Method for evaluating state of hair

Abstract

To provide a method for evaluating the state of hair, the method suitable for application to various uses.SOLUTION: A method for evaluating the state of hair includes: labeling a first functional group present on a first section of hair and to be an index of the state, with a biotin derivative specifically binding to the first functional group; and detecting the first functional group labeled with the biotin derivative. Examples of the functional group include a sulfide group, a carboxyl group, an amino group, and a carbonyl group.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for assessing the condition of hair. [Background technology]

[0002] Hair is damaged by exposure to heat from hair dryers and ultraviolet rays, and by treatments such as perms and coloring. It is known that when hair is damaged, the disulfide bonds (-SS-) of keratin are broken, increasing the number of sulfide groups (SH groups) inside the hair. If the increased number of sulfide groups can be detected, the state of damage to the hair can be known. Patent Document 1 discloses a method for diagnosing hair damage using a fluorescent substance that modifies sulfide groups. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 8-178920 Summary of the Invention [Problem to be solved by the invention]

[0004] The method of Patent Document 1 is suitable for easily determining hair damage. However, no consideration is given to the evaluation of hair conditions other than damage. If the hair condition can be evaluated without being limited to damage, it can be applied to various uses. For example, if the state of penetration of the components contained in the hair composition into the hair can be evaluated, it can be useful for the development of hair compositions.

[0005] The present invention aims to provide a method for evaluating hair condition, which is suitable for a variety of applications. [Means for solving the problem]

[0006] The present invention relates to 1. A method for assessing hair condition, comprising: labeling a first functional group present in a first cross section of the hair and capable of being an indicator of the condition with a biotin derivative that specifically binds to the first functional group; detecting the first functional group labeled with the biotin derivative; The present invention provides a method comprising the steps of: Effect of the Invention

[0007] The techniques of the present invention are suitable for application in a variety of applications. [Brief description of the drawings]

[0008] [Figure 1] 1 is a flowchart showing an example of an evaluation method of the present invention. [Diagram 2] 4 is a flowchart showing another example of the evaluation method of the present invention. [Diagram 3] 1 shows a visualized image obtained by evaluation of hair (test piece A) in Example 1. [Figure 4] 1 shows a visualized image obtained by evaluation of hair (test piece B) in Example 1. [Diagram 5] 1 shows a visualized image obtained by evaluation of hair (test piece C1) in Example 2. [Figure 6] 1 shows a visualized image obtained by evaluation of hair (test piece C2) in Example 2. [Figure 7] 1 shows a visualized image obtained by evaluation of hair (test piece C3) in Example 2. [Figure 8] 1 shows a visualized image obtained by evaluation of hair (test piece D1) in Example 3. [Figure 9] 1 shows a visualized image obtained by evaluation of hair (test piece D2) in Example 3. [Figure 10] 1 shows a visualized image obtained by evaluation of hair (test piece D3) in Example 3. [Figure 11] 1 shows a visualized image obtained by evaluation of hair (test piece E1) in Example 4. [Figure 12]1 shows a visualized image obtained by evaluation of hair (test piece E2) in Example 4. [Figure 13] 1 shows a visualized image obtained by evaluation of hair (test piece E3) in Example 4. [Figure 14] 1 shows a visualized image obtained by evaluation of hair (test piece F1) in Example 5. [Figure 15] 1 shows a visualized image obtained by evaluation of hair (test piece F2) in Example 5. [Figure 16] 1 shows a visualized image obtained by evaluation of hair (test piece F3) in Example 5. [Figure 17] 1 shows visualized images obtained by evaluation of hair (test pieces C1 to C3, G1 to G3) in Example 6. [Figure 18] 1 shows visualized images obtained by evaluating hair (test pieces H1 to H3) in a reference example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The main component of hair is protein. It is also known that various physiologically active substances accumulate in hair. However, the present inventors have studied how to detect functional groups present inside hair, rather than detecting proteins or physiologically active substances themselves, and how to detect functional groups other than sulfide groups, and have completed the present invention.

[0010] The method according to the first aspect of the present invention comprises the steps of: 1. A method for assessing hair condition, comprising: labeling a first functional group present in a first cross section of the hair and capable of being an indicator of the condition with a biotin derivative that specifically binds to the first functional group; detecting the first functional group labeled with the biotin derivative; Includes.

[0011] In a second aspect of the present invention, for example in the method according to the first aspect, the functional group is a sulfide group, a carboxyl group, an amino group, or a carbonyl group.

[0012] In a third aspect of the present invention, for example in the method according to the first or second aspect, the biotin derivative labeled for detection is used to label the first functional group, or a further compound labeled for detection is bound to the biotin derivative labeling the first functional group, and the first functional group is detected by detecting the detection label.

[0013] In a fourth aspect of the invention, such as the method according to the third aspect, the detectable label is a fluorescent label or a metal label.

[0014] In a fifth aspect of the present invention, for example in the method according to the third or fourth aspect, the detection label is a fluorescent substance that emits fluorescence with a wavelength of 500 nm or more.

[0015] In a sixth aspect of the invention, such as a method according to any one of the third to fifth aspects, the further compound is avidin or an avidin derivative.

[0016] In a seventh aspect of the present invention, such as the method of any one of the first to sixth aspects, the first cross-section is a transverse cross-section of the hair.

[0017] In an eighth aspect of the present invention, for example in the method according to any one of the first to seventh aspects, the first functional group is labeled by directly binding the biotin derivative to the first functional group.

[0018] In a ninth aspect of the present invention, the method according to any one of the first to eighth aspects, for example, further comprises visualizing the distribution of the first functional group inside the hair based on information obtained by detecting the first functional group.

[0019] In a tenth aspect of the present invention, the method according to any one of the first to ninth aspects, for example, further comprises modifying a second functional group, which is present in a second cross section of the hair and can be an indicator of the condition, by directly binding a fluorescent substance to the second functional group, detecting the second functional group modified with the fluorescent substance, and visualizing the distribution of both the first functional group and the second functional group inside the hair based on information obtained by detecting the first functional group and information obtained by detecting the second functional group.

[0020] Hereinafter, an embodiment of the present invention will be described. However, the following description is not intended to limit the present invention to a specific embodiment.

[0021] [How to evaluate hair condition] In the method for evaluating the condition of hair of this embodiment (hereinafter referred to as "evaluation method"), a first functional group (first target group) that is present in a first cross section of hair and can be an indicator of the condition of hair is labeled with a biotin derivative that specifically binds to the first functional group, and the first functional group labeled with the biotin derivative is detected. The condition of hair can be evaluated based on information obtained by detecting the first functional group.

[0022] Detection of the first functional group present in the cross section is suitable for evaluating the internal state of hair. The cross section may be a transverse section of hair, or may be an oblique cross section or a longitudinal cross section. The cross section may be a sliced ​​surface of hair formed by sectioning, or a cross section other than a sliced ​​surface, for example, a cross section formed by cutting or a cross section naturally formed by hair breakage, etc. The evaluation method of this embodiment also makes it possible to visualize the distribution of the first functional group inside the hair.

[0023] Since the biotin molecule has a side chain of valeric acid, it is relatively easy to introduce a functional group by derivatization and to control the length of the side chain. For this reason, it is possible to synthesize biotin derivatives having various reactive groups and different lengths of side chains. The ability to select a reactive group from among multiple types means that the first functional group can be selected without being limited to one specific group such as a sulfide group. In addition, biotin derivatives are usually low molecular weight compounds. This means that not only functional groups derived from proteins constituting hair, but also functional groups possessed by components contained in a hair composition that has penetrated into hair can be detected as the first functional group. In addition, being a low molecular weight compound can contribute to improving the accuracy of detection of the first functional group. The ability to select the length of the side chain means that, for example, when using a biotin derivative labeled for detection to detect the first functional group, the detection label can be selected from multiple candidates. The characteristics required for the evaluation method differ depending on the application. The evaluation method of this embodiment having the above-mentioned features, including a high degree of freedom in selection of the first functional group and the detection label, is suitable for use in a variety of applications.

[0024] Examples of the first functional group are a sulfide group (-SH), a carboxyl group (-COOH), an amino group (-NH2), and a carbonyl group (-CO-). The first functional group may be a sulfide group, a carboxyl group, an amino group, or a carbonyl group. The functional groups exemplified above are groups that are particularly useful as indicators of hair condition. However, the first functional group is not limited to the groups exemplified above.

[0025] Sulfide groups are formed, for example, by decomposition of disulfide bonds contained in keratin, the main component of hair. Sulfide groups can also be formed by decomposition of thioester bonds, which are relatively abundant in the hair cuticle. Disulfide bonds and thioesters The bonds can be decomposed by external stimuli such as heat, ultraviolet light, and hypochlorous acid in a pool, as well as hair compositions such as reducing agents used in bleach and perm I agents. However, even with the same external stimuli and hair compositions, the distribution and amount of sulfide groups formed vary depending on the quality of hair. In addition, sulfide groups are also functional groups that components contained in reducing agents may have. In other words, detecting sulfide groups makes it possible to make the following evaluations. However, the items that can be evaluated are not limited to the following examples. - Hair damage and health ·Hair resistance to external stimuli and hair compositions - Regarding the ingredients contained in hair compositions, the penetration into hair, the distribution inside the hair, and the difference in penetration and distribution due to the quality of hair - The effectiveness and mechanism of action of ingredients contained in hair compositions, and differences in effectiveness depending on hair quality

[0026] Examples of hair compositions include bleaching agents and reducing agents used in perm I agents, oxidizing agents used in perm II agents, protecting agents that protect hair from external stimuli and reducing agents, and strengthening agents that form a crosslinked structure inside hair to strengthen the hair. Examples of strengthening agents are compositions known to those skilled in the art under the names of complexing agents or plexing agents. However, hair compositions are not limited to the above examples.

[0027] Carboxyl groups are present, for example, in the side chains or terminals of proteins, which are the main components of hair. Carboxyl groups are also functional groups that may be contained in components of hair compositions. Examples of hair compositions that may contain carboxyl groups are reducing agents and strengthening agents. In other words, by detecting carboxyl groups, the following evaluations are possible. However, the items that can be evaluated are not limited to the following examples. - Hair damage and health - Regarding the ingredients contained in hair compositions, the penetration into hair, the distribution inside the hair, and the difference in penetration and distribution due to the quality of hair - The effectiveness and mechanism of action of ingredients contained in hair compositions, and differences in effectiveness depending on hair quality

[0028] Amino groups can be generated on the cross section of hair, for example, when hair is damaged and a cavity is generated in the hair, or when proteins contained in the hair are denatured. Amino groups are also functional groups that may be contained in components contained in hair compositions. An example of a hair composition that may contain amino groups is a protective agent containing amino acids or peptides. In other words, the following evaluations can be made by detecting amino groups. However, the items that can be evaluated are not limited to the following examples. - Hair damage and health - Regarding the ingredients contained in hair compositions, the penetration into hair, the distribution inside the hair, and the difference in penetration and distribution due to the quality of hair - The effectiveness and mechanism of action of ingredients contained in hair compositions, and differences in effectiveness depending on hair quality

[0029] Carbonyl groups are generated, for example, by damage to proteins. As an example, it is known that carbonylated proteins can be generated by the addition of aldehyde compounds generated by oxidative decomposition of amino acid side chains or lipid peroxidation. In hair, carbonyl groups can be generated on the cross section of hair due to damage caused by treatments such as bleaching and perming, ultraviolet rays, and heat from hair irons and dryers. In other words, the following evaluations can be made by detecting carbonyl groups. However, the items that can be evaluated are not limited to the following examples. - Hair damage and health ·Hair resistance to external stimuli and hair compositions

[0030] In this specification, a biotin derivative means a compound in which a reactive group is introduced into a biotin molecule by various derivatizations. The reactive group is a group capable of binding to a first functional group. The mode of binding is not limited, but is preferably a covalent bond. Examples of the reactive group are a maleimide group, a phenyl group in which at least one hydrogen atom is substituted with a halogen atom, a nitro group, a carboxyl group, a succinimidyl ester group, a pyridyl group, a nitropyridyl group, a carboxypyridyl group, a hydrazino group, a carboxylic acid hydrazide group, and an amino group. An example of a halogen atom is a fluorine atom. All hydrogen atoms of the phenyl group may be substituted with halogen atoms. However, the reactive group and the halogen atom are not limited to the above examples. Examples of reactive groups that can be selected according to the first functional group are shown in Table 1 below. In addition, for the maleimide group, the first functional group that is mainly bound can usually be selected from a sulfide group and an amino group by controlling the pH of the reaction system in which the first functional group is labeled.

[0031] [Table 1]

[0032] The reactive group may be introduced into the side chain of the biotin molecule, or into the end of the side chain. The number of reactive groups introduced into the biotin molecule may be, for example, 5 or less, 4 or less, 3 or less, 2 or less, or even 1.

[0033] The length of the side chain of the biotin derivative can be controlled, for example, by forming an ester bond or an amide bond using the carboxyl group of valeric acid that the biotin molecule has in the side chain, and by introducing a chemical structure serving as a spacer between the formed ester bond or amide bond and the end of the side chain. The chemical structure serving as a spacer may contain, for example, an alkylene group that may be linear or branched, and / or an oxyalkylene group that may be linear or branched. The number of carbon atoms in the alkylene group and the oxyalkylene group may be, for example, 1 to 5, 2 to 4, or even 2 to 3. The end of the side chain into which the spacer is introduced may be a reactive group.

[0034] The molecular weight of the biotin derivative is, for example, not more than 1000, and may be not more than 800, or even not more than 500. The lower limit of the molecular weight is, for example, 250 or more.

[0035] The length of the spacer in a biotin derivative having a spacer introduced into its side chain may be, for example, 5 angstroms (Å) or more and 200 Å or less, 10 Å or more and 150 Å or less, 10 Å or more and 100 Å or less, 10 Å or more and 80 Å or less, or even 10 Å or more and 60 Å or less.

[0036] Examples of biotin derivatives include N-[6-(biotinamido)hexyl]-3'-(2-pyridyldithio)propionamide, N-biotinyl-N'-(3-maleimidopropionyl)-3,6-dioxaoctane-1,8-diamine, maleimido-PEG11-biotin, N-iodoacetyl-N-biotinylhexylenediamine, (+)-biotinyl-iodoacetyl-N ... N-biotinyl-3,6-dioxaoctanediamine, 1-biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamide]butane (the first functional group is a sulfide group); N-biotinyl-3,6-dioxaoctane-1,8-diamine, N-biotinyl-3,6,9-trioxaundecane-1,11-diamine, N-biotinyl-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontane-1,35- diamine (all of which the first functional group is a carboxyl group); N-hydroxysuccinimide biotin, succinimidyl hexanoate-6-(biotinamide), N-hydroxysulfosuccinimide (NHS) ester of biotin (all of which the first functional group is an amino group); and alkoxyamine-PEG4-biotin, alkoxyamine-PEG12-biotin, biotinyl hydrazide, N''-biotinyl-6-aminohexanoyl hydrazide (all of which the first functional group is a carbonyl group). However, biotin derivatives are not limited to the above examples.

[0037] The biotin derivative may be a known compound. For example, the compounds described in JP-A-63-203683 and JP-A-63-246382 are known as biotin derivatives having a pyridyl group or a nitropyridyl group as a reactive group.

[0038] A detection label can be used to detect the first functional group labeled with a biotin derivative. The detection label may be attached to the biotin derivative used to detect the first functional group. The detection label may also be attached to a further compound (hereinafter, referred to as a "labeling compound") that is bound to the biotin derivative that labels the first functional group. In other words, in the evaluation method of this embodiment, a biotin derivative labeled for detection may be used to label the first functional group, or a labeling compound labeled for detection may be bound to the biotin derivative that labels the first functional group, and the first functional group may be detected by detecting the detection label. The use of the labeling compound may contribute to increasing the sensitivity and accuracy of the detection of the first functional group.

[0039] An example of the biotin derivative labeled for detection is a conjugate of the above-mentioned biotin derivative and a substance that can be a detection label. The biotin derivative that is a conjugate may be bound to the above-mentioned substance at the above-mentioned spacer portion, for example. The biotin derivative labeled for detection may be a conjugate of the biotin derivative and a labeling compound.

[0040] Examples of the detection label include a fluorescent label and a metal label. The fluorescent label can be made, for example, by a fluorescent substance such as a fluorescent dye. The metal label can be made, for example, by a metal species such as a metal nanoparticle. An example of a metal that can be used for the metal label is gold. The metal label may be made by gold nanoparticles. However, the detection label is not limited to the above examples.

[0041] The detection label may be made of a fluorescent substance that emits fluorescence with a wavelength of 500 nm or more. The wavelength of the fluorescence emitted by the fluorescent substance may be 520 nm or more, 530 nm or more, 540 nm or more, 550 nm or more, 565 nm or more, 575 nm or more, 600 nm or more, 620 nm or more, or even 640 nm or more. The detection label may be made of a fluorescent substance that emits fluorescence with a maximum wavelength in the above range. Hair itself has the property of emitting fluorescence in response to excitation light such as ultraviolet light (hereinafter, the fluorescence emitted by hair itself is referred to as "autofluorescence"). Autofluorescence can be a factor that reduces the sensitivity and accuracy of detection when a fluorescent substance is used as a detection label. Hair contains many components such as melanin, which has a wide absorption wavelength range, and tyrosine and tryptophan, which emit strong autofluorescence in the low wavelength range. The wavelength of the fluorescence emitted from the detection label is within the above range, which is suitable for suppressing the influence of autofluorescence in the evaluation of hair condition. Fluorescent substances that directly modify functional groups tend to emit fluorescence with a short wavelength and are easily affected by autofluorescence. On the other hand, the evaluation method of this embodiment allows for a high degree of freedom in the selection of fluorescent labels, and it is also possible to make the wavelength of the fluorescence emitted from the fluorescent label longer. This can contribute to, for example, improving sensitivity and accuracy by suppressing the effects of autofluorescence, and expanding the range of applicable applications.

[0042] Examples of the labeling compound are avidin and avidin derivatives. Examples of the avidin derivatives are streptavidin and neutravidin. Avidin and avidin derivatives are known to specifically bind to biotin derivatives. Avidin and avidin derivatives are particularly suitable for increasing the sensitivity and accuracy of detection. Examples of the detection labels that can be applied to the labeling compounds avidin and avidin derivatives, including preferred examples, are the same as the detection labels that can be applied to biotin derivatives. Examples of the avidin and avidin derivatives labeled for detection are conjugates of avidin or avidin derivatives and substances that can be detection labels. Examples of the conjugates are the Streptavidin Alexa Fluor series (manufactured by Jackson Immuno Research Laboratory), the Streptavidin DyLight series (manufactured by Vector Laboratories), and the Streptavidin Gold Colloidal Particle series (manufactured by Cytodiagnostics). The Streptavidin Alexa Fluor series and the Streptavidin DyLight series include those labeled with various fluorescent substances with different fluorescent wavelengths, such as the 594 conjugate and the 647 conjugate. The Streptavidin Gold Colloidal Particle series includes various gold nanoparticles with different particle sizes, such as 60 nm, that can be metal labeled. However, the labeled compounds and the labeled compounds labeled for detection are not limited to the above examples.

[0043] Labeling of the first functional group with a biotin derivative may be carried out in such a manner that another compound is interposed between the first functional group and the biotin derivative. However, labeling of the first functional group without the interposition of another compound is particularly suitable from the viewpoint of high freedom of selection and increasing the sensitivity and accuracy of detection. In other words, in the evaluation method of this embodiment, the first functional group may be labeled by directly binding the biotin derivative to the first functional group.

[0044] In the evaluation method of this embodiment, the first functional group may be detected using two or more types of biotin derivatives. In addition, two or more types of biotin derivatives having different reactive groups may be used to label two or more types of first functional groups, and two or more types of first functional groups labeled with biotin derivatives having different reactive groups may be detected. In this case, the detection labels may be the same or different for the detection of each first functional group. The detection labels being the same are suitable for detecting two or more types of first functional groups together. The detection labels being different are suitable for detecting each individual first functional group.

[0045] An example of a specific implementation of the evaluation method of this embodiment is shown in Fig. 1. The evaluation method in Fig. 1 includes step 11 of sectioning the hair to be evaluated, step 12 of washing the section of the hair obtained by sectioning, step 13 of treating a first cross section of the section with a first treatment solution containing a biotin derivative, step 14 of washing the section after the treatment, and step 15 of detecting the first functional group labeled with the biotin derivative on the first cross section.

[0046] In step 11, the part of the hair from which the section is taken may be a part close to the root or a part close to the tip. The part can be selected depending on the type of condition to be evaluated. Sectioning of the hair can be performed by a known method. The first cross section of the hair exposed by sectioning may be a cross section of the hair, or may be an oblique cross section or a longitudinal cross section. Evaluating the cross section of the hair is particularly suitable for evaluating, for example, the penetration state of the components contained in the hair composition into the hair. When exposing the cross section of the hair, the thickness of the section is, for example, 1 to 100 μm, and may be 1 to 50 μm, 1 to 30 μm, or 1 to 10 μm.

[0047] In step 12, foreign matter such as dust on the first cross section is removed. A buffer solution is suitable for washing the section. Examples of the buffer solution are Tris acetate buffer, phosphate buffer, and 2-morpholinoethanesulfonic acid (MES) buffer. Washing can be performed without heating. Removal of foreign matter may be performed to the extent that the first functional group can be detected with the desired sensitivity and accuracy. After washing, the section may be dried before the next step.

[0048] Step 13 can be performed, for example, by immersing the section in the first treatment solution. However, as long as the first functional group can be labeled with a biotin derivative, the method for performing step 13 is not limited. The treatment time and treatment temperature vary depending on the type of the first functional group and the biotin derivative, the shape of the section, and the type of condition to be evaluated, but are, for example, 30 minutes to 2 hours and 15 to 40°C.

[0049] The first treatment liquid is, for example, a solution in which a biotin derivative is dissolved in water or a buffer solution. Examples of the buffer solution are as described above. The first treatment liquid may contain an organic solvent such as dimethyl sulfoxide (DMSO) or 1,2-dichloroethane. When using a commercially available biotin derivative or a kit containing a biotin derivative for labeling, the first treatment liquid may be a solution recommended for the derivative or kit.

[0050] The biotin derivative contained in the first treatment liquid is usually labeled for detection.

[0051] Before immersion in the first treatment liquid, a pretreatment may be performed as necessary. An example of the pretreatment is an activation treatment in which the first functional group is activated to promote bonding with the reactive group. An example of the activation treatment is a treatment in which the carboxyl group, which is the first functional group, is activated by a water-soluble carbodiimide crosslinking agent to promote the formation of an amide bond with the amino group, which is the reactive group. The carboxyl group to be activated may be present in the cross section of the hair as, for example, the terminal of a carboxylic acid, a residue of aspartic acid, or a residue of glutamic acid.

[0052] In step 14, excess first processing solution adhering to the section is washed away. Step 14 can be performed in the same manner as step 12. A surfactant may be added to the buffer solution. An example of the surfactant is polysorbate 20.

[0053] The detection method carried out in step 15 can be selected according to the label for detection. However, as long as the first functional group labeled with a biotin derivative can be detected, the detection method is not limited. For example, a fluorescent microscope or a laser microscope can be used for detecting the fluorescent label. A confocal microscope may be used to detect the distribution of the first functional group in the thickness direction of the slice. For example, a scanning electron microscope (SEM) can be used for detecting the metal label. The detection of the first functional group by a microscope is particularly suitable for visualizing the distribution of the first functional group in the first cross section. In other words, the evaluation method of the present embodiment may further include visualizing the distribution of the first functional group inside the hair based on the information obtained by the detection of the first functional group. The visualized information, typically image information, is particularly suitable for, for example, visually grasping the state of the hair or comparing the state between slices. However, the information obtained by the detection of the first functional group may be used to evaluate the state of the hair by a method other than visualization.

[0054] Another example of a specific implementation of the evaluation method of this embodiment is shown in Figure 2. The evaluation method 2 in Figure 2 includes step 21 of sectioning the hair to be evaluated, step 22 of washing the section of the hair obtained by sectioning, step 23 of treating a first cross section of the section with a first treatment liquid containing a biotin derivative, step 24 of washing the section after treatment, step 25 of treating the first cross section of the section with a second treatment liquid containing a labeling compound, step 26 of washing the section after treatment, and step 27 of detecting a first functional group on the first cross section that has been labeled with a labeling compound via a biotin derivative.

[0055] Steps 21 to 24 can be carried out in the same manner as steps 11 to 14, respectively, except that the biotin derivative used in step 23 is generally not labeled for detection.

[0056] Step 25 can be performed, for example, by immersing the section in the second processing solution. However, as long as the first functional group can be labeled with a labeling compound, the method for performing step 25 is not limited. The processing time and processing temperature vary depending on the types of the first functional group and labeling compound, the shape of the section, and the type of condition to be evaluated, but are, for example, 30 minutes to 4 hours and 10 to 40°C.

[0057] The second treatment liquid is, for example, a solution in which a labeling compound is dissolved in water or a buffer solution. Examples of the buffer solution are as described above. The second treatment liquid may contain an organic solvent such as dimethyl sulfoxide (DMSO) or 1,2-dichloroethane. When using a commercially available labeling compound or a kit containing a labeling compound, the second treatment liquid may be a solution recommended for the labeling compound or kit.

[0058] In step 26, excess second processing solution adhering to the section is washed away. Step 26 can be performed in the same manner as step 12. A surfactant may be added to the buffer solution. An example of the surfactant is polysorbate 20.

[0059] Step 27 can be carried out in the same manner as step 15. However, the detection method is not limited as long as it is possible to detect the first functional group labeled with a labeling compound.

[0060] The evaluation method of the present embodiment may include any step other than those described above, so long as it is possible to evaluate the condition of hair.

[0061] For example, the evaluation method of this embodiment can be combined with modification of functional groups with a fluorescent substance. In one example of combination with modification of functional groups with a fluorescent substance, a second functional group (second target group) that exists in the second cross section of the hair and can be an indicator of the hair condition is detected in addition to the detection of the first functional group described above. The detection of the second functional group is performed by modifying the second functional group with a fluorescent substance. The modification can be performed by directly binding the fluorescent substance to the second functional group. For example, the distribution of both the first functional group and the second functional group inside the hair may be visualized based on the information obtained by detecting the first functional group and the information obtained by detecting the second functional group.

[0062] In other words, the evaluation method of the present embodiment may further include modifying the second functional group, which is present in the second cross section of the hair and can be an indicator of the hair condition, by directly bonding a fluorescent substance to the second functional group, detecting the second functional group modified with the fluorescent substance, and visualizing both the distribution of the first functional group and the second functional group inside the hair based on the information obtained by detecting the first functional group and the information obtained by detecting the second functional group. In one example of visualization, the distribution states of both the first functional group and the second functional group are displayed in the same image. However, the visualization mode is not limited to the above example.

[0063] The first cross section and the second cross section may be the same cross section or different cross sections. The first cross section and the second cross section may be mutually opposed cross sections of the same slice or may be cross sections of different slices.

[0064] Examples of the second functional group are the same as the examples of the first functional group. The first functional group and the second functional group may be the same or different. For the same functional group, depending on the type of functional group, the condition of the hair, and the part of the hair or the components of the hair composition that contains the functional group, detection by modification with a fluorescent substance may be more suitable. In this case, the above combination allows It is also possible to utilize the advantages of each detection method.

[0065] The fluorescent substance can be selected from those capable of modifying the second functional group. Examples of fluorescent substances that can be used to modify the second functional group are N-(9-acridinyl)maleimide (NAM) and N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM). NAM and DACM can modify sulfide groups.

[0066] The modification of the second functional group with a fluorescent substance can be carried out in the same manner as the labeling of the first functional group with a biotin derivative, except that a fluorescent substance is used instead of a biotin derivative. EXAMPLES

[0067] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0068] Example 1 The hair reduced with a reducing agent was collected from a region of about 3 cm from the root, and cut using a sliding microtome to obtain a section with a thickness of 10 μm. The cutting direction was the direction of the cross section of the hair. Next, the section was washed with Tris acetate buffer (hereinafter referred to as TAS buffer). The pH of the TAS buffer was 6.8, and it contained Trizma (trisaminomethane (hydroxymethyl)) and acetic acid as a Tris base, and also contained EDTA-2Na and sodium chloride. Next, the washed section was immersed in the first treatment solution to label the sulfide groups present on the cross section of the section. The first treatment liquid was prepared by dissolving a reagent kit (Biotin Labeling Kit-SH, DOJINDO, product code LK10) containing a biotin derivative (having a maleimide group as a reactive group) capable of specific reaction with sulfide groups in 10 μL of DMSO, and then diluting it 100-fold with sodium phosphate buffer (pH 7.4, containing CaCl2 at a concentration of 1.0 mmol / L). The immersion temperature and time were 37°C and 30 minutes, respectively.

[0069] Next, the slice was washed with TAS buffer, and then immersed in a second treatment solution containing streptavidin (Alexa Fluor 647 conjugate, product code 016-600-084, manufactured by Jackson Immuno Research Laboratory) labeled with a fluorescent substance that emits fluorescence at a maximum wavelength of 668 nm, to bind the streptavidin, which is a labeling compound, to the biotin derivative that labels the sulfide group. The temperature and time of immersion were 25°C and 2 hours, respectively. Next, the slice was washed with TAS buffer, and mounted with a mounting medium for fluorescent staining (VECTASHIELD Mounting Medium, product code H-1000, manufactured by Vector Laboratories) to obtain a test piece A for evaluation.

[0070] The hair that had been reduced with a reducing agent was collected from a region of about 3 cm from the root, and cut using a sliding microtome to obtain a section with a thickness of 10 μm. The cutting direction was the direction of the cross section of the hair. Next, the obtained test piece was treated with a treatment solution containing N-ethylmaleimide (NEM), which is known to specifically bind to sulfide groups, to block the sulfide groups contained in the section. Next, the treated section was washed in the same manner as test piece A, and treated with the first treatment solution and the second treatment solution to obtain test piece B for evaluation.

[0071] The cross sections of test piece A and test piece B were observed using a fluorescence microscope (Keyence, BZ-X810) and the results are shown in Figures 3 and 4, respectively. The excitation wavelength was 620 / 60 nm. Note that 620 / 60 nm indicates that the central wavelength of the excitation light obtained using an excitation filter is 620 nm and the bandwidth is 60 nm.

[0072] As shown in Figures 3 and 4, the distribution of fluorescence emitted from the fluorescent label was observed on the cross section of test piece A, but no fluorescence was observed on the cross section of test piece B, in which the sulfide groups were blocked by NEM. This confirmed that the sulfide groups present in the cross section of hair could be specifically detected and visualized by labeling with a biotin derivative.

[0073] Example 2 For one subject, untreated hair, hair reduced with a reducing agent, and hair oxidized with an oxidizing agent after reduction (presuming perm treatment) were each collected from a range of about 3 cm from the root and cut using a sliding microtome to make sections with a thickness of 10 μm. The cutting direction was the direction of the cross section of the hair. Next, each section was washed and treated with the first and second treatment liquids in the same manner as for test piece A in Example 1, to obtain test pieces C1 (untreated), C2 (reduced), and C3 (oxidized after reduction). The results of observing the cross sections of test pieces C1 to C3 using a fluorescent microscope in the same manner as in Example 1 are shown in Figures 5 to 7.

[0074] As shown in Figures 5 to 7, it was confirmed that sulfide groups are almost absent in the cross-section of untreated hair, except in the medulla, that they are generated in the cortex and cuticle by reduction treatment, and that they are reduced by the subsequent oxidation treatment.

[0075] Example 3 For one subject, untreated hair, hair reduced with a reducing agent, and hair oxidized with an oxidizing agent after reduction (assuming perm treatment) were each collected from a range of about 3 cm from the root, and cut using a sliding microtome to make sections with a thickness of 10 μm. The cutting direction was the direction of the cross section of the hair. Next, each section was washed and treated with the first and second treatment liquids in the same manner as for the test piece A in Example 1 to obtain test pieces D1 (untreated), D2 (reduced), and D3 (oxidized after reduction). However, for the first treatment liquid, N-biotinyl-3,6-dioxaoctane-1,8-diamine (manufactured by Thermo Fisher Scientific, 21346, having an amino group as a reactive group), which can specifically react with carboxyl groups, was diluted with MES buffer (pH 5.0) to a concentration of 50 mmol / L. After the sections were immersed in the first treatment liquid, a solution in which 1,2-dichloroethane was mixed with MES buffer to a concentration of 100 mmol / L was further added in a volume of 1 / 20 of the first treatment liquid to label the carboxyl groups present on the cross sections of the sections. The cross sections of test pieces D1 to D3 were observed under a fluorescent microscope in the same manner as in Example 1, and the results are shown in Figures 8 to 10.

[0076] 8 to 10, it was confirmed that detectable carboxyl groups were hardly present in the cross section of untreated hair except in the medulla, and that carboxyl groups thought to be derived from the reducing agent were present in the cortex and cuticle in the reduction-treated hair. It was also confirmed that the carboxyl groups in the cortex and cuticle almost disappeared due to the oxidation treatment.

[0077] Example 4 For one subject, untreated hair, hair reduced with a reducing agent, and hair oxidized with an oxidizing agent after reduction (assuming perm treatment) were each collected from a range of about 3 cm from the root, and cut using a sliding microtome to make sections of 10 μm thickness. The cutting direction was the direction of the cross section of the hair. Next, each section was washed and treated with the first and second treatment liquids in the same manner as for test piece A in Example 1 to obtain test pieces E1 (untreated), E2 (reduced), and E3 (reduced and then oxidized) for evaluation. However, for the first treatment liquid, N-hydroxysuccinimidobiotin (Thermo Fisher Scientific, 21217, having a succinimidyl ester group as a reactive group), which is capable of specific reaction with amino groups, was diluted with ultrapure water to a concentration of 10 mmol / L. The cross sections of test pieces E1 to E3 were observed under a fluorescent microscope in the same manner as in Example 1. The results of observation through a microscope are shown in FIGS.

[0078] 11 to 13, it was confirmed that a small amount of detectable amino groups exist in the cortex of untreated hair, that amino groups are generated mainly in the cortex of reduction-treated hair, and that the amino groups generated in the cortex do not disappear even by oxidation treatment. Since they do not disappear even by oxidation treatment, the generation of amino groups by reduction treatment may be due to protein denaturation.

[0079] Example 5 For one subject, untreated hair, hair reduced with a reducing agent, and hair oxidized with an oxidizing agent after reduction (assuming perm treatment) were each collected from a range of about 3 cm from the root, cut using a sliding microtome, and each was cut into a section with a thickness of 10 μm. The cutting direction was the direction of the cross section of the hair. Next, each section was washed in the same manner as the test piece A in Example 1, and treated with the first treatment liquid and the second treatment liquid to obtain test pieces F1 (untreated), F2 (reduced), and F3 (reduced and then oxidized). However, the labeled compound contained in the second treatment liquid was streptavidin (Streptavidin Gold Colloidal Particle AC-60-04-05, manufactured by Cytodiagnostics) metal-labeled with gold nanoparticles (particle diameter 60 nm). The cross sections of the test pieces F1 to F3 were observed by SEM (Hitachi High-Tech, TM3030), and the results are shown in FIGS.

[0080] 14 to 16, it was confirmed that sulfide groups are almost absent in the cross section of untreated hair, are generated in the cross section of hair by reduction treatment, and are reduced by the subsequent oxidation treatment. It was also confirmed that the use of a metal-labeled labeling compound allows detection and visualization of sulfide groups labeled with a biotin derivative.

[0081] Example 6 For one subject, untreated hair, hair reduced with a reducing agent, and hair oxidized with an oxidizing agent after reduction (assuming perm treatment) were each collected from a range of about 3 cm from the root, and cut using a sliding microtome to make sections of 10 μm thickness. The cutting direction was the direction of the cross section of the hair. Next, each section was treated with a third treatment liquid containing DACM to modify the sulfide groups present on the cross section, thereby obtaining test pieces G1 (untreated), G2 (reduced), and G3 (oxidized after reduction). The treatment with the third treatment liquid was carried out in the same manner as the treatment with the first treatment liquid in Example 1, except that the third treatment liquid was used instead of the first treatment liquid, and the immersion temperature and time were room temperature and 1 minute. For the third treatment liquid, a solution of DACM dissolved in a small amount of acetone and TAS buffer added to adjust the concentration to 0.01 mol / L was used. After treatment with the third treatment liquid, each section was washed with a TAS buffer solution and mounted with a mounting medium for fluorescent staining (Vector Laboratories, VECTASHIELD Mounting Medium, product code H-1000) to give test pieces G1 to G3.

[0082] FIG. 17 shows the result of superimposing the image information of the test pieces G1 to G3 obtained by observing the cross section with a fluorescent microscope in the same manner as in Example 1 on the image information of the test pieces C1 to C3 obtained in Example 2. As shown in FIG. 17, in the image information of the test pieces C1 to C3, the distribution of sulfide groups that cannot be confirmed in the image information of the test pieces G1 to G3 was confirmed mainly in the hair cuticle and the cortex. On the other hand, the sulfide groups remaining in the hair cuticle even after the oxidation treatment were not confirmed in the image information of the test piece C3, but were confirmed in the image information of the test piece G3. The sulfide groups generated in the hair cuticle include those derived from the decomposition of thioester bonds, and it may be advantageous to combine detection by DACM for the detection of the sulfide groups. From the above, it was confirmed that it is possible to visualize sulfide groups in different environments by combining the evaluation methods.

[0083] (Reference example: autofluorescence of hair) Hair from one subject was collected from a range of about 3 cm from the root, and was in each of the following states: untreated, permed once using a reducing agent and an oxidizing agent, and bleached once using a bleaching agent. Next, the hair in each state was cut using a sliding microtome to make sections of 10 μm thickness. The cutting direction was the direction of the cross section of the hair. The obtained sections were washed and mounted in a mounting medium for fluorescent staining (VECTASHIELD Mounting Medium, product code H-1000, manufactured by Vector Laboratories) to make test pieces H1 (untreated), H2 (permed), and H3 (bleached) for evaluating autofluorescence. The results of observing the cross sections of test pieces H1 to H3 with a fluorescent microscope in the same manner as in Example 1 are shown in FIG. 18. However, in this observation, the intensity of the excitation light was increased compared to the observation of fluorescence in each of the above examples in order to make the autofluorescence easier to understand. As shown in Figure 18, autofluorescence was observed mainly at wavelengths of 535 nm or less, particularly at wavelengths of 450-470 nm, in all of the test pieces H1 to H3. Autofluorescence was particularly strong in the cuticle of untreated and permed hair, and in the hair follicle of bleached hair. In addition, when a similar evaluation was performed with an increased number of perm and bleach treatments, a tendency was observed in which the autofluorescence became stronger with increasing number of treatments. [Industrial Applicability]

[0084] The technology of the present invention can be applied, for example, to the development of hair compositions. [Explanation of symbols]

[0085] 11~15 steps 21~27 steps

Claims

1. 1. A method for assessing hair condition, comprising: labeling a first functional group present in a first cross section of the hair and capable of being an indicator of the condition with a biotin derivative that specifically binds to the first functional group; detecting the first functional group labeled with the biotin derivative; The method includes:

2. The method of claim 1 , wherein the first functional group is a sulfide group, a carboxyl group, an amino group, or a carbonyl group.

3. using the detectably labeled biotin derivative to label the first functional group or binding a further detectably labeled compound to the biotin derivative labeling the first functional group; The method of claim 1 , wherein the first functional group is detected by detecting the detectable label.

4. The method of claim 3 , wherein the detectable label is a fluorescent label or a metal label.

5. The method according to claim 3, wherein the detection label is a fluorescent substance that emits fluorescence with a wavelength of 500 nm or more.

6. The method of claim 3 , wherein the further compound is avidin or an avidin derivative.

7. The method of claim 1 , wherein the first cross-section is a transverse cross-section of the hair.

8. The method of claim 1 , wherein the first functional group is labeled by directly binding the biotin derivative to the first functional group.

9. The method according to claim 1 , further comprising visualizing a distribution of the first functional group inside the hair based on information obtained by detecting the first functional group.

10. modifying a second functional group that is present in a second cross section of the hair and that can be an indicator of the condition by directly binding a fluorescent substance to the second functional group; detecting the second functional group modified with the fluorescent substance; visualizing the distribution of both the first functional group and the second functional group inside the hair based on information obtained by detecting the first functional group and information obtained by detecting the second functional group; The method of claim 1 further comprising:

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