremely preferably 25 to 30 minutes, after application to the keratin fibers.

After the dye has taken effect, the keratin fibers are rinsed with water to wash out the dye.

Optionally, the keratin fibers can be dried after this rinsing step. Drying can be done with an absorbent cloth, such as a towel. The towel-dried hair can optionally be dried partially or completely with a hair dryer or another heat source. It is also possible to let the keratin fibers air dry.

Further dyeing processes preferred according to the invention are characterized in that no oxidation dye precursors are used in them. Typical oxidation dye precursors are p-aminophenol, 4-amino-3-methylphenol, 4-amino-2-aminomethylphenol, 4-amino-2-(1,2-dihydroxyethyl)phenol, 4-amino-2-(diethylaminomethyl)phenol, 2-(2,5-diaminophenyl)ethanol, 2-(1,2-dihydroxyethyl)-p-phenylenediamine, N,N-bis-(2 -hydroxyethyl)-p-phenylenediamine, N-(4-amino-3-methylphenyl)-N-[3-(1H-imidazol-1-yl)propyl]amine, N,N'-bis-(2-hydroxyethyl)-N,N'-bis-(4-aminophenyl)-1,3-diamino-propan-2-ol, bis-(2-hydroxy-5-aminophenyl)methane, 1,3-bis- (2,5-diaminophen- oxy)propan-2-ol, N,N'-Bis-(4-aminophenyl)-1 ,4-diazacycloheptan, 1 ,10-Bis-(2,5-diaminophenyl)- 1 ,4,7,10-tetraoxadecan, 2,4,5,6-Tetraaminopyrimidin, 4-Hydroxy-2,5,6-triaminopyrimidin, 2-Hydro- xy-4,5,6-triaminopyrimidin, 2,3-Diamino-6,7-dihydro-1 H,5H-pyrazolo-[1 ,2-a]-pyrazol-1-on, 3-Amino- phenol, 5-Amino-2-methylphenol, 3-Amino-2-chlor-6-methylphenol, 2-Hydroxy-4-aminophenoxy- ethanol, 5-Amino-4-chlor-2-methylphenol, 5-(2-Hydroxyethyl)-amino-2-methylphenol, 2,4-dichloro-3-aminophenol, 2-aminophenol, 3-phenylenediamine, 2-(2,4-diaminophenoxy)ethanol, 1,3-bis(2,4-diaminophenoxy)propane, 1-methoxy-2-amino-4-(2-hydroxyethylamino)benzene, 1, 3-Bis(2,4-diamino- phenyl)propan, 2, 6-Bis(2'-hydroxyethylamino)-1 -methylbenzol, 2-({3-[(2-Hydroxyethyl)amino]-4- methoxy-5-methylphenyl}amino)ethanol, 2-({3-[(2-Hydroxyethyl)amino]-2-methoxy-5-methyl- phenyl}amino)ethanol, 2-({3-[(2-Hydroxyethyl)amino]-4,5-dimethylphenyl}amino)ethanol, 2-[3-Mor- pholin-4-ylphenyl)amino]ethanol, 3-Amino-4-(2-methoxyethoxy)-5-methylphenylamin, 1-Amino-3- bis-(2-hydroxyethyl)aminobenzol, Resorcin, 2-Methylresorcin, 4-Chlorresorcin, 1 ,2,4-Trihydroxy- benzol, 2-Amino-3-hydroxypyridin, 3-Amino-2-methylamino-6-methoxypyridine, 2,6-dihydroxy-3,4-di-methylpyridine, 3,5-diamino-2,6-dimethoxypyridine, 1-phenyl-3-methylpyrazol-5-one, 1-naphthol, 1,5-dihydroxynaphthalene, 2,7-dihydroxynaphthalene, 1,7-dihydroxynaphthalene, 1 ,8-Dihydroxynaphthalene, 2-(1,3-benzodioxole-5-ylamino)ethanol (1-beta-hydroxyethyl-3,4-methylenedioxyaniline), 4-hydroxyindole, 6-hydroxyindole, 7-hydroxyindole, 4-hydroxyindoline, 6-hydroxyindoline and 7-hydroxyind olin.

The compositions (Ind) and (E) used according to the invention and preferably according to the invention can - independently of one another - optionally contain further additives that can optimize their application properties. Preferred additives are in particular thickeners that ensure that the colorant according to the invention or preferred according to the invention produced from the compositions (Ind) and (E) remains better on the hair during application.

Compositions (Ind) and (E) used particularly preferably according to the invention contain - independently of one another - at least one or more hydrophilic thickeners other than cellulose, which are preferably selected from polysaccharides that can be chemically and/or physically modified. Compounds from the group of polysaccharides are particularly preferred as hydrophilic thickeners according to the invention, since the basic structures of the polysaccharides are of natural origin and biodegradable. Preferred hydrophilic polysaccharide thickeners are selected from xanthan gum, alginic acids (and their corresponding physiologically acceptable salts, the alginates), agar agar (with the polysaccharide agarose present in agar agar as the main component), starch fractions and starch derivatives such as amylose, amylopectin and dextrins, karaya gum, locust bean gum, gum arabic, pectins, dextrans and guar gum, and mixtures thereof.

In preferred embodiments, xanthan gum is included as a hydrophilic thickener with a view to reliable viscosity adjustment and residue-free application to keratin fibers and the scalp.

Compositions (Ind) and (E) which are particularly preferably used according to the invention contain, in each case based on the weight, independently of one another, at least one hydrophilic thickener in a total amount of from 0.1 to 5% by weight, preferably from 0.5 to 4% by weight, more preferably from 1 to 3.5% by weight and very particularly preferably from 1.2 to 2% by weight.

In a further preferred embodiment of the present invention, the compositions (Ind) and (E) used according to the invention contain, in each case based on their weight, independently of one another 0.1 to 3 wt.%, preferably 0.5 to 2.5 wt.%, more preferably 1.2 to 2.0 wt.%, xanthan gum.

Compositions (Ind) and (E) used particularly preferably according to the invention contain, independently of one another, at least one organic solvent which has a phenyl group in the molecule. This solvent is preferably selected from phenoxyethanol, benzyl alcohol and mixtures thereof. Surprisingly, it has been found that such aromatic solvents can have a positive effect on the dyeing results of the dyeing process according to the invention; this was observed in particular with aromatic solvents.

In a further preferred embodiment of the present invention, the compositions (Ind) and (E) used according to the invention contain, in each case based on their weight, independently of one another, 0.1 to 3% by weight, preferably 0.5 to 2.5% by weight, more preferably 0.8 to 1.0% by weight, of at least one organic solvent which has a phenyl group in the molecule. In a further preferred embodiment of the present invention, the compositions (Ind) and (E) used according to the invention contain, in each case based on their weight, independently of one another, 0.1 to 3% by weight, preferably 0.5 to 2.5% by weight, more preferably 0.8 to 1.0% by weight, of at least one organic solvent selected from phenoxyethanol, benzyl alcohol and mixtures thereof.

Compositions (Ind) and (E) which are particularly preferably used according to the invention contain, independently of one another, at least one aliphatic solvent selected from C1-C4-alkanols and C2-C4-polyols, in particular selected from ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerin and 1,3-butylene glycol, and mixtures of these solvents, but only in a total amount of 0.01-8% by weight, preferably 0.1-6% by weight, particularly preferably 0.5-4% by weight, in each case based on the weight of the compositions (Ind) or (E).

Other compositions (Ind) and (E) which are particularly preferably used according to the invention contain, independently of one another, no aliphatic solvent selected from C1-C4-alkanols and C2-C4-polyols.

Powders from the leaves of Indigofera tinctoria that are preferably used according to the invention react approximately neutrally in aqueous dispersion, with slight deviations between a slightly acidic and a slightly alkaline pH range. A 5% by weight dispersion of ground leaves of Indigofera tinctoria in deionized water has a pH of 6.6 to 7.4, preferably 6.8 to 7.2, particularly preferably 6.9 to 7.1, in each case measured at 20°C. Colorants preferred according to the invention have a pH of 2.0 to 10.0, preferably from 4.0 to 9.0, particularly preferably from 5.0 to 8.0, extremely preferably from 6.6 to less than 7.5, in each case measured at 20°C.

In a preferred embodiment of the dyeing process according to the invention, the desired pH value is set using an acid or a base. Preferred acids are selected from citric acid, lactic acid, gluconic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, galactaric acid (mucous acid), tartaric acid, malic acid, sulfuric acid and phosphoric acid, and mixtures of these acids. Preferred bases are sodium hydroxide, potassium hydroxide, arginine, lysine, monoethanolamine, triethanolamine, 2-amino-2-methylpropan-1-ol, and mixtures of these bases.

In a further preferred embodiment of the dyeing process according to the invention, the desired pH value is adjusted with the aid of a buffer system selected from a mixture of a medium-strong or weak acid with its conjugated or corresponding base (or the respective salt) and a mixture of a medium-strong or weak base with its conjugated or corresponding acid.

According to the invention, preferred suitable corresponding acid-base pairs are those which stabilize the compositions used according to the invention in the pH range from 2.0 to 10.0, preferably from 4.0 to 9.0, particularly preferably from 5.0 to 8.0, extremely preferably from 6.6 to less than 7.5, each measured at 20°C.

Particularly preferred buffer systems according to the invention are selected from

- ammonia/ammonium salt mixtures, wherein the ammonium salt is preferably selected from ammonium chloride, ammonium bromide, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate, ammonium carbonate, ammonium nitrate, ammonium acetate, ammonium glycolate, ammonium gluconate, ammonium tartrate, ammonium lactate, and mixtures of these ammonium salts, particularly preferably selected from ammonium chloride, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate and ammonium carbonate, extremely preferably selected from ammonium chloride,

- mixtures of hydrogen phosphate and dihydrogen phosphate, in particular the alkali metal salts of hydrogen phosphate and dihydrogen phosphate, particularly preferably the sodium salts and/or the potassium salts of hydrogen phosphate and dihydrogen phosphate,

- mixtures of alkali metal hydrogen carbonate with alkali metal carbonate, in particular mixtures of sodium or potassium hydrogen carbonate with sodium or potassium carbonate, - mixtures of citric acid and its salts, in particular the alkali metal citrates, in particular the sodium salts, in particular trisodium citrate,

- mixtures of tartaric acid and its salts, in particular alkali metal tartrates, in particular potassium salts, in particular potassium hydrogen tartrate,

- Mixtures of phthalic acid and its salts, in particular potassium salts, in particular potassium hydrogen phthalate,

- mixtures of lactic acid and its salts, in particular lactic acid/sodium lactate mixtures,

- Mixtures of gluconic acid and its salts, in particular gluconic acid/sodium gluconate mixtures,

- Mixtures of succinic acid and its salts, in particular the sodium salts, in particular sodium hydrogen succinate and disodium succinate, and

- mixtures of malic acid and its salts, in particular the sodium salts, in particular sodium hydrogen malate and disodium malate, and

- Ammonia/ammonium salt mixtures, wherein the ammonium salt is preferably selected from ammonium chloride, ammonium bromide, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate, ammonium carbonate, ammonium nitrate, ammonium acetate, ammonium glycolate, ammonium gluconate, ammonium tartrate, ammonium lactate, and mixtures of these ammonium salts, particularly preferably selected from ammonium chloride, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate and ammonium carbonate, extremely preferably selected from ammonium chloride.

Other buffer systems, e.g. acetic acid/sodium acetate, are also suitable in principle according to the invention. However, due to the vinegar smell, such a buffer is not acceptable for the manufacture of a cosmetic market product.

In principle, other buffer systems are also suitable.

The specialist can find out the appropriate weights of buffer salts to set the desired pH value from the relevant manuals.

Examples of implementation

The embodiments presented below are intended to explain the subject matter of the invention in more detail without limiting it thereto. The dyeing for this invention was carried out on buffalo belly hair strands (brush body tied, approx. 8 cm free hair).

Preparation of the indican-containing component i) (composition (Ind)

95 ml of demineralized water were heated to approximately 95 °C. Then, 5 grams of ground leaves of Indigofera tinctoria L * were added (5% by weight of the total dispersion) and stirred for 5 minutes at approximately 95 °C.

* The following raw materials were used:

“C30-Indigo Leaves Powder” (Indigofera tinctoria L.y powdered leaves of Indigofera tinctoria pH of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C) or

“Indigo Powder” (Indigofera tinctoria L.y, powdered leaves of Indigofera tinctoria pH value of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C)

The dispersion was then cooled to room temperature (20°C) and filtered (filter: Whatman 10340810, viscose, pore size 100 pL, approx. 5 to 10 min, vacuum pump at approx. 500 mbar).

The filtrate was a brown, opaque solution without precipitate, with a pH of approximately 7.7 (20°C). This indican-containing component i) is hereinafter referred to as "Composition (Ind)".

Treatment of hair strands with composition (Ind) does not produce coloring under standard dyeing conditions (50 ml of composition (Ind) per 1 g of hair strand, dye for 30 minutes at room temperature (20°C) with stirring, rinse for 30 seconds under running deionized water, comb 20 times at 20°C, dry with hairdryer, distance: d = 10 cm, temperature: 80 ± 5 °C, comb 20 times).

Coloring according to the invention

The aqueous composition (E) according to the invention, which contains an enzyme mixture ii) according to the invention comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91), was obtained from Novozymes. This aqueous composition (E) is referred to below as "cellulase blend".

The enzyme mixture ii) in the “Cellulase Blend” from Novozymes comprised at least one endo-1,4-beta-glucanase (EC 3.2.1.4) in a total amount of 20 - 35 wt.%, at least one beta-glucosidase (EC 3.2.1.21) in a total amount of 15 - 25 wt.%, and at least one exo-1,4-beta-D-glucanase (EC 3.2.1.91) in a total amount of 25 - 35 wt.%, as well as at least- at least one other enzyme with cellulase activity in a total amount which adds up to 100% by weight to the amounts of the other enzymes, the amounts referring to the total weight of the enzyme mixture ii).

The enzyme active substance content in the raw material used, "Cellulase Blend" from Novozymes, which was available as an aqueous composition (E), was 190 pg/pl, i.e. 19% by weight. The "Cellulase Blend" also contained, based on its weight, 66% by weight of water and 15% by weight of a sucrose/D-glucose mixture.

To prepare the dye composition according to the invention, 49.95 ml of the composition according to the invention (Ind) were mixed with 50 pL of "cellulase blend", corresponding to a composition according to the invention (E). The dye composition according to the invention thus obtained had a total enzyme content of 0.019% by weight based on its weight.

Then a buffalo belly strand was placed in this dye composition according to the invention and dyed for 30 minutes at room temperature (20° C) while stirring (liquor ratio: 50 ml for 1 g of hair strand; pH: approx. 7.7, measured at 20° C). The dye solution was then washed off the strand (see above) and the strand was dried (see above).

The addition of the “cellulase blend” resulted in a blue color tone being visible on the hair fiber again after coloring (see Table 1).

Carrying out the control staining with a non-inventive indican-containing component that had not been heat-treated

To prepare a control dye composition not according to the invention, 2.5 g of indigo powder were dispersed in 47.5 grams of demineralized water (5% by weight of the total dispersion). After stirring the dispersion for 2 minutes, a buffalo belly hair strand (brush body tied round, approx. 8 cm of free hair) was placed in the control dye composition for 30 minutes while stirring (liquor ratio: 50 grams of dye composition to 1 gram of hair strand, pH value: 6.46 - 7.29 (20°C)). The control dye composition was then washed off the strands by rinsing the strands for 30 seconds under running deionized water and combing 20 times (20°C). The hair strands were then dried with a commercially available hair dryer at a defined distance (d = 10 cm) and a defined temperature (T = 80 ± 5 °C) by combing them 20 times.

The following plant raw materials were used:

“C30-Indigo Leaves Powder” (Indigofera tinctoria L.) powdered leaves of Indigofera tinctoria pH of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C) or “Indigo Powder” (Indigofera tinctoria L. , powdered leaves of Indigofera tinctoria pH value of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C)

Colorimetric measurements

All colorimetric measurements were carried out using the Spectraflash SF 600 colorimetric device from Datacolor.

The color difference, also known as dE or AE, can be easily determined colorimetrically using a colorimeter that measures colors in the L*, a*, b* color space, for example a Datacolor colorimeter, type Spectraflash SF 600.

The L*, a*, b* color space refers to the CIELAB color space. The L value represents the brightness of the color (black-white axis); the higher the L value, the brighter the color. The a value represents the red-green axis of the system; the higher this value, the more the color is shifted towards red. The b value represents the yellow-blue axis of the system; the higher this value, the more the color is shifted towards yellow.

The color shift AE, i.e. the color difference between two (hair) colors, for each of which an L*, a*, b* value combination was determined, is calculated according to the following formula:

AE = (AL ² + Aa ² + Ab ² ) ⁰⁵

The larger the value of AE, the more pronounced the color difference.

A D65 illuminant and diffuse/8° optical configuration were used for the spectrophotometer measurements. Spectral reflectance data for each sample from 380 nm to 700 nm were converted to colorimetric data using DCI Color software. Reflectance measurements were determined for each hair sample, with the average of 4 measurements recorded.

The colour difference (AE) between two strands with different colouring treatment was calculated according to the following formula:

Lv, av, bv: colorimetric values for colored strands

Ln, an, bn: Colorimetric values for undyed strand

The differences between the control strand and the strands with heat-deactivated and subsequently enzymatically reactivated indican-containing extract, corresponding to the mixture of composition according to the invention (Ind) and composition (E) immediately after dyeing (day 0) and 14 days after dyeing, are shown in Table 1 (mean values). Table 1: Colour comparison of indigo dyeing control and heat-treated composition (Ind) with reactivation by cellulase addition 0 days and 14 days after dyeing

The conversions of the L a b values into Pantone values and color names were carried out using the website https://www.e-paint.co.uk/convert-lab.asp.

Immediately after coloring, the mean values of the color difference between the two strands differ with an AE value of 11.9.

After 14 days, the mean values of both strands differ with an AE value of 19.3. The AE value between the color change of the control strands from day 0 to day 14 is 18.5. In addition, there was also a strong shift in the color impression: the strong change in the a value describes the shift in the color tone from green to red; the strong change in the b value describes the shift in the color tone from yellow to blue. The control coloring therefore showed a very unstable coloring result, the final coloring result of which only appeared after 14 days.

The color change in the hair when dyed according to the invention from day 0 to day 14 was determined with an AE value of only 1.6, which is almost imperceptible to the human eye. There were also hardly any changes in the color tone itself.

By initially deactivating the enzyme activity present in indican-containing, indigo-producing plant parts and then adding a selected enzyme mixture with cellulase activity shortly before use, the dyeing performance of the initially non-dyeing composition (Ind) can be restored. On the other hand, the strong time-dependent color change from an initial turquoise-blue tone to a redder and darker shade is prevented. Improvement over the dyeing process known from US20040055096A1

The dyeing experiments presented below show that the enzymatic reactivation of the heat-treated indican-containing component according to the invention i) brings about an improvement in the color development ("different color impression") compared to an enzymatic reactivation with plant-specific enzymes.

Instead of the enzyme mixture ii) used according to the invention, an extract of plant-own enzymes from powdered leaves of Indigofera tinctoria was used, wherein the plant-own enzymes from powdered leaves of Indigofera tinctoria were obtained by extraction with ethanol (96 vol%).

Extraction with ethanol as the preferred solvent allows the separation of the ethanol-soluble indican from all ethanol-insoluble components of the plant, here Indigofera tinctoria. Among the ethanol-insoluble components of the plant are the plant's own cellulases.

The ethanolic extraction was carried out using a Soxhlet apparatus. For this, 15 grams of leaf powder from Indigofera tinctoria in a commercially available tea bag were placed in a 100 mL extraction thimble. The 250 mL extractor was used, a system pressure of 190-160 mbar was applied and the cooling liquid was kept at a temperature of less than 5 °C. The heating bath was set to 60 °C and 250 mL of 96 vol.% ethanol was added to the system. 50 extraction cycles were then run.

The Indigofera leaves extracted with ethanol were dried. They still contained the plant's own cellulases, but were largely free of indican. This dried extraction residue is called Indigo B powder.

Carrying out the comparison staining with a non-inventive enzyme-containing component and the heat-treated indican-containing component

This indican-containing component i) is hereinafter referred to as ‘Composition (Ind)’.

To prepare the comparative dye composition not according to the invention, 1 gram of Indigo B powder was dispersed in 49 grams of the composition (Ind), i.e. a heat-treated indican-containing component i), the preparation of which was described above. After stirring the dispersion for 2 minutes, a strand of buffalo belly hair (brush body tied round, approx. 8 cm of free hair) was placed in this comparative dye composition for 30 minutes and The dye composition was stirred (liquor ratio: 50 grams of dye composition to 1 gram of hair strand, pH value: 7.3 - 7.7, measured at 20°C). The comparison dye composition was then washed off the strands by rinsing the strands under running deionized water for 30 seconds and combing them 20 times (20°C). The hair strands were then dried with a commercially available hair dryer at a defined distance (d = 10 cm) and a defined temperature (T = 80 ± 5 °C) and combing them 20 times.

The test strands were colorimetrically measured using the method described above. The results are shown in Table 2.

Bereits direkt nach der Färbung (Tag 0) misst man für die unterschiedlich reaktivierten Strähnen einen Farbabstand zueinander von AE = 7,4. Nach 14 Tagen unterscheiden sich die Mittelwerte beider Strähnen mit einem AE-Wert von 6,7. Table 2: Color comparison of heat-treated indican component i) (Ind) with reactivation by addition of enzyme mixture according to the invention or with comparative reactivation by lndigofera-e'\ger\er\ enzyme extract (Indigo B powder), 0 days and 14 days after coloration

Immediately after coloring (day 0), a color difference of AE = 7.4 is measured for the differently reactivated strands. After 14 days, the mean values of both strands differ with an AE value of 6.7.

On the other hand, the temporal color change of both strands differs.

The color achieved with the indican-containing component i) reactivated according to the invention changed between day 0 and day 14 by only AE = 1.6, which is almost imperceptible to the human eye. There were also hardly any changes in the color tone itself.

In contrast, the coloring achieved with the indican-containing component i) reactivated with the plant's own enzyme extract changed by AE = 14.9 within 14 days. In addition, there was also a strong shift in the color impression: the strong change in the a value describes the shift in the color tone from green to red; the strong change in the b value describes the shift in the color tone from yellow to blue. The coloring of the indican-containing component i) reactivated with the plant's own enzyme extract therefore showed a very unstable coloring result, the final coloring result of which only occurred after 14 days.

Claims

Patent claims 1. Agent for the non-oxidative coloration of keratin fibers, in particular human hair, containing i) at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, furthermore ii) an enzyme mixture comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91) and iii) water. 2. Agent for non-oxidative coloring according to claim 1, characterized in that the indican-containing component i) is selected from crushed parts of an indigo-producing plant and from indican-containing extracts of an indigo-producing plant and mixtures of these indican-containing components i). 3. Agent for non-oxidative coloring according to claim 2, characterized in that the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore Ci-C4-alkanols and C2-C4-polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerin and 1,3-butylene glycol, and mixtures of these extraction agents, in particular mixtures of water and at least one Ci-C4-alkanol, mixtures of water and at least one C2-C4-polyol, particularly preferably water/ethanol mixtures. 4. Agent according to one of claims 2 or 3, characterized in that the indigo-producing plant(s) is/are selected from at least one species of the following genera: - Indigofera, in particular Indigofera tinctoria, Indigo suffruticosa, Indigofera articulata, Indigofera arrecta, Indigofera heterantha “Gerardiana”, Indigofera argentea or Indigofera longiracemosa; - Isatis, especially Isatis tinctoria (woad); - Persicaria, especially Persicaria tinctoria (dyer's knotweed); - Wrightia, especially Wrightia tinctoria; - Calanthe, especially Calanthe veratrifolia; and - Baphicacanthus cusia, synonym Strobilanthes cusia, where the indigo producing plant(s) is/are preferably selected from the genus Indigofera and in particular from Indigofera tinctoria. 5. Agent according to one of claims 1 to 4, characterized in that the indican-containing component i) contains, based on its weight, 0.01 - 6 wt.% indican. 6. Agent according to one of claims 1 to 5, characterized in that the pretreatment of the at least one indican-containing component i) is carried out at a temperature in the range from 60 °C to 200 °C by heating, in particular by heating in a solvent, furthermore by treatment with steam or by dry heating, preferably for a period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extremely preferably 5 to 10 minutes. 7. Agent according to one of claims 1 to 6, characterized in that the extracted indican is separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, so that the indican-containing component i) is in powder form. 8. Agent according to one of claims 1 to 7, characterized in that the enzyme mixture ii) contains at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4) in a total amount of 20 - 35 wt. %, at least one beta-glucosidase (E.C. 3.2.1.21) in a total amount of 15 - 25 wt. %, and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91) in a total amount of 25 - 35 wt. %, the amounts referring to the total weight of the enzyme mixture ii). 9. Agent for non-oxidative coloring according to one of claims 1 to 8, characterized in that the enzyme mixture ii) at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4) in a total amount of 20 - 35 wt. %, at least one beta-glucosidase (E.C. 3.2.1.21) in a total amount of 15 - 25 wt. %, and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91) in a total amount of 25 - 35 wt. %, and at least one further enzyme with cellulase activity in a total amount which, with the amounts of the other enzymes, adds up to 100 wt. %, wherein the amounts refer to the total weight of the enzyme mixture ii). 10. Agent according to one of claims 1 - 9, characterized in that it contains, based on its weight, an enzyme mixture with cellulase activity which contains at least one endo-1,4-beta-glucanase (EC 3.2.1.4), at least one beta-glucosidase (EC 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (EC 3.2.1.91) in a total amount of 0.0001 - 1 wt.%, preferably 0.001 - 0.1 wt.%, more preferably 0.002 - 0.05 wt.%, particularly preferably 0.003 - 0.04 wt.%, extremely preferably 0.01 - 0.03 wt.%. 11. Agent according to one of claims 1-10, characterized in that it contains, based on its weight, 19.9-95.0 wt.%, preferably 30.0-90.0 wt.%, particularly preferably 50.0-88.0 wt.%, extremely preferably 60.0-85.0 wt.%, of water. 12. Kit for the non-oxidative coloring of keratin fibers, in particular human hair, with natural dyes, which comprises the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, and ii) an aqueous composition (E) which contains an enzyme mixture comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91), characterized in that the compositions i) (Ind) and ii) (E) are designed as described in any one of claims 1-10. 13. A method for non-oxidative coloring of keratin fibers, in particular human hair, comprising the following method steps in the order given: a) providing a composition (Ind) which contains at least one indican-containing component which has been pretreated at a temperature in the range of 60 °C to 200 °C, and providing an aqueous composition (E) which contains an enzyme mixture comprising at least one endo-1,4-beta-glucanase (E.C. 3.2.1.4), at least one beta-glucosidase (E.C. 3.2.1.21) and at least one exo-1,4-beta-D-glucanase (E.C. 3.2.1.91), b) mixing the compositions (Ind) and (E) with one another to produce a colorant, c) applying the colorant obtained in step b) to the keratin fibers, wherein the application of the in step b) the dyeing agent obtained is applied to the keratin fibers preferably within 1 second to 30 minutes, particularly preferably within 10 seconds to 15 minutes after completion of step b), d) allowing to act for a time of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 15 to 30 minutes, e) rinsing the keratin fibers with water, f) optionally drying the keratin fibers. 14. Dyeing process according to claim 13, characterized in that the process is free of hydrogen peroxide and/or free of mordants and/or characterized in that the keratin fibers are dyed in a period of up to 7 days before and up to 7 days after were not treated with a mordant prior to the application of a dye according to the invention. 15. Dyeing process according to one of claims 13 or 14, characterized in that the colorant obtained in step b) is an agent according to one of claims 1 - 11.