JP5448182B2 - Method for measuring hair damage by permanent agent using keratin film - Google Patents

Description

  The present invention relates to a method for measuring the degree of hair damage by a permanent agent using a keratin film, and in particular to improvement of its diagnostic ability.

Factors that damage hair include ultraviolet irradiation, air dust, dryer heat, combing friction, excessive hair washing, perms, use of dyes and hair dyes, and the like. In recent years, research on hair damage caused by various factors has been actively conducted, and it is required to measure the degree of damage.
Since hair is a biological sample that can be easily collected and is easy to process and treat, many techniques for measuring the degree of hair damage measure changes in physical properties of the hair that accompany hair damage. Among them, the elongation stress in the hair shaft direction is effective as an index of hair strength that decreases due to damage, and is widely used as a tensile strength test (see, for example, Patent Document 1). In order to measure the degree of damage using this technique, obtain healthy hair with little damage as an initial value or a comparison target, perform damage treatment on this hair, and quantify the degree of damage by the tensile strength. Is. As a further improved technique, a technique has been developed in which the hair is torn along the longitudinal direction to determine the tear strength, and the damage degree of the hair is evaluated based on the tear strength (see, for example, Patent Document 2). However, since these measurement methods measure changes in physical properties of hair, they are limited to damage accompanied by large changes in physical properties, and there is a problem that it is impossible to detect fine initial damage that does not involve changes in physical properties. there were.

  In order to solve these problems, the present inventors have developed a method for measuring changes in the amount of protein eluted with hair damage as an index other than the physical properties of hair (for example, see Patent Document 3). ing. This method is effective to detect the initial damage of hair that is not reflected in the physical property change. However, since this method detects a secondary phenomenon caused by denaturation of hair constituent proteins, more direct damage detection has been desired.

  Thus, the present inventors have proposed a method for measuring the quantification of carbonyl groups in hair caused by hair damage using a fluorescent dye as a method for directly detecting protein denaturation occurring in the hair. (For example, refer to Patent Document 4). This method can quantify initial damage that does not reflect on the physical properties of hair, and is a highly sensitive method for determining the degree of damage by using a fluorescent dye.

  However, since all of these methods require the initial value or the healthy hair to be compared, before the sensitivity of the measurement method, variation depending on the initial value or the history of damage of the healthy hair to be compared and individual differences Was a problem. There is no way to determine how much hair is damaged in appearance, and it is necessary to prepare hair with the same degree of damage as the detection sensitivity increases. However, there is actually no prescription for controlling this, and it has been a challenge to develop a treatment method that makes the damage history and individual differences uniform while maintaining the hair constituent proteins.

  As a solution to the above-mentioned problems, the present inventors have already reported a method for extracting a keratin protein which is efficient and suitable for analysis in a state as it is (see, for example, Patent Document 5). That is, the hair is treated with a specific urea compound in the presence of a reducing agent, and the keratin of the matrix containing the microfibrils and cell interstitial substances constituting the hair cortex site and its associated protein are collected and collected. The cuticle part maintaining the shape is collected from the residue after elution. Furthermore, the present inventors also provide a method for producing a molded article such as a film or a gel composed of keratin collected by further improving this method and its related protein (see, for example, Patent Document 6). . It has already been found that a film composed of keratin and its related protein, that is, a keratin film, retains hair constituent proteins without being denatured (see, for example, Non-Patent Document 1). Furthermore, the present inventors have evaluated the damage caused by the hair treatment agent from the measurement of the eluted protein by the surfactant and hair coloring agent treatment of the keratin film (Patent Document 7).

Japanese Patent Publication No. 3-10899 JP 2002-282240 A Japanese Patent No. 3862665 WO2005 / 057211 JP 2002-114798 A JP 2002-332357 A JP 2009-300191 A

Journal of the Japanese Cosmetic Society Vol. 30, no. 1, pp. 5-9 (2006)

However, there has not yet been a specific report regarding a method for measuring the degree of hair damage by a permanent agent using a hair keratin film. In particular, the evaluation of the degree of hair damage by a permanent agent is currently largely dependent on changes in the physical properties of the hair using hair strands, and it has excellent accuracy with little variation in data due to unevenness of the permanent agent and the hair quality of the specimen. There is a high demand for measurement methods.
The present invention has been made in view of such circumstances, and provides a measurement method that can easily measure the influence of a permanent agent on hair with little variation in measured values and that is excellent in accuracy. With the goal.

As a result of intensive studies by the present inventors to achieve the above-mentioned problems, the keratin film shows the same effect as hair by applying a permanent agent, and by using the film as an application target of a permanent agent, The inventors have found that the degree of hair damage caused by the same agent can be clearly measured, and have completed the present invention.
That is, the method for measuring the degree of hair damage using a permanent agent according to the present invention is characterized by analyzing hair proteins contained in a permanent agent treated with a keratin film.

In the measurement method, it is preferable that the analysis of the hair protein is a quantitative analysis of the protein.
In the measurement method, it is preferable that the analysis of the hair protein is an SDS-PAGE analysis.
In the measurement method, it is preferable that the permanent agent is mainly composed of thioglycolic acid.

Moreover, in the said measuring method, it is suitable to analyze the keratin film processed with the permanent agent.
In the measurement method, it is preferable that the analysis of the keratin film is microscopic observation by SEM and / or SPM.

In the measurement method, it is preferable that the analysis of the keratin film is an evaluation of transparency of the keratin film.
In the measurement method, it is preferable that the analysis of the keratin film is a weight measurement of the keratin film.

  According to the method of the present invention, it is possible to easily measure the degree of hair damage caused by a permanent agent without causing variations in measured values. This makes it possible to test the durability and the like of hair when using a permanent agent with high accuracy, and thus can be expected to make a significant contribution in the development and research of related cosmetics, pharmaceuticals, quasi drugs, and the like.

It is a Tricine / SDS-PAGE development image of the component contained in the various permanent agents after making it react with the keratin film of the measurement example 1. It is the external appearance photograph, SEM image, and SPM image of the keratin film in the measurement example 2 before and after the permanent agent treatment. It is a graph which shows the elution protein mass with respect to the change of the permanent agent density | concentration of the measurement example 3. It is a Tricine / SDS-PAGE development image regarding the permanent agent of each density | concentration reacted with the keratin film of the measurement example 3. It is the graph which showed the weight change of the keratin film before and after the permanent agent process of the measurement example 3 with respect to the permanent agent concentration. It is an external appearance photograph of the keratin film after the permanent agent treatment of each concentration of Measurement Example 3.

Hereinafter, preferred embodiments of the present invention will be described.
The method for measuring the degree of hair damage by a permanent agent according to the present invention is used for evaluating the degree of hair damage to which a permanent agent is applied.
In the present invention, “hair” includes animal hair and feathers in addition to human hair. It is also possible to apply the measurement method according to the present invention to nails, skin, etc. containing keratin protein.
First, the keratin film used for the measurement method according to the present invention will be described.

<Preparation of keratin film>
In order to extract the keratin protein group which comprises it from hair, a protein denaturant is used. The protein denaturant is preferably a urea compound, and examples thereof include urea, thiourea, and derivatives thereof. It is preferable to use a mixture of one or more of these urea-based protein denaturants. More preferably, urea and thiourea are mixed and used. When using a mixture of urea and thiourea, the mixing mass ratio is preferably 5: 1 to 1: 2 (urea: thiourea). If the mixing ratio of thiourea is less than the above range, the protein denaturing action may be inferior, and if it exceeds the above range, the extraction rate of the keratin protein group tends to decrease.

The concentration of the protein denaturant in the hair sample treatment solution is preferably 30 to 70% by mass. If it is less than 30% by mass, the extraction rate of the keratin protein group tends to decrease, and even if it is used in excess of 70% by mass, the effect of improving the extraction rate by increasing the amount is not recognized, and the hair sample treatment solution In some cases, the viscosity of the resin becomes high and the workability deteriorates. Here, the “hair sample treatment solution” means a hair keratin protein solution comprising a hair sample and a protein denaturant, and a mixed solution in the production process for extracting a keratin protein group, including a reducing agent described later. .
As described above, by using a protein denaturant, it is possible to efficiently dissolve and extract a keratin protein group from hair under mild conditions.

In preparing the keratin film used in the present invention, a reducing agent is used in combination with the protein denaturing agent. Examples of the reducing agent include thioalcohols such as 2-mercaptoethanol and dithiothreitol thioglycolic acid. These 1 type (s) or 2 or more types can be used in combination.
By using a reducing agent in combination with the protein denaturant, the extraction rate of the keratin protein group can be further improved. This is because the protein modifier denatures the strong keratin fiber structure, and then the reducing agent efficiently dissociates the strong S—S bond between the keratin proteins, and recombination in the hair sample treatment liquid hardly occurs. It is thought to do.

When the reducing agent is used in combination with a protein denaturant, the hair sample treatment solution preferably contains 0.5 to 40% by mass, more preferably 1 to 20% by mass. However, it is preferably determined as appropriate depending on the solubility of the reducing agent used in the hair sample treatment solution.
When the concentration of the reducing agent is less than 0.5% by mass, there is a tendency that the strong reductive cleavage of the S—S bond between the keratin proteins is not sufficiently performed, and when the concentration exceeds 40% by mass. The solubility of the keratin protein group in the hair treatment solution may be deteriorated.

The treatment time for obtaining the hair keratin protein solution depends on the treatment temperature, but is preferably 1 to 4 days. Moreover, it is preferable that processing temperature is 20-60 degreeC. If the temperature is lower than 20 ° C., the reaction progresses slowly and the efficiency is poor. If the temperature exceeds 60 ° C., the hair sample treatment solution exhibits alkalinity, which causes side reactions such as peptide bond cleavage, substituent conversion, and crosslinking. There is a case.
The ratio of the hair sample to the hair sample treatment solution is preferably 1 to 100 mg hair sample / ml hair sample treatment solution.
After the keratin protein is sufficiently extracted from the hair sample treatment solution, the hair-extracted hair is removed by filtration to obtain a hair keratin protein solution.

In order to solidify or gel the hair keratin protein solution obtained by treating a hair sample with a protein denaturing agent and a reducing agent and filtering it, the developing solution is contacted. By this solidification or gelation, the film is formed as a film having a form suitable for the measurement method according to the present invention.
Examples of the developing solution include a modifying agent solution obtained by mixing a modifying agent such as trichloroacetic acid, guanidine hydrochloride, perchloric acid, and derivatives thereof, and a solvent such as water, physiological saline, and lower alcohol, and hydrochloric acid. And acidic solutions composed of acidic substances such as sulfuric acid, acetic acid, phosphoric acid and salts thereof, and one or more of these can be used. In the preparation of the keratin film used in the present invention, guanidine hydrochloride, a perchloric acid modifier or a perchloric acid solution obtained by mixing acetic acid and water, a guanidine hydrochloric acid solution, an acetic acid buffer solution, and an acetic acid solution as a developing solution. It is preferable that it is 1 type, or 2 or more types selected. Particularly preferred is an acetate buffer (pH 4.0) and / or an acetic acid solution.

The concentration of the denaturant solution used as the developing solution is preferably 10 to 60% by mass. The concentration of the acidic solution used as the developing solution is preferably 10 to 500 mM.
The developing solution has an action of lowering the ionic strength of the hair keratin protein solution, thereby causing a decrease in the solubility of the protein denaturant and reducing agent in the hair sample treatment solution. As a result, the solubility of the keratin protein group is reduced, and the SS bond between the keratin proteins is dissociated and the SS state is re-formed, and the SS bond is reformed. Solidification will proceed.

When preparing the keratin film used in the present invention, the Post-cast method or the Pre-cast method can be applied.
As a post-cast method, a container such as a petri dish is filled with the solution for development in advance and a hair keratin protein solution is cast into the container (forward method), or a container such as a petri dish to which a hair keratin protein solution is added in advance. In addition, a method of casting the developing solution (reverse method) may be mentioned.
The pre-cast method is a method in which a developing solution is mixed with hair keratin protein in advance, and the mixed solution is cast into a container such as a petri dish filled with water.
In the present invention, a keratin film excellent in uniformity suitable for measurement of the degree of hair damage by a permanent agent can be prepared by any application of the Post-cast method and the Pre-cast method.
In particular, an acetic acid solution can be suitably used as a developing solution used in the Pre-cast method. In the Post-cast method, an acetate buffer is suitable as a developing solution.
As a reducing agent, 2-mercaptoethanol is suitable for use in the Post-cast method, and dithiothreitol is suitable for use in the Pre-cast method.
Further, the developing solution is preferably used at a mass ratio of 10 to 10,000 times that of the hair keratin protein solution. By bringing the developing solution into contact with the hair keratin protein solution within the above range, a thin film exhibiting an appropriate thickness can be prepared.

  A thin-film hair keratin protein molded product formed in the petri dish is washed with the developing solution. The number of times of washing is not particularly limited, but is preferably about 1 to 5 times. After washing, the solution is removed, and the hair keratin protein molded product on the petri dish is dried to obtain a keratin film. The drying method is not particularly limited, and examples thereof include standing at room temperature free from dust.

  Some hair samples used for the preparation of keratin film contain a large amount of oil, and may be degreased before treatment. Although there is no limitation in the method of degreasing, For example, processing with the mixed solvent of chloroform and methanol etc. are mentioned.

<Measurement method of hair damage by permanent agent>
Then, the measuring method of the hair damage degree by the permanent agent concerning this invention is demonstrated.
The method according to the present invention uses the above-mentioned keratin film treated with a permanent agent as a measurement object, and quantitative analysis of hair proteins eluted from the film by the treatment, change in weight of the keratin film before and after the treatment, etc. It is intended to measure the degree of hair damage. In addition to the above, the method of the present invention includes directly observing changes in film permeability and structural changes in the film using a microscope or the like. Moreover, after washing | cleaning and drying a film, a hair damage degree can also be measured from the weight change and form change.

  A general permanent agent is a disulfide bond (S) that connects the polypeptide chains of hair keratin by applying a first agent containing a reducing agent (thioglycolic acid, cysteine, acetylcysteine, thiolactic acid, etc.) to the hair. -S bond) is broken to release its network structure, and then a second agent mainly composed of an oxidizing agent (bromate, hydrogen peroxide, etc.) is applied to bring the broken disulfide bond into a new position. It is to be formed. By applying this series of treatments, the hair can be fixed in a desired state.

  Since hair proteins such as keratin are structured mainly by cross-linking by the disulfide bond, when the reducing agent contained in the permanent agent is applied and the bond is cleaved, the interaction is extremely reduced. It is known that it becomes easy to elute. Moreover, the hair protein hydrolyzed by the alkali agent mix | blended with a reducing agent as an adjuvant may elute. Such elution of hair proteins is a cause of hair damage caused by the permanent agent, and it is considered that measuring the degree thereof can serve as an index for evaluating the influence of various permanent agents on the hair.

Hereinafter, a specific measurement method will be described.
First, a permanent agent is applied to a keratin film as a sample.
The permanent agent to be used is not particularly limited in terms of ingredients, including its dosage form, but from the viewpoint of using the hair protein eluted by the above reducing agent as an index, it is preferable to use one containing a reducing agent. In other words, this includes the use of only the reducing agent component contained in the permanent agent and the use of only the agent containing the reducing agent of the multi-drug type permanent agent. As the permanent agent, those using thioglycolic acid as the reducing agent are particularly preferred because of easy detection of damage.
On the other hand, it is also possible to use a permanent agent or a permanent component that does not contain a reducing agent, and measure the degree of hair damage due to other than the reducing agent. Of course, various permanent agent compositions may be used as they are.

  The amount and method of use of the permanent agent to the keratin film can be basically the same as when applied to the hair. However, depending on the content of the permanent agent to be evaluated, its component concentration, usage conditions, method, etc. It is preferable to change appropriately. For example, the degree of hair damage due to the concentration of the reducing agent can be examined by comparing the measurement results of the keratin film samples in which the concentration of the reducing agent in the applied permanent agent is changed. Moreover, if the application time and temperature of a permanent agent are changed, the hair damage degree by this condition can be examined.

  The treatment of the keratin film with the permanent agent is performed by bringing the permanent agent into contact with the keratin film contained in a petri dish or the like, that is, adding the permanent agent directly to the container (casting), as in the case of hair. Can do. The permanent agent is recovered from the container after reacting the permanent agent with the film for the time, temperature and other conditions set for the permanent agent to be used or for the hair damage measurement test. The collected permanent agent contains keratin film, that is, hair protein eluted from the hair due to the influence of the permanent agent. Therefore, by analyzing the protein in the recovered permanent agent, it is possible to measure the degree of hair damage that affects the protein.

  The analysis of the hair protein in the recovered permanent agent can be appropriately selected from known protein analysis methods, and is not particularly limited. For example, when the hair protein in a permanent agent is quantitatively analyzed and the degree of hair damage is evaluated on the basis of the amount of the eluted protein, a known method using a spectrophotometer such as an ultraviolet absorption method, a Bradford method, a Lowry method, or a BCA method is used. And the use of a colorimetric method. In the present invention, it is particularly preferable to measure the absorbance at 595 nm of the recovered permanent agent or its supernatant in accordance with the Bradford method, and convert it to a protein mass using a calibration curve prepared in advance.

Other hair protein analysis methods include electrophoresis such as SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). In the case of SDS-PAGE, for example, the recovered permanent agent or its supernatant is treated with a reducing agent as necessary, and this is developed on a polyacrylamide gel using a buffer solution to which SDS is added, and further, nitrocellulose, PVDF, etc. The protein transferred onto the membrane and captured on the membrane is detected by immunostaining or the like. Based on the detection band of the detected protein, the identification of the hair protein eluted from the permanent agent and the amount of the protein eluted can be evaluated.
In the present invention, in addition to the above, any analysis method for evaluating the eluted protein by a permanent agent such as protein identification and mass spectrometry using ionization methods such as ESI-MS and MALDI-MS is evaluated. It can be applied according to the purpose.

  Moreover, in this invention, not only the permanent agent collect | recovered after reaction with a keratin film but the keratin film after the process can also be used for the measurement of a hair damage degree. As described above, when the treatment is performed with a permanent agent, the hair protein may be eluted from the keratin film corresponding to the hair depending on the type of the permanent agent to be applied and the use conditions. Such protein elution is thought to be partly due to the disruption of the network structure of the protein constituting the hair (ie, keratin film) due to the influence of the components contained in the permanent agent. Therefore, by analyzing the change in the network structure of the protein in the keratin film before and after application of the permanent agent, the influence of the permanent agent on the hair can be evaluated.

  Examples of the method for analyzing the structure of the keratin film include a method using structure observation data of the keratin film using a microscope or the like. The microscope used here is not particularly limited. For example, a scanning electron microscope (SEM) is used for three-dimensional structure observation, and a scanning probe microscope (SPM) is used for nano-scale surface observation. It is preferable to select it according to desired information. The data obtained by microscopic observation may be used as it is for examining the degree of hair damage, or may be analyzed in more detail.

Another analysis method includes evaluation of the transparency of the keratin film.
The aforementioned keratin film used in the present invention forms a network structure by S—S bonds as in normal hair. Therefore, in a normal state, it has a translucent to opaque appearance. However, when the film is treated with a permanent agent, the S—S bond is cleaved by the permanent agent, the network structure is collapsed, and the degree of transparency of the film increases depending on the degree. The property of this keratin film can be used to evaluate the degree of hair damage caused by a permanent agent. Transparency measurement is applied according to the purpose of evaluating known analytical methods such as optical measurement such as refractive index and absorption spectrum in addition to visually observing the appearance change of keratin film before and after perm treatment and under various conditions. May be.

Furthermore, another approach using a keratin film includes analysis of film weight before and after treatment with a permanent agent.
As described above, hair proteins are eluted from the keratin film treated with a permanent agent. Therefore, the weight of the keratin film after the permanent treatment tends to be lighter than the amount before the treatment. That is, it can be evaluated that the greater the amount of the eluted hair protein, the lighter the keratin film is and the higher the degree of hair damage.
Therefore, it is possible to measure the degree of hair damage by the permanent agent before and after the permanent agent treatment, and by comparing the change in weight depending on the type and the use condition of the permanent component.
In addition, when measuring the weight of the keratin film after the permanent agent treatment, it is preferable that the film is sufficiently washed to remove the permanent agent and kept in a dry state as before the treatment.

  The measurement data of the hair protein or keratin film obtained as described above can be used to measure the degree of hair damage by a permanent agent in development and research in the fields of beauty, pharmacy, medicine, food and the like. The evaluation of the measurement result of the hair damage degree and the use thereof are performed according to the respective development and research contents, and are not particularly limited as long as the object of the present invention is not impaired.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to the following Example. In addition, the numerical value in an Example shows the mass% unless there is particular description.
First, the preparation method of the keratin film used for the present Example is shown.
(Example of preparation of keratin film)
A hair sample treatment solution was prepared by immersing 600 g of hair of a 15-year-old woman in a mixed solution containing 30% by weight of urea, 20% by weight of thiourea, 5% by weight of dithiothreitol, and 8 ml of 25 mM Tris-HCl buffer (pH 8.5). This was held at 50 ° C. for 1 to 4 days to dissolve the hair keratin protein. The powder was extracted from this solution by filtration to obtain a hair keratin protein solution. 6 ml of 100 mM acetic acid aqueous solution was added to 3.5 mg of this protein solution, mixed, and this mixed solution was gently cast into a petri dish (35 mm in diameter) filled with water. After the hair keratin protein solidified, the developing solution containing distilled water was changed several times, and the solution in the gel was replaced with distilled water. Finally, distilled water was removed, and it was sufficiently dried in a box containing silica gel to obtain a keratin film fixed in a petri dish.

<Measurement Example 1: Identification of eluted protein>
For the keratin film according to the above preparation example (fixed to a petri dish), only 1 ml of each of the following permanent agents was cast and left at 25 ° C. for 10 minutes. Thereafter, the permanent agent in each petri dish was collected and centrifuged at 12,000 g for 10 minutes at room temperature, and the supernatant was collected. The obtained supernatant was developed with Tricine / SDS-PAGE (polyacrylamide gel concentration 15%), stained with Coomassie Brilliant Blue, and then analyzed for protein bands separated on the gel. The results are shown in FIG.

(Perm formulation)
Test Example 1: Thioglycolic acid based permanent agent (first agent)
(mass%)
Ammonium thioglycolate (50%) [Reducing agent] 10.0
Diammonium dithioglycolate liquid (40%) [Reaction modifier] 8.0
Monoethanolamine [alkali] 1.0
Strong ammonia water [alkali] 2.0
Ammonium bicarbonate [alkali] 1.2
Stearyltrimethylammonium chloride (80%) [feel imparted] 0.5
Hydroxyethane diphosphonic acid [sequestering agent] 0.1
POE / POP decyl tetradecyl ether [solubilizer] 0.3
Fragrance [fragrance] 0.1
Polydimethylmethylenepiperinium chloride solution [giving elasticity] 0.5
Purified water [Purified water] Appropriate amount

Test Example 2: Thioglycolic acid based permanent agent (first agent)
(mass%)
Ammonium thioglycolate (50%) [Reducing agent] 12.0
Monoethanolamine [alkali] 1.0
Strong ammonia water [alkali] 1.5
Ammonium bicarbonate [alkali] 1.5
Stearyltrimethylammonium chloride (80%) [feel imparted] 0.5
Hydroxyethane diphosphonic acid [sequestering agent] 0.1
POE / POP decyl tetradecyl ether [solubilizer] 0.3
Fragrance [fragrance] 0.1
Polydimethylmethylenepiperinium chloride solution [giving elasticity] 0.5
Purified water [Purified water] Appropriate amount

Test Example 3: Cysteine permanent agent (first agent)
(mass%)
Ammonium thioglycolate (50%) [Reducing agent] 1.5
DL-cysteine [reducing agent] 3.5
Monoethanolamine [alkali] 1.0
Strong ammonia water [alkali] 1.0
Stearyltrimethylammonium chloride (80%) [feeling] 0.4
Hydroxyethane diphosphonic acid [sequestering agent] 0.1
POE / POP decyl tetradecyl ether [solubilizer] 0.3
Fragrance [fragrance] 0.1
1,3-butylene glycol [humectant] 1.5
Polydimethylmethylenepiperinium chloride solution [giving elasticity] 0.25
Purified water [Purified water] Appropriate amount

Test Example 4: Cysteine permanent agent (first agent)
(mass%)
Ammonium thioglycolate (50%) [Reducing agent] 1.8
DL-cysteine [reducing agent] 3.0
L-cysteine [reducing agent] 2.0
Monoethanolamine [alkali] 1.0
Strong ammonia water [alkali] 2.0
Stearyltrimethylammonium chloride (80%) [feeling] 0.4
Hydroxyethane diphosphonic acid [sequestering agent] 0.1
POE / POP decyl tetradecyl ether [solubilizer] 0.3
Fragrance [fragrance] 0.1
1,3-butylene glycol [humectant] 1.5
Polydimethylmethylenepiperinium chloride solution [giving elasticity] 0.25
Purified water [Purified water] Appropriate amount

As shown in FIG. 1, in the permanent agent of Test Example 1, bands presumed to be keratin-related proteins (KAPs) were observed at positions of 7 kDa, 14 kDa and 20 kDa.
Therefore, according to the present invention, by using a keratin film derived from hair, it is possible to confirm elution of a specific hair protein by a permanent agent.

<Measurement Example 2: Structural change of keratin film>
To the keratin film (prepared in a petri dish) according to the above preparation example, 1 ml of the permanent agent of Test Example 1 was cast and left at 25 ° C. for 0.5 minutes or 10 minutes. After each standing time, a part of the permanent agent in the petri dish was collected and then washed away. The keratin film was washed several times and dried, and the permeability was evaluated from the appearance. The evaluation is based on whether or not the characters written under the transparent petri dish in which the keratin film can be recognized are recognized. Moreover, the protein structure of the film was also observed with a scanning electron microscope (SEM) and a scanning probe microscope (SPM). As a control, the same measurement was performed on a keratin film treated with distilled water instead of the permanent agent. The results are shown in FIG.

As shown in FIG. 2, the keratin film had an opaque appearance when untreated, but the transparency gradually increased with the application of the permanent agent. That is, the change in transparency of the film was already observed when the permanent agent was added for 0.5 minutes, and became almost completely transparent when added for 10 minutes.
Moreover, from the results of SEM and SPM, it was found that the keratin film treated with the permanent agent was broken down in the network structure of the hair protein as the treatment time progressed, and the surface became smooth. It is considered that the increase in transparency of the film due to the addition of the permanent agent is accompanied by the change in the three-dimensional structure of the hair protein.
Therefore, according to the present invention, the degree of hair damage can be confirmed from the structural change of the keratin film.

<Measurement Example 3: Measurement of hair damage degree at permanent agent concentration>
For the keratin film prepared in the above preparation (fixed in a petri dish), cast 1 ml each of sodium thioglycolate solution (pH 9.4) at different concentrations of sodium thioglycolate, and let stand at 25 ° C. for 10 minutes did. The concentration of sodium thioglycolate was 0, 50, 100, 150, 200, 250, 300, 350, 400 mM.
After standing, the sodium thioglycolate solution in each petri dish was collected and centrifuged at 12,000 g for 10 minutes at room temperature, and the supernatant was collected. About each obtained supernatant, while quantifying protein amount by Bradford method (FIG. 3), Tricine / SDS-PAGE was performed like the measurement example 1 (FIG. 4).
On the other hand, for the keratin film after collecting the sodium thioglycolate solution, the weight of each film is measured after washing and drying, and the film weight measured in advance before performing the permanent treatment. The difference was calculated (weight before treatment−weight after treatment) (FIG. 5). Further, the transparency of each keratin film after the permanent agent treatment was also evaluated in the same manner as in Measurement Example 2 (FIG. 6). The results are shown in FIGS.

  As shown in FIG. 3, as the sodium thioglycolate concentration increased, it was revealed that the amount of hair protein eluted in the solution tends to increase. Also in the result of electrophoresis in FIG. 4, a band considered to correspond to a keratin-related protein (7 kDa to 20 kDa) was observed in a sample having a sodium thioglycolate concentration of 150 mM or more. In addition, a high molecular weight protein band of 100 kDa or more appeared in a sample having a sodium thioglycolate concentration of 250 mM or more.

Moreover, in the weight change of the film before and after the treatment shown in FIG. 5, the film weight after the treatment tended to decrease as the sodium thioglycolate concentration increased. This result is consistent with the result of FIG. 3, which shows that the amount of protein eluting increases in proportion to the sodium thioglycolate concentration. Furthermore, the appearance of the keratin film after the treatment shown in FIG. 6 also showed that the disintegration of the network structure of keratin became remarkable and the transparency became high with the sodium thioglycolate concentration.
Therefore, according to the present invention, using a keratin film, the degree of hair damage caused by a permanent agent can be evaluated without any variation by various means.

Claims (4)

A method for measuring the degree of hair damage by a permanent agent, wherein the hair protein contained in the permanent agent treated with a keratin film is analyzed by SDS-PAGE .   The method for measuring the degree of hair damage with a permanent agent according to claim 1, wherein the permanent agent is mainly composed of thioglycolic acid. A method for measuring the degree of hair damage with a permanent agent, characterized by analyzing the keratin film treated with the permanent agent by microscopic observation with SEM and / or SPM . A method for measuring the degree of hair damage by a permanent agent, comprising analyzing a keratin film treated with a permanent agent by evaluating the transparency of the keratin film .