METHOD FOR ESTIMATING pH INSIDE HAIR

Abstract

To provide a method for estimating pH at a specific site inside hair.SOLUTION: A method for estimating pH of a specific site inside hair, comprises: preparing the cross-sectional slice of the hair; contacting the hair or the cross-sectional slice with a solution of a pH-sensitive reagent before or after the cross-sectional slice preparation step; and observing the cross-sectional slice after contacting with the solution by an optical microscope and estimating the pH of the specific site on the basis of a color at the specific site, or irradiating the cross-sectional slice after contacting with the solution with excitation light of a specific wavelength, observing the same by a fluorescent microscope and estimating the pH of the specific site on the basis of the intensity of fluorescence of the specific wavelength at the specific site.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a method for estimating the pH inside hair.

Background Art

[0002] Hair bleaching agents, oxidative hair dyes, etc. are widely used in a two-agent type consisting of a first agent containing an alkaline agent and a second agent containing an oxidizing agent such as hydrogen peroxide. When hair is treated with such hair bleaching agents, oxidative hair dyes, etc., even if it is thoroughly washed, the alkaline agent cannot be completely removed and a certain amount remains. Therefore, it is required to measure the amount of the remaining alkaline agent.

[0003] As a method for measuring the pH inside hair, for example, the methods described in Patent Documents 1 and 2 are known. Patent Document 1 describes a hair treatment method in which hair is dyed with an oxidative hair dye and an after-treatment agent is applied to the hair to adjust the pH inside the hair to 7.5 to 9. In the examples, 1 g of hair was cryogenically pulverized to 1 mm or less with liquid nitrogen, immersed in 100 ml of distilled water at 50 °C for 10 minutes, cooled to 25 °C, and the pH of the aqueous solution was measured and used as the pH inside the hair.

[0004] Patent Document 2 describes an acidic hair dye composition in which the pH of hair after dyeing is 5.5 to 6. In the examples, 1 g of hair cut to about 5 mm was frozen with liquid nitrogen, pulverized, 2 g of purified water was added to 0.5 g of hair powder to form a paste-like aqueous solution, and the pH of the aqueous solution was measured and used as the pH inside the hair.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] In Patent Documents 1 and 2, since the pH in the hair is obtained by adding hair powder to water to form a paste-like aqueous solution, the measured pH is different from the pH inside the hair itself. Further, in the methods of Patent Documents 1 and 2, it is impossible to measure the pH of specific sites inside the hair, such as the cuticle (hair cuticle), cortex (hair cortex), and medulla (hair medulla). Therefore, an object of the present invention is to provide a method for estimating the pH of a specific site inside the hair. [Means for Solving the Problems]

[0007] As a result of intensive studies to solve the above problems, the present inventors focused on a cross-sectional slice of hair after contacting it with a solution of a pH-sensitive reagent, and found that the pH for each specific site can be estimated over the entire cross-sectional slice of the hair based on the color at the specific site or the intensity of fluorescence at a specific wavelength at the specific site, thereby completing the present invention.

[0008] That is, the present invention includes the following inventions. [1] A method for estimating the pH of a specific site inside the hair, comprising: a step of preparing a cross-sectional slice of the hair; a step of contacting the hair or the cross-sectional slice with a solution of a pH-sensitive reagent, respectively, before or after the step of preparing the cross-sectional slice; and observing the cross-sectional slice after contacting the solution with an optical microscope, and estimating the pH of the specific site based on the color at the specific site, or irradiating the cross-sectional slice after contacting the solution with excitation light of a specific wavelength and observing it with a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence at the specific wavelength at the specific site.

[0009] [2] A method for estimating the pH of a specific site inside the hair, comprising: a step of preparing a cross-sectional slice of the hair; Thereafter, a step of bringing a solution of a pH-sensitive reagent into contact with the cross-sectional slice, and observing the cross-sectional slice after bringing the solution into contact with an optical microscope, and estimating the pH of the specific site based on the color at the specific site, or irradiating the cross-sectional slice after bringing the solution into contact with excitation light of a specific wavelength and observing with a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site, a method comprising the steps.

[0010] [3] The pH-sensitive reagent is a pH-sensitive fluorescent dye, irradiating the cross-sectional slice after bringing the solution into contact with excitation light of a specific wavelength and observing with a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site, the method according to [1] or [2].

[0011] [4] Irradiating the cross-sectional slice after bringing the solution into contact with excitation light of a specific wavelength and observing with a fluorescence microscope, measuring the intensity of fluorescence of the first wavelength at the specific site, irradiating the cross-sectional slice after bringing the solution into contact with excitation light of the same or different wavelength as the specific wavelength and observing with a fluorescence microscope, measuring the intensity of fluorescence of the second wavelength at the specific site, estimating the pH of the specific site based on the intensity ratio of the fluorescence intensity of the first wavelength and the fluorescence intensity of the second wavelength, the method according to [3]. [Advantages of the Invention]

[0012] By the method for estimating the pH of a specific site inside a hair of the present invention, the pH of each specific site inside the hair can be estimated over the entire cross-sectional slice of the hair. [Brief Description of the Drawings]

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0014] Based on an embodiment of the present invention (hereinafter referred to as "the present embodiment"), the present invention will be described below.

[0015] The method for estimating the pH of a specific site inside the hair of the present embodiment is a step of preparing a cross-sectional slice of the hair, a step of bringing a solution of a pH-sensitive reagent into contact with the hair or the cross-sectional slice, respectively, before or after the step of preparing the cross-sectional slice, and observing the cross-sectional slice after bringing the solution into contact with an optical microscope, and estimating the pH of the specific site based on the color at the specific site, or irradiating the cross-sectional slice after bringing the solution into contact with excitation light of a specific wavelength and observing with a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site, the method comprising.

[0016] <Step of preparing a cross-sectional slice of hair> A cross-sectional slice of hair can be prepared according to a conventional method. Examples of the thickness of the cross-sectional slice include 2 to 50 μm, preferably 5 to 40 μm, and more preferably 10 to 30 μm. By having a thickness within this range, the intensity of fluorescence of a specific wavelength at a specific site of the hair can be observed.

[0017] The surface of the cross-sectional slice of hair is preferably smooth. For this purpose, it is preferable to embed and cut the hair with some solid substance in advance. Examples of the solid substance include ice or resins used for preparing slices of transmission electron microscopes. In the step of bringing the solution of the pH-sensitive reagent into contact with the cross-sectional slice of hair, since the solid substance can be removed as ice or water, ice is preferable as the solid substance. Examples of the above resin include epoxy resin, polyester resin, methacrylate resin, acrylic resin, melamine resin, polyacrylamide resin, etc. Embedding in the resin can be carried out according to the conventional method depending on the characteristics of each resin. Also, a method can be used in which the hair is placed on celluloid, sandwiched and fixed while dissolving the celluloid plate with a solvent such as ethyl acetate, and then a cut surface is formed.

[0018] To form the cut surface of the sample, a knife or the like usually used for preparing slices of transmission electron microscopes can be used. To form a smoother cut surface, it is preferable to use a diamond knife. More specifically, after trimming using a glass knife, a grinder, or a single-edge razor degreased with acetone, the surface can be planed with a diamond knife. As the cutting device, for example, a cryomicrotome equipped with a glass knife (such as "HistoCore NANOCUT R" manufactured by Leica Microsystems and "Electronic Sample Freezing Device EF-12" manufactured by Nippon Microtome Laboratory) can be used.

[0019] <Before or after the step of preparing the cross-sectional slice, the solution of the pH-sensitive reagent is brought into contact with the hair or the cross-sectional slice, respectively> In this step, before or after the step of preparing the cross-sectional slice, the solution of the pH-sensitive reagent is brought into contact with the hair or the cross-sectional slice, respectively. It is also possible to bring the solution of the pH-sensitive reagent into contact with the hair before the adjustment step of the cross-sectional slice, but preferably, the solution of the pH-sensitive reagent is brought into contact with the cross-sectional slice after the step of preparing the cross-sectional slice. By bringing an appropriate amount of the solution of the pH-sensitive reagent into contact with the hair or the cross-sectional slice, the solution of the pH-sensitive reagent penetrates into the interior of the hair or the interior of the cross-sectional slice. In the step of preparing the cross-sectional slice, when the hair is cut by embedding it in ice, it is preferable because the solution of the pH-sensitive reagent penetrates into the interior of the cross-sectional slice of the hair from the side surface of the cross-sectional slice of the hair.

[0020] As the pH-sensitive reagent used in this embodiment, any reagent can be used as long as it can observe the intensity of fluorescence at a specific wavelength at a specific site of the hair. Specifically, for example, a universal pH reagent or the like, and a pH-sensitive fluorescent reagent can be used. Examples of the pH-sensitive fluorescent reagent include 4-(naphthalen-2-yl)-1H-imidazo[4,5-c]pyridine·TFA (LysoGlow84, Reference 1: Mar. Drugs, 2015, 13, 920-935), a rhodamine-based pH probe: SiRpH5 and Me-pEPPR (Reference 2: J. Am. Chem. Soc., 2018 May 9;140(18):5925-5933; Chemistry and Education, 68(7), 306-309), BCECF AM (Reference 3: J. Appl. Bacteriol, 79, 4, 399-408(1995)), ageladine A (Reference 4: Mar. Drugs, 2012, 10, 223-233), pHrodo (Reference 5: ASCB, 2013, Poster B1994), pH-sensitive melamine formaldehyde-pyronin nile blue fine particles (Reference 6: Sensors, 2010, 20, 5243), and the like. Preferably, a pH-sensitive fluorescent reagent in which the intensity of fluorescence at the first wavelength and the intensity of fluorescence at the second wavelength differ depending on the pH can be mentioned. Examples of this pH-sensitive fluorescent reagent include LysoGlow84, SiRpH5 and Me-pEPPR, BCECF AM, etc., and more preferably LysoGlow84. These pH-sensitive fluorescent reagents can more accurately estimate the pH using the ratio of the intensities of the first and second fluorescences without being affected by the difference in the concentration of the pH-sensitive fluorescent reagent on the specific site.

[0021] When using, for example, a universal pH reagent as the pH-sensitive reagent, a cross-sectional slice contacted with the solution of the universal pH reagent can be observed with an optical microscope, and the pH of the specific site can be estimated based on the color at the specific site. Specifically, using an optical microscope image of a cross-sectional slice not contacted with a solution such as a universal pH reagent as a blank control, subtracting the blank control from the optical microscope image of a cross-sectional slice contacted with the solution of the universal pH reagent, the pH can be estimated based on the color of the specific site. Since this method is easy to distinguish colors, it is preferably lighter hair. In the case of black hair, since the color difference of the cross-sectional slice contacted with the solution of the universal pH reagent is not large compared to the blank control, it is not easy to estimate the pH.

[0022] <A step of irradiating a cross-sectional slice after contacting the solution with excitation light of a specific wavelength, observing with a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site> When using a pH-sensitive fluorescent reagent as the pH-sensitive reagent, a cross-sectional slice contacted with the solution of the pH-sensitive fluorescent reagent is irradiated with excitation light of a specific wavelength and observed with a fluorescence microscope, and the pH of the specific site can be estimated based on the intensity of fluorescence of the specific wavelength at the specific site. Specifically, assuming that a certain amount of the solution of the pH-sensitive fluorescent reagent has penetrated into the cross-sectional slice of the hair, it is irradiated with excitation light of a specific wavelength and observed with a fluorescence microscope, and the pH of the specific site can be estimated based on the intensity of fluorescence of the specific wavelength at the specific site.

[0023] It is preferable to use a pH-sensitive fluorescent reagent in which the intensity of fluorescence of the first wavelength and the intensity of fluorescence of the second wavelength differ depending on the pH. Examples of this pH-sensitive fluorescent reagent include LysoGlow84, SiRpH5, Me-pEPPR, BCECF AM, etc., and more preferably LysoGlow84. Specifically, after the solution is brought into contact, the cross-sectional slice is irradiated with excitation light of a specific wavelength and observed with a fluorescence microscope. The intensity of fluorescence at the first wavelength at the specific site is measured. The cross-sectional slice after the solution is brought into contact is irradiated with excitation light of the same or different wavelength as the specific wavelength and observed with a fluorescence microscope. The intensity of fluorescence at the second wavelength at the specific site is measured. A method for estimating the pH of the specific site based on the intensity ratio of the fluorescence intensity at the first wavelength and the fluorescence intensity at the second wavelength can be mentioned.

[0024] Since this method estimates pH based on the fluorescence intensity, there is little influence due to the color of the hair, and the pH inside the hair can be estimated for hair of any color.

Example

[0025] The present invention will be described below with reference to examples, but the present invention is not limited to the examples.

[0026] <pH-Sensitive Fluorescent Dye Solution> pH-sensitive fluorescent dye: 1 g of 4-(naphthalen-2-yl)-1H-imidazo[4,5-c]pyridine·TFA (LysoGlow84, manufactured by Funakoshi) was dissolved in 0.1 mL of dimethyl sulfoxide (DMSO, manufactured by Sigma-Aldrich) to obtain a pH-sensitive fluorescent dye solution.

[0027] <Calibration pH-Sensitive Fluorescent Dye Solution> The above pH-sensitive fluorescent dye solution was dropped into 1 mL of a 0.1 M phosphoric acid aqueous solution with a pre-prepared pH of 7 or 9 and stirred well to obtain a calibration pH-sensitive fluorescent dye solution.

[0028] Reference Example 1 <Ratio of Fluorescence Intensity at 400 nm to Fluorescence Intensity at 440 nm (Blank Coefficient K)> A calibration pH-sensitive fluorescent dye solution was dropped onto a blank slide glass without placing a cross-sectional slice of hair, and a cover glass was placed over it. Immediately, an excitation filter (BP330 - 385, manufactured by Olympus, transmitting light with wavelengths in the range of 330 - 380 nm), two types of absorption filters (HM band-pass filter 400 nm, HM band-pass filter 440 nm, manufactured by Asahi Spectra Co., Ltd., transmitting light with wavelengths in the range of 400 nm or 440 nm), a dichroic mirror (T387lp, manufactured by Chroma Technology Corp, transmitting light with wavelengths of 387 nm or more), and a high-pressure mercury lamp as a light source were attached to a fluorescence microscope (BX2-FL, manufactured by Evident), and the obtained slide glass was installed. Using the absorption filters of 400 nm and 440 nm, the exposure time was fixed (34 ms), and imaging was performed with a CCD camera. The fluorescence intensity at 400 nm and the fluorescence intensity at 440 nm were measured. The ratio of the obtained fluorescence intensity at 400 nm to the fluorescence intensity at 440 nm was calculated to obtain the blank coefficient K.

[0029] Comparative Example 1 <Fluorescence Observation of Cross-Sectional Slices of Untreated Hair> An untreated hair bundle made from hair collected at a beauty salon was wrapped with ice, and a cryomicrotome (「HistoCore NANOCUT R」manufactured by Leica Microsystems GmbH and 「Electronic Sample Freezing Device EF-12」manufactured by Nippon Microtome Laboratory Co., Ltd.) equipped with a glass knife was used to prepare cross-sectional slices of hair with a thickness of 20 μm. The obtained cross-sectional slices of untreated hair were placed on a slide glass, a cover glass was placed over them, and purified water was poured in from the side gaps. The obtained slide glass was immediately installed in a fluorescence microscope. Subsequently, using the absorption filters of 400 nm and 440 nm, the exposure time was fixed (34 ms), and imaging before fluorescence staining was performed with a CCD camera. After imaging, it was dried at room temperature, then the above pH-sensitive fluorescent dye solution was applied and left at room temperature for 24 hours. Subsequently, purified water was poured in again, and it was immediately attached to a fluorescence microscope equipped with a CCD camera. Using the absorption filters of 400 nm and 440 nm, the exposure time was fixed (34 ms), and imaging after fluorescence staining was performed with a CCD camera.

[0030] The measurement results of the fluorescence intensity ratio of untreated hair are shown in Fig. 2. Fluorescence photographs were taken at wavelengths of 400 nm and 440 nm before and after fluorescence staining. The obtained photographs were imported into a personal computer, and the fluorescence intensity of each part of the binarized image was quantified in the range of 0 to 255 using image analysis software (ImageJ, manufactured by National Institutes of Health). For the obtained numerical values, the filter was changed at the same position for measurement, and the ratio of the two was calculated using the obtained values. This ratio was multiplied by the coefficient K of the blank obtained with the pH-sensitive fluorescent dye solution for calibration at pH 7 and 9 described above to correct the value of the ratio. After that, for each wavelength, the post-staining one was subtracted from the pre-staining one to obtain an image of the fluorescence intensity (C1) after subtraction at 400 nm and an image of the fluorescence intensity (C2) after subtraction at 440 nm. Thereafter, the image of C2 was divided by the image of C1 to obtain a division image (D). Thereafter, using the image processing software, an intensity profile in the equatorial direction of the observation image of the hair cross-section was obtained. The ratio (R) of the fluorescence intensity at 400 nm and the fluorescence intensity at 440 nm at each position was determined.

[0031] <Estimation of pH based on the relationship between the fluorescence intensity ratio (R) and pH> Fig. 1 is Fig. 3D of Reference 1 (Mar. Drugs, 2015, 13, 920 - 935). It shows the ratio of the fluorescence intensities of LysoGlow84 at 400 nm and 440 nm depending on pH. Based on this graph, what was obtained for the relationship between the ratio (R) of the fluorescence intensities at 400 nm and 440 nm and pH is shown in Table 1.

[0032]

Table 1

[0033] Based on the relationship between the ratio (R) of the fluorescence intensities at 400 nm and 440 nm in Table 1 and pH, and assuming that the pH values at both ends sandwiching the fluorescence intensity ratio are linear functions, the pH of the measurement site was estimated from the obtained corrected ratio values. The results are shown in Figure 2 and Table 1. When calculating the pH of the part corresponding to the cortex, the pH of the cortex part of the untreated hair was 6.24 (R = 0.72).

[0034] Example 1 <Estimation of the internal pH of hair after treatment with an aqueous solution of an oxidizing agent containing monoethanolamine> 20 mL of purified water was added with 30.3 g of 35% hydrogen peroxide solution and stirred, and an 80% aqueous solution of monoethanolamine was added to adjust the pH to 10, thereby preparing an aqueous solution of an oxidizing agent containing monoethanolamine.

[0035] An untreated hair bundle made from hair collected at a beauty salon was immersed in an aqueous solution of an oxidizing agent containing monoethanolamine at a bath ratio of 1:50 (w / v), and after 10 minutes or 30 minutes, it was pulled out. Then, it was rinsed with running water for 20 seconds, an aqueous solution of 1% sodium dodecyl sulfate was applied at 1:1 (w / v), and rubbing and washing were performed for 30 seconds. After that, it was rinsed again with running water for 20 seconds, the surface moisture was blotted with a towel, and it was completely dried using a dryer to obtain a hair sample. The obtained hair sample was wrapped with ice, and a cross-sectional slice of the hair with a thickness of 20 μm was prepared using a cryomicrotome (「HistoCore NANOCUT R」manufactured by Leica Microsystems and 「Electronic Sample Freezing Device EF-12」manufactured by Nippon Microtome Laboratories) equipped with a glass knife.

[0036] In the same manner as in Comparative Example 1, the pH of the cortex part was estimated. The results are shown in Figure 3 and Table 2. At a treatment time of 10 minutes, it was 9.39 (R = 1.44), and at 30 minutes, it was 9.78 (R = 1.46). Compared with the untreated hair, the pH increased significantly, and the pH also increased slightly with the increase in the treatment time.

[0037] Example 2 <Estimation of the pH inside hair after treatment with an aqueous oxidizing agent solution containing aqueous ammonia> Instead of the 80% aqueous monoethanolamine solution, 28% aqueous ammonia was used to prepare an aqueous oxidizing agent solution containing aqueous ammonia with a pH of 10 in the same manner. In the same manner as in Example 1, a cross-sectional section of hair with a thickness of 20 μm was prepared. Subsequently, in the same manner as in Comparative Example 1, the pH of the cortex portion was estimated. The results are shown in FIG. 4 and Table 2. At a treatment time of 10 minutes, the pH was 9.16 (R = 1.42), and at 30 minutes, it was 9.0 (R = 1.4).

[0038]

Table 2

[0039] The embodiments and examples disclosed this time are illustrative in all respects and not restrictive. The scope of the present invention is shown not by the above description but by the claims, and includes all modifications within the meaning and scope equivalent to the claims.

Claims

1. A method for estimating the pH of a specific site inside a hair, comprising: a step of preparing a cross-sectional slice of the hair; a step of bringing a solution of a pH-sensitive reagent into contact with the hair or the cross-sectional slice, respectively, before or after the step of preparing the cross-sectional slice; and observing the cross-sectional slice after the solution has been brought into contact therewith under an optical microscope, and estimating the pH of the specific site based on the color at the specific site, or irradiating the cross-sectional slice after the solution has been brought into contact therewith with excitation light of a specific wavelength, observing it under a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site.

2. A method for estimating the pH of a specific site inside a hair, comprising: a step of preparing a cross-sectional slice of the hair; subsequently, a step of bringing a solution of a pH-sensitive reagent into contact with the cross-sectional slice; and observing the cross-sectional slice after the solution has been brought into contact therewith under an optical microscope, and estimating the pH of the specific site based on the color at the specific site, or irradiating the cross-sectional slice after the solution has been brought into contact therewith with excitation light of a specific wavelength, observing it under a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site.

3. The method according to claim 1 or 2, wherein the pH-sensitive reagent is a pH-sensitive fluorescent dye, and irradiating the cross-sectional slice after the solution has been brought into contact therewith with excitation light of a specific wavelength, observing it under a fluorescence microscope, and estimating the pH of the specific site based on the intensity of fluorescence of the specific wavelength at the specific site.

4. irradiating the cross-sectional slice after the solution has been brought into contact therewith with excitation light of a specific wavelength, observing it under a fluorescence microscope, and measuring the intensity of fluorescence of a first wavelength at the specific site; irradiating the cross-sectional slice after the solution has been brought into contact therewith with excitation light of the same or a different wavelength as the specific wavelength, observing it under a fluorescence microscope, and measuring the intensity of fluorescence of a second wavelength at the specific site; and estimating the pH of the specific site based on the intensity ratio of the fluorescence intensity of the first wavelength to the fluorescence intensity of the second wavelength.

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