Novel method for evaluating hair roughness and gloss in experimental animal

Abstract

The present invention relates to a novel method for evaluating hair roughness and gloss in an experimental animal and, more specifically, to a method for evaluating hair roughness and gloss in an experimental animal, the method comprising the steps of: inducing hair roughness by ultraviolet irradiation; inducing hair growth after administration of the test material; and evaluating a change in hair roughness by analyzing with a program. The method for evaluating hair roughness and gloss of the present invention can clearly evaluate hair gloss in an animal test by using a program. In the existing visual evaluation, only skilled researchers can perform the evaluation, but in the present invention, anyone can perform the evaluation by utilizing a program, thereby expanding the scope of the evaluator. In addition, the accuracy of evaluation can be improved by controlling an environment in which an error may occur by photographing hair under the same conditions (curvature, distance, and illuminance). Therefore, the present invention can be usefully used for preclinical animal test evaluation of hair-related health functional foods.

Description

A novel method for evaluating hair roughness and gloss in laboratory animals

[0001] The present invention relates to a novel method for evaluating hair roughness and gloss in laboratory animals, and more particularly, to a method for evaluating hair roughness and gloss in animals, comprising the steps of: inducing hair roughness by ultraviolet irradiation; inducing hair growth after administering a test substance; and evaluating changes in hair roughness by analyzing with a program.

[0002] Hair plays a variety of roles in the human body, including protecting the head, influencing one's appearance, and maintaining head temperature. While not essential for life, it is a vital part of the body, serving as a barometer of health and determining one's appearance. However, in modern society, hair health is frequently deteriorating due to natural factors such as environmental pollution and strong UV rays, as well as physiological factors like stress and hormonal imbalances. This has created a pressing need for technological development to prevent and treat hair loss.

[0003] As of 2022, the domestic hair care market size exceeded KRW 1.5 trillion, and the domestic hair loss market is estimated to be worth KRW 4 trillion, with annual growth. Furthermore, according to a report by the Health Functional Food Association, self-medication has recently become a significant trend, leading to rapid growth in interest in and the market for hair health functional foods.

[0004] With growing interest in hair health, the National Institute of Food and Drug Safety Evaluation (NIFDA) has published a guide to evaluating the functionality of health functional foods (a guide for complainants) titled "Hair Health." Hair health biomarkers include antioxidant activity, anti-inflammation, cell proliferation, hair follicle cycle regulation, and clinical evaluation. Clinical evaluations are further categorized into changes in hair luster, elasticity, and density. While visual and electron microscopic evaluation methods for human hair luster are specifically defined, a clear method for evaluating hair luster in animal testing has not yet been established.

[0005] The hair growth cycle of C57BL / 6 mice consists of three stages: the anagen phase, when hair grows most actively; the catagen phase, when hair begins to decline; and the telogen phase, when hair stops growing or enters the resting phase. After about 45 days of age, the hair follicles of C57BL / 6 mice enter the second telogen phase, and the body surface of the mice in the telogen phase turns pink. Since each mouse has the same hair follicle cycle during this period, the effects of drugs that regulate the hair follicle cycle, that is, induce the transition from the telogen to the anagen phase, can be measured. For this reason, C57BL / 6 mice can be useful for experiments examining changes in the hair cycle.

[0006]

[0007] Accordingly, the inventors of the present invention selected 6-week-old C57BL / 6 mice, which are in the resting phase of the hair growth cycle, which is the best time to observe hair growth changes, and completed the present invention by newly establishing a method for evaluating hair roughness and gloss at the animal testing stage.

[0008] The problem to be solved by the present invention is to provide a new method for evaluating hair roughness and gloss at the animal testing stage, and specifically, to provide a method for evaluating animal hair roughness and gloss, which includes a step of inducing hair roughness by ultraviolet irradiation and a step of evaluating changes in hair roughness by analyzing the hair using a program after hair growth.

[0009] In order to solve the above problem, the present invention provides a method for evaluating animal hair roughness and gloss, comprising the steps of (a) inducing hair roughness by ultraviolet irradiation; (b) inducing hair growth after administering a test substance; and (c) evaluating changes in hair gloss, wherein the step of evaluating changes in hair gloss in step (c) comprises the steps of: i) extracting and fixing skin at a hair-grown area; ii) taking a photograph of the hair; and iii) analyzing the taken photograph.

[0010] The fixation in step i) above may be to spread out the removed skin and then fix it flat on a cylindrical tool.

[0011] The analysis of the above step iii) may be performed by measuring the pixel value according to the hair gloss area and gloss after cropping the photograph in the same way and analyzing it using a program.

[0012] By measuring the above pixel values ​​and analyzing them with a program, pixel values ​​0 to 150 can be analyzed as rough, 151 to 300 as normal, and 301 or higher as glossy.

[0013] The ultraviolet irradiation in step (a) above is ultraviolet-B (UV-B) at 40 to 60 mJ / cm 2 It can be investigated by concentration.

[0014] The administration in step (b) above may be one or more selected from the group consisting of oral administration, skin application, subcutaneous injection, and device application.

[0015] The step of removing hair from the animal may be additionally included prior to the above step (a).

[0016] The above animal may be a mouse.

[0017] The hair roughness and gloss evaluation method of the present invention utilizes a program to clearly assess hair gloss in animal testing. While conventional visual evaluations can only be performed by experienced researchers, the program allows anyone to perform the evaluation, expanding the scope of evaluators. Furthermore, by photographing hair under identical conditions (curvature, distance, and illumination), the accuracy of the evaluation can be improved by controlling the environment where errors may occur. Therefore, the present invention can be usefully utilized in preclinical animal testing of hair-related health functional foods.

[0018] Figure 1 shows a series of processes for obtaining an animal, removing hair, inducing roughness, inducing hair growth for a certain period of time, and evaluating the degree of improvement in hair luster.

[0019] Figure 2 shows the process of removing and photographing mouse skin to evaluate hair roughness and gloss.

[0020] Figure 3 shows a roughness and gloss visual evaluation grading table, Figure 4 shows a roughness and gloss program evaluation grading table, and Figure 5 shows a visual evaluation and program evaluation correlation prediction table.

[0021] Figure 6 is a photograph showing the skin of a mouse's back after 7 days of UV-B irradiation, which serves as basis for setting the UV-B concentration.

[0022] Figure 7 shows the induction of hair roughness after UV-B irradiation, with UV-B concentrations of 0, 40, and 60 mJ / cm 2The evaluation was conducted by A. A represents the results of the visual evaluation, B represents the results of the program evaluation, and C represents the correlation between the visual evaluation and the program evaluation.

[0023] Figure 8 shows the improvement in hair luster after ingestion of the test substance. Negative control group, 60 mJ / cm 2 , 60 mJ / cm 2 + The evaluation was conducted by dividing the test substance group into groups. A represents the results of the visual evaluation, B represents the results of the program evaluation, and C represents the correlation between the visual evaluation and the program evaluation. In this case, minoxidil was used as the test substance to improve hair shine.

[0024] The inventors of the present invention selected 6-week-old C57BL / 6 mice, which are in the resting phase of the hair growth cycle, which is the best time to observe hair growth changes, and developed a new method for evaluating hair roughness and gloss during the animal testing stage.

[0025] The present invention was designed based on references showing that UV irradiation causes hair to become rough (VF MONTEIRO et. al, 2003) and damages hair follicles (Jennifer Gherardini et. al, 2019). To clearly show the difference in hair roughness in C57BL / 6 mice, hair roughness was induced with UV. To ensure that UV irradiation does not directly affect the skin or hair but damages new hair follicles, sufficient UV irradiation was performed until new hair grew out of the skin after hair removal. UV irradiation was 40 to 200 mJ / cm 2 At this time, the UV concentration was 80 mJ / cm 2 If it exceeds 40 to 60 mJ / cm, it is confirmed that the skin of the mouse, etc. is damaged or hair growth is not normal. 2 An experiment to improve hair shine was conducted by setting it to (Example 1).

[0026] Accordingly, the present invention relates to a method for evaluating animal hair roughness and gloss, comprising, from one aspect, (a) a step of inducing hair roughness by ultraviolet irradiation; (b) a step of inducing hair growth after administering a test substance; and (c) a step of evaluating hair gloss changes, wherein the step of evaluating hair gloss changes in step (c) comprises: i) a step of removing and fixing skin from a hair-grown area; ii) a step of taking a photograph of the hair; and iii) a step of analyzing the taken photograph.

[0027] In the present invention, the ultraviolet irradiation in step (a) is ultraviolet-B (UV-B) at 40 to 80 mJ / cm 2 It can be investigated in concentrations of 40 to 60 mJ / cm, preferably 2 It can be investigated in concentration. In one embodiment of the present invention, the UV-B concentration is 60 mJ / cm 2 When investigated, it was confirmed that hair gloss was reduced and roughness was best induced (Example 2).

[0028] In the present invention, the administration in step (b) may be oral administration, skin application, subcutaneous injection, or device application, but is not limited thereto. The device application may involve applying a device that increases product absorption when the product is applied, or may involve providing microstimulation through current, such as microcurrent acupuncture, to alleviate hair roughness by promoting blood circulation. In one embodiment of the present invention, the experiment was conducted using oral administration.

[0029] In the present invention, the skin extracted in step i) can be spread out and then flattened onto a cylindrical tool. The cylindrical tool has a high surface friction and can therefore secure hair without a separate adhesive. When photographing without removing the skin, the light intensity and distance from the camera are not consistent, and the curvature indicating gloss varies from individual to individual. Therefore, by completely spreading out the extracted skin and then flattening it, the curvature of the hair can be matched.

[0030] In the present invention, after the photo taken in step iii) is cropped identically, the pixel values ​​according to the hair gloss area and gloss can be measured and analyzed using a program. In one embodiment of the present invention, pixel values ​​of 0 to 150 are evaluated as rough, 151 to 300 as normal, and 301 or higher as glossy.

[0031] In the present invention, a step of removing hair from an animal may be additionally included prior to step (a), preferably removing hair from the animal's back, but is not limited thereto.

[0032] In the present invention, the animal is not limited to a mouse or a rat, but is preferably a mouse, more preferably a C57BL / 6 mouse.

[0033] The above C57BL / 6 mouse has black body hair and natural hair loss occurs during the resting phase. Since melanin synthesis is well synchronized with the hair growth cycle, it is easy to visually estimate the hair cycle. It also has the characteristics of having similar conditions to human hair in terms of histology and immunology.

[0034]

[0035] Hereinafter, the composition and effects of the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0036]

[0037] <Experimental Method>

[0038] 1. Animal husbandry and experimental setup

[0039] Five-week-old male C57BL / 6 mice were purchased from Orient Bio Co., Ltd. and underwent a one-week acclimation period. The experimental animals were maintained under conditions with a temperature of 24 ± 0.5°C, a humidity of 55–65%, and a 12-hour light / dark cycle automatically controlled, with free access to laboratory animal food.

[0040] After a one-week acclimation period, mice were anesthetized with isoflurane and the hair on the back was primarily removed using animal clippers. A hair removal agent was applied to the shaved area and left on for 3 minutes, and then any fine hair remaining on the skin was removed secondarily. After the skin was stabilized for one day (24 hours) after hair removal, 6-week-old mice with pink telogen hair on the shaved back were weighed and divided into groups of 6. To induce roughness due to intrinsic aging of newly growing hair follicles, ultraviolet-B (UV-B) was irradiated for 7 days, the period before the hair grows to the skin surface, to induce roughness.

[0041] The negative control group and the test substance were administered orally at 0.1 ml each, five times a week, at regular intervals for three weeks until hair growth began. The negative control group used distilled water, and the test substance was minoxidil, administered at a dose of 5 mg / kg / day. The series of processes for obtaining the animals, shaving them, inducing roughness, inducing hair growth for a certain period, and evaluating the degree of improvement in hair luster are shown in Figure 1.

[0042]

[0043] 2. Skin extraction and skin photography to evaluate hair roughness and shine

[0044] After the experiment, mice were anesthetized with carbon dioxide gas, and the entire skin on the dorsal area where hair had grown was peeled off. If the mouse skin was left on to take pictures, the light illumination and distance from the camera would not be consistent, and the curvature indicating hair luster would vary from individual to individual. Therefore, the peeled skin was completely spread out and fixed to a cylindrical tool to match the curvature of the hair. The hair of all individuals was photographed under the same conditions (curvature, distance, and illumination), and the photographed hair was evaluated visually and by software. The process of removing the mouse skin and taking pictures to evaluate hair roughness and luster is shown in Figure 2.

[0045]

[0046] 3. Visual evaluation of hair roughness and shine

[0047] Hair roughness and gloss were visually observed using photographs taken of the hair. Experienced researchers (skilled enough to assess hair roughness and gloss area) conducted a blind test and calculated the average score based on the roughness and gloss visual evaluation grading scale in Figure 3. The grades were divided into three categories: Rough, Normal, and Glossy.

[0048]

[0049] 4. Hair Roughness and Shine Program Evaluation

[0050] After cutting the photographed hair identically, the hair gloss was measured in pixel values ​​using a program (I-MAXPLUS, Korea). The program measured the pixel values ​​according to the hair gloss area and gloss level, and analyzed them as the average value of all pixels according to the roughness and gloss program evaluation grade table in Fig. 4. The grades were evaluated based on the pixel value range, and were divided into three grades: Rough for pixel values ​​0 to 150, Normal for pixel values ​​151 to 300, and Glossy for pixel values ​​301 or higher.

[0051] Additionally, the correlation between visual evaluation and program evaluation was predicted according to the visual evaluation and program evaluation correlation prediction table of Fig. 5.

[0052]

[0053] 5. Statistics

[0054] Statistical analysis was performed using the IBM SPSS statistics 25.0 program, and significance was confirmed at a hypothesized mean difference of 5% (p<0.05). After the normality test, significance was confirmed using an independent-samples t-test (parametric method) depending on whether normality was satisfied.

[0055]

[0056] [Example 1] Setting the UV-B concentration

[0057] Figure 6 shows the basis for the UV-B concentration setting. After irradiating the mouse with UV-B for 7 days, the skin on the back was observed and the UV-B concentration was 80 mJ / cm. 2 If the UV-B concentration exceeds 40 mJ / cm, the skin of the mouse may be damaged or hair growth may not occur normally, making it impossible to accurately evaluate the roughness. Based on this, the UV-B concentration was set at 40 mJ / cm 2 and 60 mJ / cm 2 The experiment was conducted by setting it to .

[0058]

[0059] [Example 2] Confirmation of hair roughness induction after UV-B irradiation

[0060] Figure 7 shows the induction of hair roughness after UV-B irradiation, with UV-B concentrations of 0, 40, and 60 mJ / cm 2 The evaluation was conducted by A. A represents the results of the visual evaluation, B represents the results of the program evaluation, and C represents the correlation between the visual evaluation and the program evaluation.

[0061] As a result, both visual evaluation and program evaluation confirmed that the gloss decreased and roughness was induced after UV-B irradiation compared to before UV-B irradiation. In addition, R 2 It was confirmed that there was a correlation between visual evaluation and program evaluation, as the value was close to 1.

[0062] At this time, UV-B concentration 40 mJ / cm 2 By confirming that the induction of hair roughness was not statistically significant, UV-B concentration was 60 mJ / cm 2 It was confirmed that it was the most suitable.

[0063]

[0064] [Example 3] Confirmation of improved hair shine after ingestion of test substance

[0065] Figure 8 shows the improvement in hair luster after ingestion of the test substance. Negative control group, 60 mJ / cm 2 , 60 mJ / cm 2 + The evaluation was conducted by dividing the test substance group into groups. A represents the results of the visual evaluation, B represents the results of the program evaluation, and C represents the correlation between the visual evaluation and the program evaluation. In this case, minoxidil was used as the test substance to improve hair shine.

[0066] As a result, both visual and program evaluations confirmed that the gloss was improved in the group that took minoxidil after UV-B irradiation. In addition, R 2 It was confirmed that there was a correlation between visual evaluation and program evaluation, as the value was close to 1.

Claims

1. (a) A step of inducing hair roughness by ultraviolet irradiation; (b) a step of inducing hair growth after administering a test substance; and (c) A method for evaluating animal hair roughness and gloss, including a step of evaluating hair gloss changes, The step of evaluating the change in hair gloss in step (c) above is: i) A step of removing the skin from the hair-raising area, spreading it out, and then fixing it flat on a cylindrical tool; ii) a step of taking a photograph of the hair; and iii) A method for evaluating animal hair roughness and gloss, characterized by including a step of measuring pixel values ​​according to hair gloss area and glossiness after cutting the photographed image in the same manner, and analyzing the pixel values ​​of 0 to 150 as rough, 151 to 300 as normal, and 301 or more as glossy using a program.

2. In the first paragraph, the ultraviolet irradiation of step (a) is ultraviolet-B (UV-B) at 40 to 60 mJ / cm. 2 A method for evaluating the roughness and gloss of animal hair, characterized by examining it in terms of concentration.

3. A method for evaluating animal hair roughness and gloss, characterized in that in the first paragraph, the administration in step (b) is at least one selected from the group consisting of oral administration, skin application, subcutaneous injection, and device application.

4. A method for evaluating animal hair roughness and gloss, characterized in that the method further comprises a step of removing animal hair prior to step (a) in the first paragraph.

5. A method for evaluating animal hair roughness and gloss, characterized in that the animal in paragraph 1 is a mouse.

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