WO2008092841A1

WO2008092841A1 - Method of detecting previous straightening by the action of an alkaline agent

Info

Publication number: WO2008092841A1
Application number: PCT/EP2008/051007
Authority: WO
Inventors: Marine Peron; Joel Bover
Original assignee: L'oreal
Priority date: 2007-01-29
Filing date: 2008-01-29
Publication date: 2008-08-07
Also published as: FR2911778B1; FR2911778A1

Abstract

The present invention relates to a method of de tecting keratin fibre(s) that have undergone a previous alkaline straighten ing or smoothing treatment. The method of the invention comprises bringing the keratin fibre(s) into contact with an extraction solution free from thiourea or one of its derivatives and co mprising at least urea or one of its derivatives, mixed with at leas t one reducing agent other than thiourea, in effective conditions for dissolving keratino us proteins, and the qualitative and/or quantitative assay of the protei ns in said extraction solution, and a solution heating stage after immersion of the keratin fibres.

Description

METHOD OF DETECTING PREVIOUS STRAIGHTENING BY THE ACTION OF

AN ALKALINE AGENT

The present invention relates to a method for quickly detecting, on keratin fibres, previous treatment, notably of the straightening or smoothing type, by alkaline technology. It also aims to propose a kit that can be used for applying said method.

By "keratin fibres", we mean, according to the invention, fibres of human or animal origin such as the hair, body hair or bristles, eyelashes, wool, angora, cashmere or fur. Although the invention is not limited to particular keratin fibres, reference will be made more particularly to the hair. Hair consists of proteins to 85-90%. These proteins belong to two main groups: the intermediate filaments or keratin, and KAP (Keratin Associated Proteins). Overall, the protein composition of hair is rich in cysteine in its dimeric form: cystine.

The properties of shaping of hair result notably from its protein architecture.

Thus, permanent shaping of hair generally makes use of chemo-cosmetologic effects, which occur at the level of the polypeptide chains of the hair proteins. These chains contain sulphur-containing amino acids, including cysteine in its dimeric form

(disulphide): cystine (about 15% in g/100g of total amino acids). These disulphide bridges can be reduced to thiols, then reoxidized after shaping, so as to make said shaping permanent. This reaction constitutes one of the cosmetic principles of permanent changes of hair shape.

According to a first technique, the disulphide bridge is opened temporarily through the action of a reducing agent, generally a sulphite in the form of an alkaline salt or a thiol such as thioglycolic acid. This rupturing action has the effect of breaking the disulphide bridges, thus ensuring flexibility and a capacity for movement of the polypeptide chains relative to one another. The bridges are then reformed at other points after the desired shaping, by fixation with an oxidizing agent.

A second technique comprises carrying out an operation called lanthionization, by means of a composition containing a base belonging to the hydroxide class. It leads to replacement of some of the cystines with lanthionines (monosulphide linkages -CH₂-S- CH₂). This operation of lanthionization first involves a reaction of β-elimination on the cystine, by means of a hydroxide ion, then reacting the dehydroalanine thus obtained with a thiol group. In contrast to the first technique, which employs a reducing agent, this lanthionization technique does not require a fixation stage. Formation of the lanthionine bridges is irreversible. This technique therefore provides, in a single stage, straightening or smoothing of keratin fibres.

Accordingly, the two techniques mentioned above, although leading to the same result, namely permanent shaping, affect the structure of the hair in different ways. In particular, a professional in the area of hair care knows that the causticity of the alkaline derivatives employed according to the second technique may, in some cases, have a significant effect on the condition of the hair, notably making it much more brittle. This brittleness is accompanied by a loss of substances and can even lead to breaking of the hair, notably if it is exposed to successive treatments of this type.

Methods already exist for characterizing previous alkaline straightening, notably by detecting the presence of lanthionine within the keratin fibres. Said detection is carried out either by determination of lanthionine after acid hydrolysis and amino acid analysis, or by analysis by confocal Raman spectroscopy. Moreover, several methods for collecting the proteins have already been proposed. Thus, for extracting the cortical proteins, it is known that it is possible to release the proteins from the hair in an aqueous urea solution, then cut the disulphide bridges with 2-mercaptoethanol or thioglycolic acid (MacLaren J.A. & Kilpatrick D. J., Aust. J. Biol. Sci., 21, 805-813 [1968]). More recently, document JP 2002-114798 A proposed combining particular urea compounds in the presence of a reducing agent. The method of collection more particularly described envisages eluting and collecting the keratin proteins from the microfibrils and the matrix making up the cortical portion of the hair by treating the latter with a mixture of urea and thiourea, at a ratio between 5/1 and 1/2, and collecting the cuticle portion, with its form preserved, from the elution residue. Patent application EP 1 671 622 describes a method of detecting previous alkaline straightening of the hair using a solution containing urea and thiourea. This method has the drawback that it uses thiourea.

It is clear that all of these techniques, although very reliable in diagnostic terms, prove to be very demanding in terms of application, and therefore costly. Therefore the professional still has a need for a means that is reliable, quick, and does not require elaborate scientific apparatus, for identifying the "past history" of hair for the best possible assessment of the compatibility of subsequent treatment(s) with its present condition, notably with respect to proteins.

In the sense of the invention, the protein condition of keratin fibres denotes their protein concentration according to a qualitative scale, rather than quantitative, in other words their protein richness. The present invention therefore aims to propose a useful, rapid method for detecting whether a keratin fibre such as the hair has already been exposed to an alkaline treatment such as straightening by alkaline technology, regardless of the degree of sensitization of said keratin fibre.

Usually, hair described as "sensitized" is hair that has undergone at least one oxidizing treatment, for example during colouring or decolouring, in contrast to natural hair.

Thus, the present invention relates to a method of detecting keratin fϊbre(s) that have undergone a previous alkaline straightening or smoothing treatment comprising at least bringing the keratin fϊbre(s) into contact with an extraction solution that is free from thiourea or one of its derivatives and includes at least urea or one of its derivatives, mixed with at least one reducing agent other than thiourea, in effective conditions for dissolving keratinous proteins, and the qualitative and/or quantitative assay of proteins in said extraction solution, and a stage of heating the solution after immersion of the keratin fibres therein at a temperature above 80⁰C. The present invention relates, according to another of its aspects, to a kit that can be used for characterizing the protein condition of keratin fibre(s), and notably for detecting, for keratin fibre(s), previous treatment by alkaline technology, said kit comprising at least a solution free from thiourea or one of its derivatives and containing urea or one of its derivatives, a reducing agent other than thiourea or its derivatives, at least one compound for use as a colorimetric indicator for qualitative and/or quantitative assay of keratinous proteins.

In the sense of the invention, the expression "qualitative assay of proteins" denotes an assay that aims to characterize the relative abundance of the proteins without accurately evaluating their concentration. In other words, the assay enables its user to estimate the protein richness of the extraction solution. A quantitative assay aims, in contrast, to determine the exact protein concentration in the extraction solution, as illustrated in the examples presented below.

Advantageously, the method according to the invention makes it possible to characterize the existence of previous treatment by alkaline straightening regardless of what sensitization treatment or treatments the hair in question has undergone previously or subsequently. The method according to the invention proves effective on all kinds of hair, namely natural or sensitized.

Furthermore, it allows previous alkaline straightening to be detected without employing thiourea or its derivatives.

Solution for extracting keratinous proteins

As stated previously, the mixture employed for extracting the proteins is a solution free from thiourea or its derivatives, combining at least urea or one of its derivatives and a reducing agent other than thiourea. In the sense of the invention, derivatives of thiourea or of urea refer to their derivatives of the N-alkylated and N-hydroalkylated type. These alkylated units can be of Ci to Cio and preferably Ci to C₄ and can, if necessary, be substituted, provided that said substituents are not able to affect the reactivity of the urea or thiourea.

More particularly, urea or its derivatives are generally present at a rate from 20 to 80 wt.%, in particular from 30 to 70 wt.% relative to the total weight of the extraction solution.

These compounds are combined with an effective amount of at least one reducing agent other than thiourea or its derivatives.

In the sense of the invention, a reducing agent denotes a compound that is able to cut the disulphide bridges in keratin fibres and, more particularly, in order to reduce them.

Thus, notably, derivatives of the thiol, phosphine and sulphite type are suitable.

According to a particular embodiment of the invention, it is a reducing agent of the thiol type. Such agents are notably selected from dithiothreitol, thiogly colic acid or thiolactic acid and their ester and amide derivatives, notably glycerol monothioglycolate, cysteamine and its Ci-C₄ acylated derivatives such as N-acetyl-cysteamine or N-propionyl- cysteamine, cysteine, N-acetyl-cysteine, thiomalic acid, pantetheine, 2,3- dimercaptosuccinic acid, sulphites or bisulphites of an alkali metal or alkaline-earth metal, N-(mercaptoalkyl)-ω-hydroxyalkylamides such as those described in patent application EP-A-354 835, N-mono or N,N-di-alkyl-mercapto-4-butyramides such as those described in patent application EP-A-368 763, aminomercaptoalkylamides, such as those described in patent application EP-A-432 000, derivatives of N-(mercaptoalkyl)succinamic acids and of N-(mercaptoalkyl)succinimides such as those described in patent application EP-A-465 342, alkylamino mercaptoalkylamides such as those described in patent application EP-A- 514 282, azeotropic mixture of 2-hydroxypropyl thioglycolate and (2-hydroxy-l- methyl)ethyl thioglycolate such as those described in patent application FR- A-2 679 448, mercaptoalkylaminoamides such as those described in patent application FR- A-2 692 481, N-mercaptoalkylalkanediamides such as those described in patent application EP-A-653 202, as well as derivatives of formamidine sulphinic acid such as those described in application PCT/USOl/43124, filed by the applicant. This reducing agent is of course used at a concentration sufficient to obtain the desired effect, namely reduction of the disulphide bridges. For obvious reasons, said amount can vary significantly depending on the nature of the reducing agent in question.

In particular, said concentration can be of the order of 0.01 to 2 M, in particular 0.03 to 0.5 M and more particularly 0.04 to 0.1 M. 2-Mercaptoethanol, dithiothreitol (DTT), thioglycolic acid, one of their derivatives or mixtures thereof, are more particularly suitable as reducing agent.

It is also possible to envisage combining, with this reducing agent, one or more compounds known to have the capacity of reinforcing its effectiveness. As an illustration, said compounds can be in the form of an SiO₂ZPDMS (polydimethylsiloxane) mixture, dimethylisosorbitol, pyrrolidone, N-alkylpyrrolidone, thiamorpholinone, alkyleneglycol alkyl ethers, dialkyleneglycol, C3-C6 alkane diols or mixtures thereof.

Said compounds can be present at a rate from 0.001 to 10 wt.% relative to the weight of the composition.

In addition to urea or its derivatives, and the reducing compound, the solution intended for dissolving the keratinous proteins can also contain a surfactant of the non- ionic, anionic, cationic or amphoteric type. It can notably comprise alkylsulphates, alkylbenzenesulphates, alkylethersulphates, alkylsulphonates, alkylbetaines, oxyalkylenated alkylphenols, alkanolamides of fatty acids, esters of oxyalkylenated fatty acids, as well as oxyalkylenated fatty alcohols and alkylpolyglucosides.

The extraction solution can also include one or more acidifying or alkalizing agents and in particular TRIS (TRIS[hydroxymethyl]aminomethane). Of course, these agents are selected in such a way as not to adversely affect the desired reactivity with respect to the keratinous proteins.

As stated previously, the method according to the invention involves bringing keratin fibres into contact with an extraction solution according to the invention.

Generally, a sample such as a thin lock of hair, corresponding to about ten hairs, is sufficient for carrying out the method according to the invention.

More precisely, the weight ratio of the keratin fibres to be analysed to the extraction solution varies from 0.2 to 100 mg/ml, notably from 1 to 50 mg/ml and more particularly is of the order of 10 mg/ml.

The quantity of keratin fibres is generally of the order of 50 mg. As for the volume of the extraction solution, it is generally of the order of 5 ml.

The thin lock of keratin fibres is brought into contact with the extraction solution, generally by direct immersion in a container, of the tube or dish type for example, containing said solution. The keratin fibres can advantageously be cut up beforehand into sections of reduced size, from 2 to 3 mm for example. This thin lock is generally taken by cutting from the hair that is to be characterized for previous straightening treatment by alkaline technology.

The inventors demonstrated that the reactivity of the extraction solution is increased significantly if the reaction of solubilization of the proteins is carried out at a temperature greater than or equal to 100⁰C. It is to be understood that said temperature must be compatible with the hair and must not cause its destruction.

Said heating can be applied by any conventional method. It can thus be heating on a water bath, with a bottle heater, microwave heating, ultrasonic heating and/or using a hair dryer.

Of course, the time required for extraction can vary significantly from a minute, or even a second, to several hours depending on the conditions adopted, notably with respect to temperature, for carrying it out.

In general, extraction of the proteins takes around 15 minutes, when the reaction is carried out at 100⁰C on a water bath, or some seconds when microwave heating is used.

This heating can moreover be carried out with simultaneous agitation of the vessel containing the keratin fibres and the extraction solution. Measurement or evaluation of the relative abundance of the proteins dissolved in the extraction solution can of course be performed using various methods.

Thus, we can envisage characterizing the abundance of proteins in the solution by a method of determination of the physical type, for example light scattering or turbidity, of the chemical or physicochemical type, or of the immunological type. However, since this test is more particularly intended for hairdressing professionals, it is advantageous to combine this method of extraction with detection by colorimetry, notably based on the method of Bradford or Lowry. Such tests are commonly used for protein assay.

According to this alternative, all or a portion of the treatment solution, which may contain proteins in dissolved form, can be put in contact with an effective amount of a compound, also called "colorimetric indicator " hereinafter, which is able to interact with the latter and display said interaction by a colorimetric method. It can either be the appearance of a colour, or a change, or even disappearance, of its original colour.

This bringing into contact of the extraction solution with a colorimetric indicator can in particular be performed by putting a defined sample of said solution in a solution containing the colorimetric indicator. The bringing into contact of the colorimetric indicator with some or all of the extraction solution may require the prior dilution of said extraction solution. This option depends of course on the nature of the colorimetric indicator and is generally specified in the instructions for use of the colorimetric indicator. We may also envisage an embodiment according to which the colorimetric indicator is fixed, notably by adsorption, on a solid support for example of the imaging strip type, for example of pH paper. The bringing into contact is then effected by impregnation of the support with some or all of the extraction solution.

The intensity of colour obtained following bringing into contact has the further advantage that it can in parallel be an indicator of the protein concentration, provided corresponding calibration is available with the coloured indicator used.

According to a particular embodiment of the invention, the indicator is moreover based on an acid solution of Coomassie blue G-250, which can vary in terms of shades of colour over a colour range from 465 μm to 585 μm, when it is combined with proteins.

As an illustration of a commercial colorimetric test suitable for the invention, we may particularly mention that distributed by the company BIO RAD under the name BIO RAD PROTEIN ASSAY^®, a commercial kit for assaying proteins based on Bradford's method.

For the reasons mentioned previously, this detecting method of the colorimetric type is particularly interesting in terms of ease of manipulation and quick read-out. Furthermore, it may be desirable to have, in parallel, a colorimetric standard for assessing the relative abundance of proteins. This standard can be provided by a mixture of keratinous proteins of a defined concentration and in purified form, notably extracts of natural hair. However, in the absence of such a reference, it is possible to use any other protein as a relative standard. For obvious reasons, it is advantageous to select a reference protein that will give a depth of colour close to that of the protein to be determined.

Proteins of the bovine serum albumin or bovine γ-albumin type, which are proteins commercially available in purified forms, are suitable, notably, as standard proteins. As will be seen from the examples presented below, the detection of previous alkaline straightening or smoothing treatment is characterized by the presence of a much reduced amount, or even none at all, of proteins in dissolved form. Conversely, in the absence of such a prior history, the method according to the invention makes it possible to characterize, or even quantify, the presence of a significant amount of keratinous proteins in the extraction solution. Said presence can be reflected, in the case of a means of detection by colorimetry, by the display of a colour whose intensity is proportional to the quantity of dissolved proteins.

The invention also relates to a kit that can be used for characterizing the protein condition of keratin fibres, and notably can be used for characterizing previous straightening or smoothing treatment by alkaline technology, comprising at least: a solution free from thiourea or its derivatives and comprising at least urea or derivatives thereof a reducing agent other than thiourea, and at least one compound for use as a colorimetric indicator for qualitative and/or quantitative assay of the keratinous proteins extracted.

All of the constituents making up the solution intended for extracting the proteins can be packaged according to a first variant in one and the same container, or according to a second variant in separate containers.

According to this second alternative, the urea solution on the one hand, and the reducing agent on the other hand, are packaged separately. In this case, the respective dedicated containers can be configured so as to be suitable for mixing all of their components prior to carrying out the test. For example, it can be envisaged that these two containers are combined so as to form a single container at the moment of carrying out the test.

According to a first embodiment, the container with the solution free from thiourea or its derivatives and comprising urea or its derivatives and, if necessary, the reducing agent is configured so as to be suitable for the immersion of keratin fibres. In this case, it can be provided with a closure of the detachable cap type, intended to be removed at the moment of carrying out the test.

According to another embodiment, the kit can additionally include a separate container for receiving the urea solution, the reducing agent and the keratin fibres in an unspecified order.

Whatever the embodiment, the container intended for immersion of the thin lock during execution of the test is compatible with heating at a temperature above 80⁰C, and even of the order of 100⁰C. It can for example be configured for the water-bath, bottle heater, or microwave type of heating. With regard to the colorimetric indicator, it is generally packaged in solution in its own specific container.

All of the containers making up the kit are closed during packaging. The closures are arranged so as to allow easy opening at the moment of carrying out the test and, if applicable, controlled release of their contents, for example by fitting a dropping nozzle. This last-mentioned option is notably of interest for the stage of detection by colorimetry.

The kit according to the invention also includes, advantageously, instructions for use, which the user is to follow for carrying out the test.

In general, the procedure that is to be followed can involve the following stages: bringing keratin fibres, more particularly a thin lock of hair, generally precut to sections of 2 to 3 mm, into contact with the urea solution and the reducing agent, these two components of the extraction solution having been mixed together beforehand, or they are mixed in the presence of said keratin fibres, exposure of the container with the aforesaid mixture to a source of heat, if necessary with stirring, so as to heat the extraction solution to a temperature above 80⁰C or even 100⁰C for a specified period, for example about 15 minutes for heating at 100⁰C on a water bath, or a few seconds for microwave heating. if necessary, dilution of the extraction solution, at the end of said heating stage, introduction of a given amount of this solution in the container with the colorimetric indicator or conversely of a given amount of the colorimetric indicator in said extraction solution, and reading of the result, referring to a colorimetric standard, if applicable. Said colorimetric standard can be supplied in the kit or can be packaged with the kit. We can also envisage providing the kit with a colour chart to permit qualitative and/or quantitative assay of the proteins extracted in relation to the intensity of colour obtained at the end of the test.

The kit according to the invention can also contain reference keratin fibres to guide the user on the size of the sample of keratin fibres that should be taken, and notably of the hair to be tested. The kit can be for single use, or conversely it can include sufficient amounts of reagents for the characterization of several thin locks.

Finally, the kit can be designed to be used without reference proteins to be tested in parallel. In fact, the absence of proteins in the case of alkaline treatment is reflected in absence of a signal in comparison with the abundant presence of proteins in the absence of previous alkaline treatment. According to this option, reading of the result can be envisaged as being reflected instantly by a blue/brown or yes/no or +/- message, giving an unambiguous result respectively for an alkaline/non-alkaline previous history of the fibres tested.

EXAMPLES

All of the tests described below were carried out using the following keratin fibres, treated by the following straightening technologies:

The hair used in the examples is either Caucasian Hair, or African hair.

TECHNOLOGY FOR ALKALINE STRAIGHTENING (TA):

TAG: Alkaline straightening treatment based on Guanidine carbonate. TAL: Alkaline straightening treatment based on Lithine (lithium hydroxide).

TAS: Alkaline straightening treatment based on Soda (sodium hydroxide)

TECHNOLOGY FOR NON-ALKALINE STRAIGHTENING (TT): TT = Thiol Treatment for straightening based on thioglycolic acid Cysteine = Straightening treatment based on cysteine

Urea = Straightening treatment based on urea

EXTRACTION SOLUTION

The reducing solution used at the rate of a volume of 5 ml per test is prepared from the following compounds at the stated concentrations:

- Urea (7M) marketed by BIORAD reference: 1610730.

- Thiourea (2M) marketed by FLUKA reference: 88810,

- DTT (Dithiothreitol) (0.05M) marketed by SIGMA reference: D-9779,

- TRIS (0.05M) marketed by ALDRICH reference: T8,760-2 - Milli-Q water.

METHOD OF DETECTION

As for the method of detection used for characterizing the dissolved proteins, it is a colorimetric method based on Bradford's method (Bradford, Anal. Biochem. 72, 248, 1976).

More particularly, the test used is the Bio-Rad Protein^® test marketed by the company BIO RAD and using Coomassie Blue^® as colorimetric indicator. The calibration protein used is that recommended by the test, namely the protein from bovine serum albumin (BSA) marketed by CALBIOCHEM under the commercial reference 12659.

METHOD OF EXTRACTION

The extraction procedure followed for all of the tests is as follows: A thin lock of hair fibres (corresponding to about 50 mg) is cut into 2 to 3 mm sections with scissors and is brought into contact with 5 ml of the extraction solution described previously. This bringing into contact is carried out at a temperature of 100⁰C, which is provided by heating the extraction medium on a water bath for 15 minutes. The whole is kept stirred by means of a magnetized bar throughout extraction.

At the end of extraction, the proteins in the supernatant are assayed by means of the Bio Rad Protein Assay^® commercial colorimetric kit.

This is carried out by adding 30 μL of the supernatant to 5 ml of Bradford's colorimetric reagent (diluted beforehand to 1/5 and filtered according to the instructions given in the BIO RAD kit). The colour is left to develop for a few minutes. The results are read as follows: if a colour develops (ranging from brown to blue), the hair has not undergone alkaline straightening, - if there is little if any colour change, the hair has undergone alkaline straightening.

Based on a colour chart previously established in relation to concentrations of proteins obtained with various treatments previously carried out on hair, it is possible to read the result visually and answer the question of whether or not there is a previous history of alkaline straightening for the hair tested.

Based on a reference range of proteins, it is possible to quantify the proteins in the extraction solutions and calculate a degree of extraction relative to the mass of hair used. The results are shown in the tables given below. The results are expressed in the tables in g of proteins extracted per 100 g of dry hair, and referring to the reference protein used: bovine serum albumin (BSA).

Example 1: Protein extraction tests were carried out using a thiourea-free extraction solution that contained urea (invention) and an extraction solution comprising urea and thiourea (comparative) on hair that had previously undergone straightening according to alkaline technology or thiol technology, the hair being held on a water bath at 100⁰C for 15 minutes. The results represent the degree of extraction of the proteins, expressed in g of proteins per gram of hair and relative to a range of the reference protein used: BSA (bovine serum albumin), carried out in the extraction solution

These results show that the thiourea-free solution of the invention is just as effective as the comparative composition comprising urea and thiourea. Moreover, the discrimination with respect to alkaline straightening treatment is maintained.

Example 2: A thiourea-free composition illustrating the invention and comprising urea 8M and dithiothreitol 0.05M was used for extracting the proteins from different types of hair straightened in different ways (25 mg of hair for 15 minutes on a water bath with stirring at 100⁰C): The results presented in the following tables show that the composition of the invention makes it possible to discriminate previous alkaline treatment from all of the other treatments tested: thiol (TT and cysteine) or urea straightening treatments, as well as natural untreated hair.

Thus, in Table 1 below, all the tests show that the extraction solution does not change colour but remains brown as initially before extraction of the hair (degree of extraction relative to the range of BSA is less than or equal to 4%). In Table 2, all the tests show that the extraction solution, initially brown, changes colour to blue (degree of extraction relative to the range of BSA is greater than or equal to 6%). TABLE 1

TABLE 2

Claims

1. Method of detecting keratin fϊbre(s) that have undergone previous alkaline straightening or smoothing treatment comprising at least bringing the keratin fϊbre(s) into contact with an extraction solution free from thiourea or one of its derivatives and comprising at least urea or one of its derivatives, mixed with at least one reducing agent other than thiourea, in effective conditions for dissolving keratinous proteins, and the qualitative and/or quantitative assay of the proteins in said extraction solution, and a solution heating stage after immersion of the keratin fibres therein at a temperature above 80⁰C.

2. Method according to Claim 1, characterized in that it includes the qualitative assay of the dissolved proteins in the extraction solution.

3. Method according to Claim 2, characterized in that urea or its derivatives is present at a rate from 20 to 80 wt.%, in particular from 30 to 70 wt.% relative to the total weight of said extraction solution.

4. Method according to any one of the preceding claims, characterized in that said solution includes at least one reducing agent of the thiol type.

5. Method according to Claim 4, characterized in that said reducing agent is selected from thioglycolic acid or thiolactic acid and their ester and amide derivatives, notably glycerol monothioglycolate, cysteamine and its C1-C4 acylated derivatives such as N-acetyl-cysteamine or N-propionyl-cysteamine, cysteine, N-acetyl-cysteine, thiomalic acid, pantetheine, 2,3-dimercaptosuccinic acid, sulphites or bisulphites of an alkali metal or alkaline-earth metal, N-(mercaptoalkyl)-ω-hydroxyalkylamides, N-mono- or N,N-di- alkylmercapto-4-butyramides, aminomercaptoalkylamides, derivatives of N- (mercaptoalkyl)succinamic acids and of N-(mercaptoalkyl)succinimides, alkylaminomercaptoalkylamides, the azeotropic mixture of 2-hydroxypropyl thioglycolate and (2-hydroxy-l-methyl)ethyl thioglycolate, mercaptoalkylaminoamides, N- mercaptoalkylalkanediamides, and derivatives of formamidine sulphinic acid.

6. Composition according to any one of the preceding claims, characterized in that said reducing agent is selected from 2-mercaptoethanol, dithiothreitol (DTT), thioglycolic acid, one of their derivatives or mixtures thereof.

7. Method according to any one of the preceding claims, characterized in that said reducing agent is present at a rate from 0.01 to 2 M, in particular from 0.03 to 0.5 M and more particularly from 0.04 to 0.1 M in the extraction solution.

8. Method according to any one of the preceding claims, characterized in that the keratin fibres and the extraction solution are brought into contact at a rate of 0.2 to 100 mg, and more particularly at a rate of 1 to 50 mg and notably at a rate of about 10 mg of keratin fibres of extraction solution.

9. Method according to any one of the preceding claims, characterized in that the keratin fibres are immersed in the extraction solution and the whole is heated at a temperature greater than or equal to 100⁰C.

10. Method according to any one of the preceding claims, characterized in that protein assay is performed by a method of chemical, physical, physicochemical or immunological assay.

11. Method according to any one of the preceding claims, characterized in that protein assay is performed by bringing some or all of the extraction solution into contact with at least one compound capable of reacting with said proteins and of displaying said interaction by a colorimetric method.

12. Kit that can be used for characterizing the protein condition of keratin fibre(s), comprising at least: a solution free from thiourea or one of its derivatives and comprising at least urea or one of its derivatives, at least one reducing agent other than thiourea or one of its derivatives, and at least one compound for use as a colorimetric indicator for qualitative and/or quantitative assay of the keratinous proteins extracted.

13. Kit according to Claim 12, characterized in that the reducing agent is as defined in Claims 4 to 7.

14. Kit according to either one of Claims 12 or 13, characterized in that it further comprises a container that is to receive the urea solution, the reducing agent and the keratin fibres.

15. Kit according to any one of Claims 12 to 14, characterized in that it further comprises a colorimetric standard and/or a colour chart permitting qualitative and/or quantitative assay of the proteins extracted in relation to the intensity of the colour obtained at the end of the test.