A method for constructing a melanin evaluation model and its application

Abstract

The present invention belongs to the technical field of efficacy evaluation of black hair products, and specifically relates to a method for constructing a melanin evaluation model and its application. The method for constructing the melanin evaluation model comprises the following steps: S1, obtaining and culturing in vitro pig skin: selecting pig back skin tissue, using a puncher to punch out in vitro pig skin with a diameter of 10 mm, placing it in a Transwell upper chamber and culturing it at a gas-liquid interface in a carbon dioxide incubator; S2, model establishment: using an oxidant to treat the in vitro pig skin to obtain a melanin evaluation model. The present invention solves the problems that the existing model for evaluating the efficacy of black hair products cannot simulate the complex human hair follicle microenvironment, cannot truly reflect its clinical effect, and has a long modeling time. The construction method is simple to operate, the result is intuitive, and it can be widely used in the efficacy evaluation of raw materials and products with black hair effects.

Description

Technical Field

[0001] The invention belongs to the technical field of hair-blackening product efficacy evaluation, and specifically relates to a method for constructing a melanin evaluation model and an application thereof. Background Art

[0002] In addition to normal aging, the reasons for gray hair are also affected by genetic factors, diseases, lack of nutrition, oxidative stress, mental stress and other factors. Therefore, gray hair exists in people of all ages. In daily life, gray hair is a problem that deeply troubles many people and affects people's image and mental state.

[0003] Hair dye can directly change gray hair to other colors, but it is only a temporary solution and does not address the root cause. After a period of time, the hair needs to be dyed again. Therefore, more and more people are beginning to pay attention to "white to black" black hair products that can prevent and treat endogenously. However, the efficacy evaluation of black hair products in the prior art mainly includes: (1) cell models such as human skin fibroblasts and melanoma cells cultured in vitro; (2) animal models such as black guinea pigs and C57BL / 6 mice.

[0004] A hair-blackening composition containing plant exosomes, a preparation method and an application thereof disclosed in a Chinese patent with publication number CN116889543A, is evaluated for its hair-blackening efficacy by using the effect of the composition on melanin in melanoma cells. A hair-blackening composition, a preparation method and an application thereof disclosed in a Chinese patent with publication number CN112220719A, is also evaluated for its hair-blackening efficacy by using the effect of the composition on melanin in melanoma cells.

[0005] However, single cells or simple cell co-culture models cannot fully simulate the complex microenvironment of human hair follicles, and the culture time is limited, especially the physiological process of melanin production, transportation and the entire chronic effect of hair follicle cycle renewal. Cell tests often focus on rapid response efficacy indicators such as cell viability, proliferation, and melanin production, while the evaluation of potential cytotoxicity, allergic reactions, and long-term safety of black hair product ingredients may not be comprehensive. At the same time, two-dimensional cell culture can only simulate cell behavior in a planar environment, and cannot reflect the three-dimensional spatial interaction between cells and the integrity of the signal transduction pathway. The structure of the hair follicle is three-dimensional, and the melanin production and transmission inside it are highly spatially organized. It is difficult to reproduce this three-dimensional dynamic process with simple cell culture. In addition, when evaluating the efficacy of black hair products at the cellular level, it is impossible to accurately understand the specific mechanism of action of the product ingredients on cells and the related signal pathways. Cell tests are generally short-term tests, which are difficult to reflect the cumulative effect of the product under long-term use. Especially for those black hair products that require long-term use to show the effect, cell tests may not truly reflect the clinical effect of long-term action.

[0006] The hair-blackening composition and its preparation method and application disclosed in the Chinese patent with publication number CN116392420A and the hair-blackening composition, hair-blackening essence and its application disclosed in the Chinese patent with publication number CN112263520A all use black guinea pigs for modeling and evaluate their hair-blackening effects. However, the modeling process takes up to 40 days, which is a long time; at the same time, the cultivation of live animals does not conform to the principle of animal welfare and the 3R principle of replacement, reduction and optimization.

[0007] The Chinese patent with publication number CN117517597A discloses a method for evaluating the efficacy of drugs and health foods in darkening hair, which includes establishing a zebrafish melanin inhibition model and then detecting the melanin content in the zebrafish head. However, zebrafish are low-level aquatic organisms, and their living environment is completely different from that of humans. They also do not have the hair follicle structure of mammals, so they cannot simulate the complex microenvironment of human hair follicles. Summary of the invention

[0008] In view of the problems that the existing models for evaluating the efficacy of hair blackening products cannot simulate the complex human hair follicle microenvironment, cannot truly reflect their clinical effects and take a long time to build models, the present invention proposes a method for constructing a melanin evaluation model and its application. The construction method is simple to operate, and the constructed melanin evaluation model has intuitive results and can be widely used in the efficacy evaluation of hair blackening products. It is an ideal model for predicting the efficacy of hair blackening products.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a method for constructing a melanin evaluation model, comprising the following steps:

[0011] S1. Obtaining and culturing ex vivo pig skin: Select pig back skin tissue, use a puncher to punch out ex vivo pig skin with a diameter of 10 mm, place it in the upper chamber of Transwell and perform gas-liquid interface culture in a carbon dioxide incubator;

[0012] S2. Model establishment: Use oxidants to treat ex vivo pig skin to obtain a melanin evaluation model.

[0013] Preferably, the pig back skin tissue in S1 is derived from any one of the Tibetan miniature pig, Bama miniature pig or Wuzhishan miniature pig, preferably the Tibetan miniature pig.

[0014] Preferably, the pig back skin described in S1 is cleaned and sterilized before use.

[0015] Preferably, the culture conditions in S1 are: temperature 37±1, CO2 content 5±1%, and relative humidity 90±5%.

[0016] Preferably, the lower chamber of the Transwell in S1 is a DMEM culture medium containing a mixture of 8-12% FBS (fetal bovine serum) and 0.8-1.2% penicillin-streptomycin.

[0017] Preferably, the lower chamber of the Transwell in S1 is filled with 1.5 mL of DMEM culture medium containing a mixture of 10% FBS and 1% penicillin-streptomycin.

[0018] Preferably, S1 is specifically as follows: remove the hair from the back of a pig, use a puncher to punch out 10 mm diameter ex vivo pig skin, spray and apply 75 vol% alcohol, then wash in PBS (phosphate buffered saline), and after sterilization, place in the upper chamber of a Transwell and perform gas-liquid interface culture in a carbon dioxide incubator.

[0019] Preferably, the oxidant in S2 is at least one of hydrogen peroxide, hydroquinone and urea peroxide, and the treatment is performed 2-3 times, each treatment lasts 20-24 hours, the treatment is performed once every 2-3 days, and the culture medium in the lower chamber is replaced once every 2-3 days.

[0020] Preferably, the oxidant in S2 is hydrogen peroxide, and the treatment is performed 3 times, each treatment lasts 24 hours, and the treatment is performed once every 2 days, and the Transwell lower chamber culture medium is replaced once every 2 days.

[0021] Preferably, the concentration of hydrogen peroxide in S2 is 0-10 mM (mmol / L).

[0022] Preferably, the concentration of hydrogen peroxide in S2 is 0.5-2 mM, preferably 2 mM.

[0023] The second aspect of the present invention provides an application of the above-mentioned construction method in the evaluation of black hair products.

[0024] The hair blackening products include but are not limited to hair blackening care products, functional raw materials, auxiliary raw materials, hair blackening medicines, and hair blackening health foods.

[0025] The third aspect of the present invention provides a method for evaluating black hair products, comprising the following steps:

[0026] Step 1: Divide the ex vivo pig skin into a negative control group, a model control group and a test sample group, and establish a melanin evaluation model according to the above construction method;

[0027] Step 2: administering medication to the model ex vivo pig skin and collecting ex vivo pig skin samples;

[0028] Step 3: respectively measuring the expression levels of relevant genes, catalase activity and melanin quantity of the ex vivo pig skin samples;

[0029] Step 4: Evaluate whether the hair darkening product has the effect of darkening hair based on the results of relevant gene expression levels, catalase activity and melanin quantity.

[0030] Preferably, the step 2 specifically comprises: subjecting the model ex vivo pig skin to drug treatment for 3 consecutive days, followed by incubation for 48 hours, and collecting ex vivo pig skin samples.

[0031] Preferably, the related genes are TGF-β, MITF, TYR, TYRP1, DCT and KIT.

[0032] The primer sequences of the TGF-β, MITF, TYR, TYRP1, DCT and KIT genes are shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036] Preferably, the method for determining the expression level of the relevant genes is: using TRIzol reagent to separate and obtain total RNA from an in vitro pig skin sample, and performing quantitative and purity detection on the extracted RNA; adding 1 μg of total RNA to a 20 μL total volume liquid containing random primers, which contains SuperScript reverse transcriptase III, and performing reverse transcription of the relevant RNA according to the operating instructions of the reverse transcription instrument; using oligonucleotide primers to perform PCR amplification, and fluorescently quantifying the gene expression levels of TGF-β, MITF, TYR, TYRP1, DCT, and KIT.

[0037] Preferably, the method for determining the peroxidase activity is: using a lysis solution to lyse the in vitro pig skin sample and then using a CAT detection kit to perform the determination.

[0038] Preferably, the method for determining the amount of melanin is: performing paraffin sectioning on the in vitro pig skin sample, performing Masson-Fontana staining on the paraffin section, and observing the amount of melanin.

[0039] The normal coloring process of hair is closely related to the synthesis and transport of melanin. Compared with epidermal melanocytes, hair follicle melanocytes are sensitive to oxidative damage. The free radicals (ROS) produced in this process damage the expression and activity of catalase in hair follicle melanocytes. At the same time, the gene expression levels of enzymes related to melanin synthesis are affected, thereby reducing the amount of melanin production. The accumulation of excessive ROS induces cell apoptosis. MITF, KIT, TYR, TYRP1, and DCT are related genes in the melanin synthesis pathway. TGF-β is involved in multiple signaling pathways during hair follicle development, including regulating epithelial and mesenchymal components. It plays an important role in the development of hair follicles, cell differentiation, extracellular matrix formation, and angiogenesis. KIT is an important member of the tyrosinase receptor protein family. Its activation can cause the activation of multiple downstream signaling pathways, including the Ras-Raf-MAPK-MITF signaling pathway. KIT elevation may cause the elevation of downstream MITF and then the elevation of tyrosinase family gene expression levels. MITF: It is a basic helix-loop-helix leucine zipper transcription factor that can bind to the M in the promoter region. -box motif binds to regulate the gene expression of the tyrosinase family, thereby regulating the production of melanin; TYR, TYRP1, DCT: There are three types of tyrosinase gene family that play a role in catalyzing the production of melanin, namely TYR, TYRP1 and DCT, of which TYRP1 and DCT play a catalytic role in the last few steps of controlling the type of melanin produced by melanocytes, and TYR is a key rate-limiting enzyme in the melanin synthesis signal pathway, which regulates the formation of melanin by participating in the hydrocarbonization and oxidation of L-DOPA (Levodopa, L-DOPA) to form dopaquinone (Dopaquinone, DOPA-quinone), and its expression and activity level determine the speed and type of melanin production. Therefore, in the present invention, the phenotype of the melanin evaluation model is characterized by measuring the expression level of genes in the catalase activity and the melanin synthesis signal. In addition, since Masson-Fontana staining can intuitively display melanin, Masson-Fontana staining is also used as an indicator to characterize the phenotype of the melanin evaluation model in the present invention.

[0040] Preferably, the hair blackening product has hair blackening effect: compared with the negative control group, the expression level of related genes in the model control group is decreased, the activity of catalase is decreased, and the amount of melanin is decreased, indicating that the melanin evaluation model is successfully constructed; compared with the model control group, the expression level of related genes in the test sample group is increased, the activity of catalase is increased, and the amount of melanin is increased, indicating that the hair blackening product has hair blackening effect.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The present invention successfully constructed an in vitro pig skin model for melanin evaluation using hydrogen peroxide, which is a model of melanin regulation disorder caused by oxidative stress of in vitro pig skin under sterile air-liquid interface culture conditions. The present invention innovatively induces oxidative damage in the in vitro skin of pigs with melanin-rich hair close to the human body, simulating the mechanism of hair graying and whitening caused by endogenous stress and environmental stress factors with chronic oxidative damage as the main effect.

[0043] 2. The method for constructing the melanin evaluation model of the present invention is simple to operate and takes a short time. The pig skin and hair follicles cultured in vitro of the present invention have many similarities with human skin and hair follicles in terms of structure, physiology, hair follicle growth cycle and cross-antigens. Therefore, the results obtained using the in vitro pig skin model are more credible and are an ideal model for predicting the efficacy of black hair products.

[0044] 3. In the present invention, the production of melanin in the hair blackening product is characterized by measuring the expression level of genes in the melanin synthesis signal pathway, and the alleviating effect of the hair blackening product on oxidative damage in the melanin evaluation model is characterized by measuring the activity of catalase. Masson-Fontana staining can display melanin more intuitively than other staining methods. The present invention can comprehensively evaluate the hair blackening efficacy of the hair blackening product from the physical and chemical, molecular levels and histopathological aspects. The evaluation method is simple and the results are intuitive, providing a scientific and effective method for the study of the efficacy of hair blackening products.

[0045] 4. The method of the present invention can replace live animals and human skin and be directly used in hair darkening efficacy tests of chemicals, cosmetics, medicines and other hair darkening products. The method based on ex vivo animal skin also complies with the 3R principle of reducing, optimizing and replacing live animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the first preliminary test of the effect of 1 mM hydrogen peroxide on the stained ex vivo pig skin sections. The image has a magnification of 40x.

[0047] Figure 2 This is the first preliminary test of the effect of 2mM hydrogen peroxide on the stained ex vivo pig skin sections. The image has a magnification of 40x.

[0048] Figure 3 This is the first preliminary test of the effect of 10mM hydrogen peroxide on the stained ex vivo pig skin sections. The image has a magnification of 40x.

[0049] Figure 4 This is the second preliminary test of the effect of 0 mM hydrogen peroxide on the staining of ex vivo pig skin sections at a magnification of 40x.

[0050] Figure 5This is the second preliminary test of 0.5 mM hydrogen peroxide staining on ex vivo pig skin sections at a magnification of 40x.

[0051] Figure 6 This is the second preliminary test of 1 mM hydrogen peroxide on ex vivo pig skin sections stained with a magnification of 40x.

[0052] Figure 7 This is the second preliminary test of the effect of 2mM hydrogen peroxide on the stained ex vivo pig skin sections at a magnification of 40x.

[0053] Figure 8 and Fig. 9 The images are 100x magnification images of stained ex vivo pig skin sections after drug treatment in the NC group.

[0054] Fig.10 and Fig.11 The images are 100x magnification images of the stained ex vivo pig skin sections after drug treatment in the MC group.

[0055] Fig.12 and Fig.13 The images are stained ex vivo pig skin sections after drug treatment in the TA group at a magnification of 100x. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0057] Example 1 Preliminary test to determine the hydrogen peroxide induction concentration:

[0058] (1) The hair-free area on the back of a Tibetan miniature pig was removed, and a 10 mm diameter ex vivo pig skin was punched out using a cork puncher. The ex vivo pig skin was sprayed with 75 vol% alcohol and then washed in PBS. After aseptic treatment, the ex vivo pig skin was placed in the upper chamber of a Transwell and cultured at the gas-liquid interface in a carbon dioxide incubator. The culture conditions were: temperature 37±1, CO2 content 5±1%, and relative humidity 90±5%; the lower chamber of the Transwell contained 1.5 mL of DMEM culture medium containing a mixture of 10% FBS and 1% penicillin-streptomycin.

[0059] (2) The first preliminary test: The experimental group was: hydrogen peroxide with a concentration of 3% was diluted to three concentrations of 1 mM, 2 mM, and 10 mM using the DMEM medium in step (1). After the in vitro pig skin was adaptively cultured for 18-24 hours in step (1), the Transwell lower chamber medium was replaced with a DMEM medium containing different concentrations of hydrogen peroxide. The treatment was performed once every 2 days, and the Transwell lower chamber medium was replaced every 2 days for 3 times. The control group was treated with PBS in the same way as the experimental group. After the treatment, the activity of the in vitro pig skin cells was determined by the MTT method and the change in the amount of melanin was observed by Masson-Fontana staining. The results are shown in Tables 2 and Figure 1-3 .

[0060] (3) Second preliminary test: The same as the first preliminary test, except that the experimental group was: the hydrogen peroxide with a concentration of 3% was diluted to four concentrations of 0 mM, 0.5 mM, 1 mM, and 2 mM using the DMEM medium in step (1); after the treatment, Masson-Fontana staining was performed to observe the changes in the amount of melanin, and qPCR was performed to determine the expression levels of related genes. The results are shown in Tables 3-4 and Figure 4-7 .

[0061] Table 2 Relative activity of isolated porcine skin cells (Mean±SD)

[0062] Hydrogen peroxide concentration (mM) Relative activity (%) 0 100.00±0.05 1 88.24±0.15 2 81.55±0.28 10 45.11±2.21

[0063] Table 3 Relative content of MITF (Mean±SD)

[0064] Hydrogen peroxide concentration (mM) Relative content 0 1.00±0.22 0.5 1.10±0.04 1 1.10±0.08 2 0.79±0.10#

[0065] Note: # indicates that the difference is statistically significant compared with 0 mM hydrogen peroxide (p<0.05).

[0066] Table 4 KIT relative content (Mean±SD)

[0067] Hydrogen peroxide concentration (mM) Relative content 0 1.00±0.13 0.5 1.04±0.09 1 1.08±0.14 2 0.66±0.13#

[0068] Note: # indicates that the difference is statistically significant compared with 0 mM hydrogen peroxide (p<0.05).

[0069] Example 2 Construction of melanin evaluation model and evaluation of black hair products

[0070] 1. Test materials

[0071] 1.1. Ex vivo pig skin: Source: Skin tissue left over from the Tibetan miniature pig experiment.

[0072] 1.2. Culture medium: DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin mixture (Lot: 2500251P).

[0073] 1.3. Test sample: PBS solution containing 2% acetyltyrosine, acetyltyrosine (CAS No.: 537-55-3, purchased from Merck, product number: A0202000).

[0074] 1.4. Test conditions: incubator temperature 37±1℃, relative humidity 90±5%, CO2 content 5±1%.

[0075] 1.5. Test groups and test reagents

[0076] 1.5.1. Experimental grouping settings, see Table 5.

[0077] Table 5

[0078]

[0079]

[0080] 1.5.2 Test reagents

[0081] Hydrogen peroxide solution: Shanghai Aladdin Biochemical Technology Co., Ltd. (D1021036);

[0082] Masson-Fontana staining kit: Shanghai Shangbao Biotechnology Co., Ltd. (R22077);

[0083] CAT detection kit: Shanghai Bio-Tech Biotechnology Co., Ltd. (S0051).

[0084] 2. Test steps:

[0085] 2.1. Use a cork puncher to punch out 10 mm diameter ex vivo pig skin, spray and smear with 75 vol% alcohol, wash in PBS, and place in the upper chamber of Transwell for air-liquid interface culture in a carbon dioxide incubator after sterilization; the lower chamber of Transwell contains 1.5 mL of DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin mixture;

[0086] 2.2. Treat the ex vivo pig skin (modeling) with 2 mM hydrogen peroxide, once every 2 days (PBS was used in the NC group), replace the Transwell lower chamber culture medium every 2 days, and treat 3 times to obtain a melanin evaluation model;

[0087] 2.3. The model ex vivo pig skin was treated with drugs. The NC group was treated with PBS, the M group was treated with PBS, and the TA group was treated with the test sample. The drug volume was 50 μL.

[0088] 2.4. After 3 consecutive days of drug treatment, continue to incubate for 48 hours, collect the ex vivo pig skin samples, and divide the ex vivo pig skin samples into 3 equal parts;

[0089] 2.5. Use TRIzol reagent to separate and obtain total RNA from the first in vitro pig skin sample, and perform quantitative and purity tests on the extracted RNA; add 1 μg of total RNA to a 20 μL total volume liquid containing random primers, which contains SuperScript reverse transcriptase III, and perform reverse transcription of relevant RNA according to the reverse transcription instrument operating instructions; use oligonucleotide primers to perform PCR amplification, and perform fluorescence quantitative analysis of the gene expression levels of TGF-β, MITF, TYR, TYRP1, DCT, and KIT;

[0090] 2.6. After the second ex vivo pig skin sample was lysed with lysis buffer, the catalase activity was determined using a CAT detection kit;

[0091] 2.7. The third ex vivo pig skin sample was sectioned into paraffin and stained with Masson-Fontana.

[0092] 3. Data Analysis

[0093] Data are expressed as mean ± standard deviation, and SPSS was used for statistical analysis. One-way analysis of variance (ANOVA) and least significant difference (LSD) test were used to analyze the differences between different groups; P < 0.05 was considered statistically significant. All mathematical and statistical images were generated by EXCEL and GraphPadPrism software.

[0094] 3.1. Masson-Fontana melanin staining results are shown in Figure 8-13 .

[0095] 3.2. The results of catalase activity test are shown in Table 6.

[0096] Table 6 Catalase relative activity (Mean±SD)

[0097] Group Relative content (%) NC 100±0.23 MC 89.29±1.73# TA 99.32±0.33*

[0098] Note: # indicates that the difference is statistically significant compared with the negative control (NC) (p<0.05); * indicates that the difference is statistically significant compared with the model control group (MC) (p<0.05).

[0099] 3.3. The qPCR test results are shown in Tables 7-12.

[0100] Table 7 MITF relative content (Mean±SD)

[0101] Group Relative content NC 1.00±0.09 MC 0.21±0.06# TA 1.70±0.05*

[0102] Note: # indicates that the difference is statistically significant compared with the negative control (NC) (p<0.05); * indicates that the difference is statistically significant compared with the model control group (MC) (p<0.05).

[0103] Table 8 TYR relative content (Mean±SD)

[0104] Group Relative content NC 1.00±0.14 MC 0.17±0.02# TA 1.67±0.19*

[0105] Note: # indicates that the difference is statistically significant compared with the negative control (NC) (p<0.05); * indicates that the difference is statistically significant compared with the model control group (MC) (p<0.05).

[0106] Table 9 TYRP1 relative content (Mean±SD)

[0107] Group Relative content NC 1.00±0.03 MC 0.51±0.05# TA 1.03±0.03*

[0108] Note: # indicates that the difference is statistically significant compared with the negative control (NC) (p<0.05); * indicates that the difference is statistically significant compared with the model control group (MC) (p<0.05).

[0109] Table 10DCT relative content (Mean±SD)

[0110] Group Relative content NC 1.00±0.09 MC 0.2±0.03# TA 1.48±0.05*

[0111] Note: # indicates that the difference is statistically significant (p<0.05) compared with the negative control (NC); * indicates that the difference is statistically significant (p<0.05) compared with the model control group (M).

[0112] Table 11 KIT relative content (Mean±SD)

[0113] Group Relative content NC 1.00±0.13 MC 1.44±0.29 TA 1.21±0.26

[0114] Note: # indicates that the difference is statistically significant compared with the negative control (NC) (p<0.05); * indicates that the difference is statistically significant compared with the model control group (MC) (p<0.05).

[0115] Table 12 Relative content of TGF-β (Mean±SD)

[0116] Group Relative content NC 1.00±0.12 MC 0.24±0.01# TA 1.25±0.11*

[0117] Note: # indicates that the difference is statistically significant compared with the negative control (NC) (p<0.05); * indicates that the difference is statistically significant compared with the model control group (MC) (p<0.05).

[0118] 4. Conclusion:

[0119] (1) In Masson-Fontana melanin staining, the melanin in the hair follicles of pig skin in the hydrogen peroxide treatment group was significantly reduced, while the melanin in the hair follicles of the test samples was significantly increased after treatment.

[0120] (2) In the catalase activity test, hydrogen peroxide treatment significantly reduced the catalase activity of pig skin tissue lysate (p < 0.05), while after treatment with the test sample, the catalase activity was significantly increased (p < 0.05).

[0121] (3) In the qPCR experiment, the test samples failed to cause changes in the expression level of the KIT gene, indicating that the test samples did not cause the increase in hair follicle melanin by regulating the KIT gene-related pathway. Hydrogen peroxide modeling reduced the expression of MITF, TYR, TYRP1, DCT and TGF-β (p < 0.05), while the expression levels of the above genes increased significantly after administration of the test samples (p < 0.05). This shows that the test samples can significantly increase the expression of melanin production-related genes MITF, TYR, TYRP1, and DCT after hydrogen peroxide damage (p < 0.05); at the same time, they can also significantly increase the expression of TGF-β, a gene that is very important for the maintenance of melanin stem cells after hydrogen peroxide damage (p < 0.05).

[0122] In summary, it can be considered that the tested samples have the effect of blackening hair.

[0123] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing a melanin evaluation model, characterized in that: The following steps are involved: S1. Obtaining and culturing ex vivo pig skin: Select pig back skin tissue, use a puncher to punch out ex vivo pig skin with a diameter of 10 mm, place it in the upper chamber of Transwell and perform gas-liquid interface culture in a carbon dioxide incubator; S2. Model establishment: using oxidants to treat ex vivo pig skin to obtain a melanin evaluation model; The pig back skin tissue in S1 is derived from any one of the Tibetan miniature pig, Bama miniature pig or Wuzhishan miniature pig; The oxidant in S2 was hydrogen peroxide, which was treated 3 times, each treatment lasting 24 hours, and once every 2 days. The culture medium in the lower chamber of Transwell was replaced every 2 days. The hydrogen peroxide concentration in S2 is 2 mM.

2. The construction method according to claim 1, characterized in that: The lower chamber of the Transwell in S1 is filled with DMEM culture medium containing a mixture of 8-12% FBS and 0.8-1.2% penicillin-streptomycin.

3. Application of the construction method according to any one of claims 1-2 in the evaluation of UFA products.

4. A method for evaluating black hair products, characterized in that: The following steps are involved: Step 1: Divide the ex vivo pig skin into a negative control group, a model control group and a test sample group, and establish a melanin evaluation model according to the construction method according to any one of claims 1-2; Step 2: The model ex vivo pig skin was treated with drugs, the negative control group used PBS, the model control group used PBS, and the test sample group used the test sample, the drug volume was 50 μL, and after 3 consecutive days of drug treatment, the incubation was continued for 48 hours, and the ex vivo pig skin samples were collected; Step 3: respectively measuring the expression levels of related genes, catalase activity and melanin amount of the in vitro pig skin samples, wherein the related genes are TGF-β, MITF, TYR, TYRP1 and DCT; Step 4: Evaluate whether the hair blackening product has the hair blackening effect based on the results of relevant gene expression levels, catalase activity and melanin quantity: the hair blackening product has the hair blackening effect: compared with the negative control group, the relevant gene expression levels in the model control group were significantly decreased, the catalase activity was significantly decreased, and the melanin quantity was significantly decreased, indicating that the melanin evaluation model was successfully constructed; compared with the model control group, the relevant gene expression levels in the test sample group were significantly increased, the catalase activity was significantly increased, and the melanin quantity was significantly increased, indicating that the hair blackening product has the hair blackening effect.

5. The method for evaluating dark hair products according to claim 4, characterized in that: The method for determining the peroxidase activity is as follows: using a lysis solution to lyse the in vitro pig skin sample and then using a CAT detection kit to determine the activity.

6. The method for evaluating hair darkening products according to claim 4, characterized in that: The method for determining the amount of melanin is as follows: performing paraffin sectioning on an in vitro pig skin sample, performing Masson-Fontana staining on the paraffin section, and observing the amount of melanin.

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