Evaluation method for combined efficacy of in-vitro hair-blacking raw materials or products and application of evaluation method

Abstract

The invention relates to the technical field of product efficacy evaluation, and particularly discloses an in-vitro hair-blacking raw material or product combination efficacy evaluation method which is simple and visual in result, can be widely applied to efficacy evaluation of hair-blacking raw materials or products, is an ideal method for predicting the efficacy of hair-blacking raw materials or products, and has good application prospects. The evaluation method can replace living animal and human skin and is directly used for hair-blacking effect tests of hair-blacking products such as chemicals, cosmetics and medicines. The method established based on the in-vitro animal skin also conforms to the 3R principle of reducing, optimizing and replacing living animal experiments.

Description

Technical Field

[0001] This application relates to the field of product efficacy evaluation technology, and in particular to a method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations and its application. Background Technology

[0002] Gray hair is a common appearance concern across all age groups, and its formation mechanism involves multiple factors such as genetic susceptibility, chronic diseases, nutritional imbalance, oxidative stress, and mental stress.

[0003] The existing efficacy evaluation system for hair darkening products mainly relies on two types of models: one is an in vitro two-dimensional culture system based on melanoma cells and skin fibroblasts, such as Chinese patents with publication numbers CN116889543A and CN112220719A; the other is animal models such as black-spotted guinea pigs and zebrafish, such as Chinese patents with publication numbers CN116392420A and CN117517597A.

[0004] However, these models have significant technical limitations: cell models are limited by a two-dimensional culture environment and cannot simulate the spatial structural characteristics of the three-dimensional microenvironment of human hair follicles, especially the three-dimensional interaction between melanocytes and keratinocytes and the dynamic process of pigment transport. Existing cell experiments mostly focus on short-term melanin synthesis indicators and lack systematic evaluation of safety parameters such as hair follicle cyclic regeneration, long-term cumulative effects of components, cytotoxicity, and sensitization. As for animal models, zebrafish lack hair follicle structure, making it difficult to simulate the human hair growth mechanism, while guinea pig models have a long modeling period of up to 40 days and involve ethical controversies. Summary of the Invention

[0005] The purpose of this application is to overcome the shortcomings of the prior art and provide a method for evaluating the efficacy of hair-darkening raw materials or product combinations in vitro, and its application therein. The evaluation method of this application is simple, yields intuitive results, and can be widely used for evaluating the efficacy of hair-darkening raw materials or products, making it an ideal method for predicting the efficacy of hair-darkening raw materials or products.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations, including the following steps: S1. The antioxidant model of keratinocytes and the melanin production model of melanocytes were used to test the samples respectively; If the test results of the antioxidant model of keratinocytes and the melanin production model of melanocytes are both negative, it can be directly determined that the sample to be tested does not have the effect of darkening hair; otherwise, proceed to step S2. S2. The melanin migration model of keratinocyte-melanocyte co-culture was used to test the sample to be tested after step S1. If the test result of the melanin migration model of keratinocyte-melanocyte co-culture is negative, it is directly determined that the sample does not have the effect of darkening hair; if the test result is positive, proceed to step S3. S3. The in vitro pigskin evaluation model is used to test the sample to be tested after step S2. If the test result of the in vitro pigskin evaluation model is negative, it is directly determined that the sample does not have a hair-darkening effect; if the test result is positive, the sample has a hair-darkening effect.

[0007] In the technical solution of this application, multiple models mentioned above are used to test the sample, which can simulate the graying and whitening mechanism of hair. Furthermore, the evaluation method provided in this application is simple to operate and quick to implement. This evaluation method can replace live animals and human skin, and can be directly used for hair-darkening efficacy testing of hair-darkening products such as chemicals, cosmetics, and pharmaceuticals. The method established based on ex vivo animal skin also conforms to the 3R principle of reducing optimization and replacing live animal experiments.

[0008] This application utilizes a three-tiered model—an antioxidant model of keratinocytes, a melanin production model of melanocytes, and a melanin migration model based on co-culture of keratinocytes and melanocytes—to achieve a progressive evaluation system from simple to complex and from single-cell to co-culture, offering the following advantages: 1) Simple operation and short cycle, suitable for large-scale primary screening of raw materials and products; 2) Multi-model combination evaluation comprehensively reflects the hair follicle microenvironment from anti-oxidation and melanin production to melanin migration; 3) Improve the accuracy and predictability of evaluations to ensure that the selected raw materials or products are more likely to be effective in clinical practice.

[0009] Animal experiments typically require dozens of animals to be continuously housed for at least 8 weeks for downstream testing and analysis, which is less costly and time-efficient than in vitro evaluation methods. The cost of in vitro methods is approximately five times that of the method described in this application, and the time cost is approximately twice that of the method described in this application. Furthermore, the method described in this application, as a preliminary screening method, can provide data support for clinical research while effectively meeting animal welfare requirements.

[0010] Among them, the antioxidant model of keratinocytes is used to preliminarily screen raw materials or products, quickly determine whether they have the potential antioxidant and hair follicle environment protection capabilities, and reduce costs and experimental workload.

[0011] Using a melanocyte model of melanin production, we can further verify the direct promoting effect on melanin production in pre-screened raw materials or products, providing mechanistic support.

[0012] A melanin migration model using keratinocyte-melanocyte co-culture was employed to simulate the hair follicle microenvironment, comprehensively evaluate melanin production and migration, and reflect the overall efficacy.

[0013] This application uses a three-tiered model: an antioxidant model of keratinocytes, a melanin production model of melanocytes, and a melanin migration model based on co-culture of keratinocytes and melanocytes. This sequential approach can gradually eliminate ineffective raw materials and improve the accuracy and predictability of the final evaluation.

[0014] Preferably, in step S1, the antioxidant model of keratinocytes is first used to test the sample, and then the melanin production model of melanocytes is used to test the sample.

[0015] Using the above-mentioned testing method can better achieve initial screening of raw materials and products, thereby improving the accuracy of evaluation.

[0016] In large-scale raw material screening, the antioxidant model of keratinocytes is used to test the samples first, which helps to improve screening efficiency and reduce costs. When the sample quantity is limited or the cost is not significantly different, the antioxidant model of keratinocytes and the melanin production model of melanocytes can also be tested in parallel to shorten the overall evaluation cycle.

[0017] In some specific embodiments, the sample to be tested can be a single raw material or a product of multiple raw materials.

[0018] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the method for testing the sample to be tested using a keratinocyte antioxidant model includes the following steps: 1) Cultured keratinocytes were divided into negative control group, model control group, positive control group and experimental group. Keratinocytes from the negative control group and model control group were cultured in maintenance culture medium, keratinocytes from the positive control group were cultured in maintenance culture medium with added vitamin C derivative, and keratinocytes from the experimental group were cultured in maintenance culture medium with added test sample. Then the model control group, positive control group and experimental group were cultured in culture medium containing oxidant. 2) Disrupt the keratinocytes of the negative control group, model control group, positive control group and experimental group after the treatment in step 1), and detect the superoxide dismutase activity (SOD), catalase activity (CAT) and malondialdehyde content (MDA) of each group of cells. In cases where the superoxide dismutase (SOD) and catalase (Cal) activities in the model control group were lower than those in the negative control group (p<0.05), the malondialdehyde (MDA) content in the model control group was higher than that in the negative control group (p<0.05), and the SOD and Cal were higher than those in the model control group (p<0.05), while the MDA content was lower than that in the model control group, if the SOD and Cal were higher than those in the experimental group and the MDA content was lower than that in the model control group, and the difference was statistically significant (p<0.05), then the test result was positive; otherwise, it was negative.

[0019] This application involves testing the sample under test using an antioxidant model of keratinocytes to determine whether the sample exhibits antioxidant activity, in order to conduct subsequent experiments to confirm whether it has a hair-darkening effect.

[0020] If at least two of the following indicators in the sample are significantly increased: superoxide dismutase activity (SOD), malondialdehyde (MDA) content, and catalase activity (CAT), the sample is considered to have antioxidant properties. If only one indicator is increased, the sample is considered not to have antioxidant properties.

[0021] The keratinocyte antioxidant model (S1) can quickly eliminate obviously ineffective raw materials or products (ensuring no potential effective ingredients are missed). The keratinocyte antioxidant model is simple to operate, has a short cycle, and requires few consumables. It can reduce the amount of experiments and costs before the S3 model, making it suitable for large-scale initial screening. Furthermore, it can improve the accuracy of the test. The S1 and S2 models provide mechanistic indicators, ensuring that raw materials or products entering the S3 model are more likely to show true efficacy, thereby improving the accuracy and reliability of the final evaluation.

[0022] This application selects superoxide dismutase (SOD) activity, which reflects the cell's ability to scavenge superoxide free radicals and is a key enzyme for antioxidant protection; malondialdehyde (MDA) content, which reflects the level of lipid peroxidation damage and directly measures the degree of cellular oxidative damage; and catalase (CAT) activity, which measures the cell's ability to decompose hydrogen peroxide and ensure cellular homeostasis. These three indicators can comprehensively evaluate the antioxidant capacity of keratinocytes, are easy to operate, and have a high correlation with melanin production, making them an effective initial screening tool for the potential of hair-darkening raw materials.

[0023] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the method for testing the sample to be tested using a melanin production model of melanocytes includes the following steps: 1) Melanoma cells were divided into negative control group, model control group, positive control group and experimental group. Melanoma cells from the negative control group and model control group were cultured in maintenance culture medium, while melanoma cells from the positive control group were cultured in maintenance culture medium with rapamycin added. Melanoma cells from the experimental group were cultured in maintenance culture medium with the test sample added. Then, the model control group, positive control group and experimental group were cultured in culture medium containing oxidant. There was a significant difference between the model control group and the negative control group (p<0.05), and there was a significant difference between the positive control group and the experimental group (p<0.05). 2) The melanoma cells of the negative control group, model control group, positive control group and experimental group treated in step 1) were co-cultured with a culture medium containing oxidant, then the cells were collected by centrifugation, the cells were lysed, the supernatant was obtained, and the absorbance of the supernatant was measured. If the tyrosinase activity in the model control group is lower than that in the negative control group (p<0.05) and the tyrosinase activity in the positive control group is higher than that in the model control group (p<0.05), the test result is positive if the tyrosinase activity in the experimental group is higher than that in the model control group and the difference is statistically significant (p<0.05); otherwise, the result is negative. or, 2) The melanoma cells of the model control group, positive control group and experimental group treated in step 1) were co-cultured with a medium containing H2O2, then the cells were collected by centrifugation, NaOH solution was added to dissolve the cells to obtain cell slurry, the cell slurry was heated to lyse the melanosomes, the absorbance value of the cell slurry was measured, and the melanin content was determined. If the melanin content in the model control group is lower than that in the negative control group (p<0.05), and the melanin content in the positive control group is higher than that in the model control group (p<0.05), then if the melanin content in the experimental group is higher than that in the model control group and the difference is statistically significant (p<0.05), the test result is positive; otherwise, it is negative.

[0024] If both tyrosinase activity and melanin content in the sample are significantly increased, it proves that the sample has a melanin production effect. If only one indicator is increased, it is considered that the sample does not have a melanin production effect.

[0025] This application will conduct a melanin production model test on the sample to be tested using melanocytes to observe whether it improves the inhibition of tyrosinase caused by oxidative damage and whether it increases the melanin content in the damaged cells, so as to conduct subsequent experiments to confirm whether it has a hair-darkening effect.

[0026] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the method for testing the sample to be tested using a melanin migration model based on keratinocyte-melanocyte co-culture includes the following steps: 1) Digest melanoma cells and keratinocytes to obtain a single-cell mixed suspension, and divide the cells into negative control group, model control group, positive control group and experimental group. The cells of the negative control group and model control group were cultured in maintenance culture medium, the cells of the positive control group were cultured in maintenance culture medium with rapamycin added, and the cells of the experimental group were cultured in maintenance culture medium with the sample to be tested added. Then the cells of the model control group, positive control group and experimental group were cultured in culture medium containing oxidant. 2) Cells from the negative control group, model control group, positive control group, and experimental group after step 1) were subjected to immunofluorescence staining to determine the amount of melanin migration; If the melanin migration in the model control group is lower than that in the negative control group, and the melanin migration in the positive control group is higher than that in the model control group, and the melanin migration in the experimental group is higher than that in the model control group with a significant difference (p<0.05), then the test result is positive; otherwise, it is negative.

[0027] This application uses a melanin migration model based on the co-culture of keratinocytes and melanocytes in the sample to be tested, to observe whether it increases melanin transfer after oxidative damage.

[0028] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the molar concentration of the oxidant in the method of testing the sample using an antioxidant model of keratinocytes is 0.125~2 mmol / L, preferably 0.5 mmol / L.

[0029] The oxidant used in this application can be a concentration within the above-mentioned mass concentration range as the modeling concentration for the antioxidant model of keratinocytes. The preferred molar concentration of the oxidant is 0.5 mmol / L, which can better model the antioxidant model of keratinocytes.

[0030] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the molar concentration of the oxidant in the method for testing the sample using a melanocyte melanin production model is 0.125 ~ 0.5 mmol / L, preferably, the mass concentration of the oxidant is 0.5 mmol / L.

[0031] The oxidant used in this application within the above-mentioned mass concentration range can significantly inhibit tyrosinase activity, thereby significantly inhibiting melanin formation. The preferred molar concentration of the oxidant is 0.5 mmol / L, which better inhibits melanocyte proliferation and thus better establishes a melanin production model of melanocytes.

[0032] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the molar concentration of the oxidant in the method of testing the sample using a melanin migration model of keratinocyte-melanocyte co-culture is 0.1~0.5 mmol / L, preferably, the mass concentration of the oxidant is 0.5 mmol / L.

[0033] Using the above-mentioned concentration of oxidant, melanin transfer can be significantly inhibited, thus better establishing a melanin migration model in keratinocyte-melanocyte co-culture.

[0034] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the maintenance culture medium is DMEM medium containing 1% fetal bovine serum.

[0035] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the method for testing the sample to be tested using an in vitro pig skin evaluation model includes the following steps: 1) Take skin tissue from the back of pigs, separate the detached pig skin, and divide it into a negative control group, a model control group, and an experimental group. Then, place the detached pig skin of each group in a culture medium for culture. Treat the detached pig skin of the model control group and the experimental group with an oxidant. 2) The negative control group, model control group and experimental group after step 1) were administered PBS, the experimental group was administered the test sample, and the negative control group was administered PBS. Isolated pig skin samples were collected, paraffin sections were prepared from the isolated pig skin samples, the paraffin sections were stained, and the amount of melanin in the hair follicles of the isolated pig skin samples was observed.

[0036] The sample to be tested after steps S1 and S2 is then subjected to step S3 for measurement. The melanin in the hair follicle of the isolated pig skin sample is observed. If the melanin is significantly increased, the sample to be tested can be considered to have a hair-darkening effect.

[0037] In some specific implementations, in step S1, the levels of SOD and CAT in the sample to be tested increase, while MDA decreases; tyrosinase activity increases, and melanin content increases; in step S2, the amount of melanin that migrates into the keratinocyte increases significantly; and in step S3, the number of melanin in the hair follicles increases, proving that the hair-darkening raw materials or product combinations have hair-darkening effects.

[0038] The porcine skin and hair follicles cultured in vitro in this application 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 by using the in vitro porcine skin evaluation model are more reliable and are an ideal model for predicting the efficacy of hair darkening products.

[0039] The keratinocyte antioxidant model of this application assesses antioxidant capacity by measuring the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in keratinocytes, thus providing a favorable microenvironment for melanin production. Furthermore, the melanin production model of melanocytes verifies the direct promoting effect of raw materials or products on melanin production by measuring melanin production and related enzyme activities. This stepwise screening strategy effectively reduces inefficient experiments and costs, avoids inefficient experiments and misjudgments that may occur when directly performing step S2 testing, and improves the accuracy of the final evaluation.

[0040] In a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the oxidant includes H2O2.

[0041] As a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the excised pig skin is derived from the back skin of any one of the following: Tibetan miniature pigs, Bama miniature pigs, or Wuzhishan miniature pigs.

[0042] In a preferred embodiment of the method for evaluating the efficacy of the in vitro hair-darkening raw materials or product combinations described in this application, the keratinocytes include HaCaT; And / or, the melanocytes include B16 or A875.

[0043] This application provides the application of the above-mentioned method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations in evaluating hair-darkening products.

[0044] As a preferred embodiment of the application described in this application, the hair-darkening product includes at least one of hair-darkening shampoos and conditioners, hair-darkening medicines, and hair-darkening foods.

[0045] Compared with the prior art, this application has the following beneficial effects: This application provides a method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations and its application, involving single-cell culture, multi-cell co-culture, and ex vivo skin models. The evaluation method of this application is simple, yields intuitive results, and can be widely used for evaluating the efficacy of hair-darkening raw materials or products. It is an ideal method for predicting the efficacy of hair-darkening raw materials or products. Furthermore, the evaluation method of this application can replace live animals and human skin, and can be directly used for hair-darkening efficacy testing of chemicals, cosmetics, pharmaceuticals, and other hair-darkening products. The method based on ex vivo animal skin also conforms to the 3R principle of reducing optimization and replacing live animal experiments. The method of this application aims to establish a comparable and reproducible evaluation system through in vitro multi-model combinations to improve the accuracy and reliability of raw material and product efficacy prediction. When using existing single-model evaluations, some known effective raw materials or products may be misjudged as ineffective; however, using the multi-model combination evaluation of this application can significantly reduce misjudgments and improve the accuracy of predicting hair-darkening efficacy. Attached Figure Description

[0046] Figure 1 The fluorescence image is of the negative control (NC) in Example 1; Figure 2 The fluorescence spectrum is shown for H2O2 with a molar concentration of 0.1 mM in Example 1. Figure 3 The fluorescence image is shown when the molar concentration of H2O2 in Example 1 is 0.5 mM. Detailed Implementation

[0047] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0048] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0049] This application provides a method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations, including the following steps: S1. The antioxidant model of keratinocytes and the melanin production model of melanocytes were used to test the samples respectively; If the test results of the antioxidant model of keratinocytes and the melanin production model of melanocytes are both negative, it can be directly determined that the sample to be tested does not have the effect of darkening hair; otherwise, proceed to step S2. S2. The melanin migration model of keratinocyte-melanocyte co-culture was used to test the sample to be tested after step S1. If the test result of the melanin migration model of keratinocyte-melanocyte co-culture is negative, it is directly determined that the sample does not have the effect of darkening hair; if the test result is positive, proceed to step S3. S3. The in vitro pigskin evaluation model is used to test the sample to be tested after step S2. If the test result of the in vitro pigskin evaluation model is negative, it is directly determined that the sample does not have a hair-darkening effect; if the test result is positive, the sample has a hair-darkening effect.

[0050] Example 1 This embodiment provides a method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations, including the following steps: S1. The antioxidant model of keratinocytes and the melanin production model of melanocytes were used to test the samples in sequence. S11, Antioxidant test of keratinocytes: 1. Experimental materials: 1.1 Cell line: Human immortalized keratinocytes HaCaT, source: BeiNa Biotechnology, passage number: 28.

[0051] 1.2 Complete culture medium: MEM medium containing 10% fetal bovine serum (FBS) (FBS, Lot: 2500251P).

[0052] 1.3 Maintenance culture medium: DMEM medium containing 1% fetal bovine serum (FBS, Gibco, Lot: 2500251P).

[0053] 1.4 Test conditions: Incubator temperature 37±1℃, humidity 90±5%, CO2 5±1%.

[0054] 1.5 Sample to be tested: Mentholatum 50 Hui Black Hair Activating Essence, batch number 20230710.

[0055] 1.6. Experimental Grouping and Reagents: 1.6.1 Experimental group settings are shown in Table 1.

[0056] Table 1 1.6.2 Test reagents: Hydrogen peroxide solution: Shanghai Aladdin Biochemical Technology Co., Ltd. (D1021036).

[0057] BCA protein concentration assay kit: Shanghai Beyotime Biotechnology Co., Ltd. (111922230316).

[0058] Superoxide dismutase (SOD) kit: Shanghai Beyotime Biotechnology Co., Ltd. (20230718).

[0059] Malondialdehyde (MDA) reagent kit: Shanghai Beyotime Biotechnology Co., Ltd. (081722230316).

[0060] Catalase (CAT) Assay Kit: Shanghai Beyotime Biotechnology Co., Ltd. (20230615).

[0061] The effects of different concentrations of H2O2 on the levels of SOD, MDA, and CAT in keratinocytes (Mean ± SD) were previously investigated, as shown in Tables 2 to 4.

[0062] Table 2. Effects of hydrogen peroxide on intracellular SOD levels in keratinocytes. * indicates a statistically significant difference compared to the negative control group (NC) (p<0.05).

[0063] Table 3. Effect of hydrogen peroxide on MDA content in keratinocytes (Mean±SD) * indicates a statistically significant difference compared to the negative control group (NC) (p<0.05).

[0064] Table 4. Effects of hydrogen peroxide on intracellular CAT levels in keratinocytes (Mean ± SD) Subsequently, a concentration of 0.5 mmol / L of hydrogen peroxide was selected as the modeling concentration for the subsequent efficacy test of the hydrogen peroxide-induced keratinocyte model.

[0065] 2. Experimental Procedure: 2.1 Resuspend the cells in complete culture medium and seed them into 6-well plates, and incubate for 18-24 h.

[0066] 2.2 The three test sample groups (TA) and positive control group (PC) with different concentrations were incubated for 24 h. The original culture medium was discarded, and the samples were washed once with PBS. Except for the negative control group, DMEM containing 0.5 mmol / L H2O2 was added to each group for 0.5 h. The culture medium was then replaced with the corresponding culture medium for each group and incubated for 48 h.

[0067] 2.3 Collect cells, lyse them, and test the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and catalase (CAT) activity according to the kit instructions.

[0068] 3. Results: 3.1 SOD activity test (as shown in Table 5): Table 5. Effects of the test substances on intracellular SOD levels Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05). * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0069] Compared with the model group, the relative SOD content in the sample group increased by 21.91%, 28.34%, and 19.09% at test concentrations of 0.002 mg / mL, 0.001 mg / mL, and 0.0005 mg / mL, respectively, and the differences were statistically significant (p<0.05).

[0070] 3.2 MDA content test (as shown in Table 6): Table 6. Effects of test substances on intracellular MDA levels. Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05). * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0071] Compared with the model group, the relative MDA content in the sample group decreased by 40.01%, 42.85%, and 24.07% at test concentrations of 0.002 mg / mL, 0.001 mg / mL, and 0.0005 mg / mL, respectively, and the differences were statistically significant (p<0.05).

[0072] 3.3 CAT content test (as shown in Table 7): Table 7. Effects of the test substances on intracellular CAT activity. Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05). * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0073] Compared with the model group, the relative CAT content in the sample group increased by 31.89%, 24.78%, and 21.12% at test concentrations of 0.002 mg / mL, 0.001 mg / mL, and 0.0005 mg / mL, respectively, and the differences were statistically significant (p<0.05).

[0074] 4. Conclusion: Under the conditions of this experiment, the test samples showed antioxidant activity at test concentrations of 0.002 mg / mL, 0.001 mg / mL, and 0.0005 mg / mL, and subsequent experiments can be conducted to confirm whether they have a hair-darkening effect.

[0075] S12, Melanin production test in melanocytes: 1. Experimental materials: 1.1 Cell line: Human melanoma cells (A875), derived from Kunming Cell Bank, passage number: 12.

[0076] 1.2 Complete culture medium: DMEM medium containing 10% fetal bovine serum (FBS, Gibco, Lot: 2500251P).

[0077] 1.3 Maintenance culture medium: DMEM medium containing 1% fetal bovine serum (FBS, Gibco, Lot: 2500251P).

[0078] 1.4 Test conditions: Incubator temperature 37±1℃, humidity 90±5%, CO2 5±1%.

[0079] 1.5 Sample to be tested: Mentholatum 50 Hui Black Hair Activating Essence, batch number 20230710.

[0080] 1.6 Experimental Groups and Reagents (as shown in Table 8): Table 8 (1) Source of rapamycin: Shanghai Aladdin Biochemical Technology Co., Ltd.; (2) Source of hydrogen peroxide: Shanghai Aladdin Biochemical Technology Co., Ltd. (D1021036); (3) L-DOPA: Lot: 20230925.

[0081] The effects of different concentrations of H2O2 on the inhibition rate of tyrosinase, the inhibition rate of melanin synthesis, the relative content of MITF, KIT, and TYR were investigated in the previous study (MITF-F and MITF-R were used to detect the relative content of MITF; KIT-F and KIT-R were used to detect the relative content of KIT; TYR-F and TYR-R were used to detect the relative content of TYR; and 18S internal reference gene 18S was detected using 18S-F and 18S-R). The specific detection methods are as follows: I. Total RNA extraction: 1) Collect cells, add 1 ml of Trizol solution, mix by pipetting to fully lyse the cells, and let stand for 5 min; 2) Add 200 μl of chloroform, shake vigorously for 30 seconds to ensure full contact between the aqueous and organic phases, and let stand at room temperature for 2 minutes; 3) Centrifuge at 14,000g for 15 minutes at 4℃. It can be seen that it separates into three layers, with RNA in the upper aqueous phase. Transfer it to another new RNase-free EP tube. 4) Precipitate RNA: Add an equal volume of isopropanol, mix gently and thoroughly, and let stand at room temperature for 10 minutes; 5) Centrifuge at 14,000g for 10 min at 4℃, collect the RNA precipitate, and discard the supernatant; 6) Wash twice with 75% ethanol and air dry in a clean bench; 7) Add 20-60 μl of DEPC water to dissolve the precipitate.

[0082] II. Total RNA purity and integrity testing: 1) Purity test: Take 1 μl of RNA sample, dilute it 50 times, and measure the OD value on the BioPhotometer plus nucleic acid and protein analyzer. If the ratio of OD260 / OD280 is greater than 1.8, it indicates that the prepared RNA is relatively pure and free from protein contamination.

[0083] 2) Total RNA integrity test: Take 3 μl of RNA sample, perform 1.5% agarose gel electrophoresis at 160V for 15 min, and observe the 5S rRNA, 18S rRNA and 28S rRNA bands of total RNA using a gel imaging system. If the three bands are intact, it can be proved that the total RNA extraction is relatively complete.

[0084] III. Reverse Transcription: 1) Prepare the following solutions in RNase-free PCR tubes (as shown in Table 9): Table 9 2) Incubate the above 20 μl reaction solution at 25 °C for 10 min; 3) Keep warm at 42℃ for 30 minutes; 4) Keep warm at 85℃ for 5 seconds.

[0085] IV. Quantitative PCR: 1. Detect sequence fragment size: Internal reference fragment: 18s-112bp; Target fragments: MITF-170bp; KIT-156bp; TYR-140bp; 2. Designed primers (as shown in Table 10): Table 10 3. Reaction system (as shown in Table 11): Table 11 4. Reaction conditions: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 32 s reading, 40 cycles; Melting curve analysis: temperature 60℃-95℃; Each sample was repeated 3 times; Quantitative PCR instrument: ABI PRISM ® 7500 Sequence Detection System.

[0086] The results are shown in Tables 12 to 16.

[0087] Table 12 Tyrosinase Inhibition Rate * indicates a statistically significant difference compared to the negative control group (NC) (p<0.05).

[0088] Table 13 Melanin Synthesis Inhibition Rate * indicates a statistically significant difference compared to the negative control group (NC) (p<0.05).

[0089] All tested concentrations significantly inhibited tyrosinase activity, but cell proliferation was inhibited starting at 0.5 mmol / L. Among the tested concentrations, 0.25 mmol / L significantly inhibited melanin synthesis, but cell proliferation was inhibited starting at 0.5 mmol / L. Therefore, the subsequent test concentration was set at 0.5 mmol / L.

[0090] Table 14 Relative Content of MITF Table 15 Relative Content of KIT Table 16 Relative Content of TYR * indicates a statistically significant difference compared to the negative control group (NC) (p<0.05).

[0091] The tested concentration of 0.5 mmol / L significantly inhibited the expression of KIT and TYR. Since cell proliferation was inhibited at a concentration of 0.5 mmol / L, but cell death was obvious at 1 mmol / L, no further tests were conducted. Therefore, the concentration for subsequent tests was set at 0.5 mmol / L.

[0092] 2. Experimental Procedure: 2.1 Tyrosinase activity: 2.1.1. Seed the cells into 6-well plates at a density of 1×10⁶ cells / well. 5 / 2mL / well, incubate for 18-24 h; 2.1.2 Replace with culture medium containing 1% FBS and incubate for 12 h; 2.1.3 Remove the original culture medium, add 0.5 mmol / L H2O2 to the model group, add rapamycin and 0.5 mmol / L H2O2 to the positive control group, and add culture medium containing different concentrations of the test substance and 0.5 mmol / L H2O2 to the sample group. Culture continuously for 3 days, changing the medium once in between. 2.1.4 After exposure, remove the culture medium, wash twice with PBS, collect cells from each well with a cell scraper, and centrifuge at 10,000 rpm / min and 4°C to collect the cells. 2.1.5 Add 200 μL of cell lysis buffer (containing 1% Triton X-100), and lyse the cells by repeated freeze-thaw cycles. Centrifuge at 10,000 rpm / min and 4°C for 20 min to obtain the supernatant. 2.1.6 Add 150 μL of supernatant from each group to a 96-well plate, then quickly add 20 μL of L-DOPA solution. After shaking, measure the absorbance at a wavelength of 405 nm. After reacting at 37℃ for 30 min, measure the absorbance again.

[0093] 2.2 Melanin content: 2.2.1. Seed the cells into 6-well plates and incubate for 18-24 h; 2.2.2 Remove the original culture medium and add culture medium containing different concentrations of the test substance and 0.5 mmol / L H2O2. Incubate continuously for 3 days, changing the medium once in between. 2.2.3 After exposure, remove the culture medium, wash once with PBS, collect cells from each well with a cell scraper, count the cells, and centrifuge at 1000 rpm / min to collect the cells; 2.2.4. Add a 1 mol / L NaOH solution (containing 10% DMSO) to dissolve the cells and obtain cell sap; 2.2.5. Heat the cell sap at 80℃ for 30 min to lyse the melanosomes; 2.2.6. Take cell sap and measure the absorbance at a wavelength of 490 nm.

[0094] 3. Results: 3.1 Tyrosinase activity (as shown in Table 17): Table 17 Effects of test substances on tyrosinase activity Note: # indicates a statistically significant difference compared to the blank control (BC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0095] Compared with the model group, the relative tyrosinase activity in the sample group was increased by 83.00%, 82.80%, and 55.47% at test concentrations of 0.020 mg / mL, 0.010 mg / mL, and 0.005 mg / mL, respectively, and the differences were statistically significant (p<0.05).

[0096] 3.2 Melanin content (as shown in Table 18): Table 18 Effects of the test substances on melanin content Note: # indicates a statistically significant difference compared to the blank control (BC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0097] Compared with the model group, the relative melanin content in the sample group increased by 42.61%, 35.14%, and 17.31% at test concentrations of 0.020 mg / mL, 0.010 mg / mL, and 0.005 mg / mL, respectively, and the differences were statistically significant (p<0.05).

[0098] 4. Conclusion: The test substance significantly improved tyrosinase inhibition caused by hydrogen peroxide damage at all three concentrations and increased the melanin content in cells after damage.

[0099] S2, Melanin migration assay in keratinocyte-melanocyte co-culture: 1. Experimental materials: 1.1 Cell line: Human melanoma cells (A875); Source: Kunming Cell Bank; Passage: 3.

[0100] Human immortalized keratinocytes (HaCaT); Source: Shanghai Jingkang Biotechnology Co., Ltd.; Generation: 29.

[0101] 1.2 Culture medium: DMEM medium containing 10% fetal bovine serum.

[0102] 1.3 Test conditions: Incubator temperature 37±1℃, humidity 90±5%, CO2 5±1%.

[0103] 1.4 Sample to be tested: Mentholatum 50 Hui Black Hair Activating Essence, batch number 20230710.

[0104] 1.5. Experimental Groups and Reagents (as shown in Table 19): Table 19 (1) Source of rapamycin: Shanghai Aladdin Biochemical Technology Co., Ltd.; (2) Source of hydrogen peroxide: Shanghai Aladdin Biochemical Technology Co., Ltd. (D1021036); (3) Keratin 10 antibody source: Abogen (Shanghai) Trading Co., Ltd.; (4) gp 100 antibody source: Abogen (Shanghai) Trading Co., Ltd.

[0105] Previous experiments investigated the effects of different concentrations (0.1 mmol / L, 0.5 mmol / L) of H2O2 on melanin transfer.

[0106] The fluorescence image of the negative control (NC) is shown below. Figure 1 As shown; the fluorescence spectrum when the mass concentration of H2O2 is 0.1 mmol / L is as follows. Figure 2 As shown; the fluorescence spectrum when the mass concentration of H2O2 is 0.5 mmol / L is as follows. Figure 3 As shown, all tested concentrations significantly inhibited melanin transfer, specifically by a significant reduction in the area where red and green overlap (displayed as yellow).

[0107] 2. Experimental Procedure: 2.1. HaCaT and A875 cells in the logarithmic growth phase were digested with trypsin and then cultured in DMEM complete medium at a density of 5 × 10⁶ cells / year at a ratio of 4:1. 4 A single-cell suspension of HaCatT / A875 per mL was seeded into a 24-well plate containing cell spreaders and incubated at 37°C and 5% CO2 for 24 h. 2.2 Based on the previous experimental results, a hydrogen peroxide concentration of 0.5 mmol / L was selected for subsequent experiments. Specifically, on the day of the experiment, 0.5 mmol / L hydrogen peroxide prepared with 1% serum concentration DMEM medium was added to the sample group, and an equal volume of medium was added to the negative control group. After half an hour, the medium in all wells was discarded, and the sample was washed three times with PBS. 2.3. Then, 0.02%, 0.01%, and 0.005% test samples prepared with 1% serum concentration DMEM medium were added to the test wells, an equal amount of medium was added to the blank wells, and the positive drug (rapamycin) was added to the model control group and cultured for another 48 hours. 2.4 After the culture was completed, 4% PFA was added for fixation and immunofluorescence staining was performed. The images were taken under a fluorescence microscope and analyzed using the colocalization finder in ImageJ.

[0108] 3. Results: 3.1. Co-culture immunofluorescence staining analysis of melanin migration, as shown in Table 20: Table 20 Effects of the test substances on melanin migration Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0109] Compared with the model group, the melanin migration activity of the sample group was increased by 0.19, 0.24, and 0.13 at test concentrations of 0.005 mg / mL, 0.010 mg / mL, and 0.020 mg / mL, respectively, and the differences were statistically significant (p<0.05).

[0110] 4. Conclusion: Compared with the control group, the model group showed a significant decrease in immunofluorescence colocalization ( p <0.05), the co-localization of the positive drug rapamycin by immunofluorescence was significantly increased compared with the model group ( p <0.05). All three concentrations tested significantly increased melanin transfer following hydrogen peroxide damage.

[0111] The conclusions of S1-S2 are: Regarding the test product Mentholatum 50 Hair Darkening Activating Essence: In step S11, the activity of SOD and CAT increases while the activity of MDA decreases significantly, indicating that the substance has antioxidant properties.

[0112] In step S12, the activity of tyrosinase in this substance increased significantly, and the content of melanin increased significantly.

[0113] In step S2, the overlap rate of melanin and keratinocytes was significantly increased in this substance.

[0114] Since the experiments in steps S1-S2 were all positive, we proceed to step S3 for verification.

[0115] S3. Evaluation Model for Isolated Pigskin and Evaluation of Hair Darkening Products: 1. Test materials: 1.1. Source of excised pig skin: Skin tissue left over from experiments with Tibetan miniature pigs.

[0116] 1.2 Culture medium: DMEM high glucose medium containing a mixture of 10% FBS and 1% penicillin and streptomycin (Lot: 2500251P).

[0117] 1.3 Sample to be tested: Mentholatum 50 Hui Black Hair Activating Essence, batch number 20230710.

[0118] 1.4 Experimental conditions: Incubator temperature 37±1℃, relative humidity 90±5%, CO2 content 5±1%.

[0119] 1.5. Experimental Groups and Reagents: 1.5.1 The test group settings are shown in Table 21.

[0120] Table 21 1.5.2 Test reagents: Hydrogen peroxide solution: Shanghai Aladdin Biochemical Technology Co., Ltd. (D1021036); Masson-Fontana staining kit: Shanghai Shangbao Biotechnology Co., Ltd. (R22077); 2. Experimental Procedure: 2.1. Use a punch to punch out 10 mm diameter detached pigskin, spray it with 75 vol% alcohol, wash it in PBS, aseptically process it and place it in the upper chamber of the Transwell for gas-liquid interface culture in a carbon dioxide incubator; the lower chamber of the Transwell contains complete culture medium, which is DMEM high glucose medium containing a mixture of 10% FBS and 1% penicillin and streptomycin. 2.2. Use 2mM hydrogen peroxide to treat isolated pig skin (for modeling), once every 2 days (PBS was used in the NC group), and change the Transwell lower chamber medium every 2 days. Repeat the treatment 3 times to obtain the isolated pig skin evaluation model. 2.3. Drug treatment was performed on the isolated pig skin of the model. PBS was used in the NC group, PBS was used in the M group, and the test sample was used in the TA group. The drug volume was 50 μL. 2.4 After three consecutive days of drug treatment, continue incubation for 48 hours, collect isolated pig skin samples, and divide the isolated pig skin samples into three equal parts. 2.6. Paraffin sections were prepared from isolated pig skin samples, and Masson-Fontana staining was performed on the paraffin sections.

[0121] 3. Results: Masson-Fontana melanin staining showed a significant increase in melanin in the hair follicles.

[0122] 4. Conclusion: In Masson-Fontana melanin staining, the hydrogen peroxide-treated group showed a significant reduction in melanin in the hair follicles of pig skin, while the treated sample showed a significant increase in melanin in the hair follicles.

[0123] Based on the conclusions of step two, it can be concluded that the tested sample has a hair-darkening effect.

[0124] The Mentholatum 50 Megumi Hair Darkening Activating Essence demonstrated its hair-darkening effect in actual application (clinical trials verified the product's hair-darkening effect. After 8 weeks of product use, the improvement rate of gray hair percentage was 12.2%, and after 12 weeks, the improvement rate of gray hair percentage was 30.3%). This indicates that the actual application effect of the Mentholatum 50 Megumi Hair Darkening Activating Essence is consistent with the evaluation results using the method described in this application, demonstrating that the evaluation results using this method have better accuracy.

[0125] Example 2 1. Except for the test samples, the details of each test are the same as in Example 1. The information of the test samples is as follows: Sample to be tested: Bortezomib, catalog number: MCE, HY-10227.

[0126] 2. Results: As shown in Tables 22-27.

[0127] Table 22 Effects of test substances on intracellular SOD levels Note: # indicates a statistically significant difference (p<0.05) compared with the negative control group (NC); * indicates a statistically significant difference (p<0.05) compared with the model control group (M); the above data were processed using SPSS software.

[0128] Compared with the model group, the relative SOD content in the sample group decreased by 16.83%, 60.75%, and 62.3% at test concentrations of 2 nM, 1 nM, and 0.5 nM, respectively, and the differences were statistically significant (p<0.05).

[0129] Table 23 Effects of test substances on intracellular CAT activity Note: # indicates a statistically significant difference (p<0.05) compared with the negative control group (NC); * indicates a statistically significant difference (p<0.05) compared with the model control group (M); the above data were processed using SPSS software.

[0130] Compared with the model group, the relative CAT content in the sample group increased by 244.5%, 175.61%, and 190.2% at test concentrations of 2 nM, 1 nM, and 0.5 nM, respectively, and the differences were statistically significant (p<0.05).

[0131] Table 24 Effects of test substances on intracellular MDA content Note: # indicates a statistically significant difference (p<0.05) compared with the negative control group (NC); * indicates a statistically significant difference (p<0.05) compared with the model control group (M); the above data were processed using SPSS software.

[0132] Compared with the model group, the relative MDA content in the sample group decreased by 18.58%, 18.58%, and 9.76% at test concentrations of 2 nM, 1 nM, and 0.5 nM, respectively, and the differences were statistically significant (p<0.05).

[0133] Table 25 Effects of test substances on tyrosinase activity Note: # indicates a statistically significant difference compared to the blank control (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0134] Compared with the model group, the relative tyrosinase activity in the sample group was increased by 86.03%, 30.91%, and 6.22% at test concentrations of 0.8 nM, 0.4 nM, and 0.2 nM, respectively, and the differences were statistically significant (p<0.05).

[0135] Table 26 Effects of the test substances on melanin content Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0136] Compared with the model group, the sample group showed a 46.76%, 30.88%, and 6.42% increase in relative melanin activity at test concentrations of 0.8 nM, 0.4 nM, and 0.2 nM, respectively, with significant differences (p<0.05).

[0137] Table 27 Effects of the test substances on melanin migration Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0138] Compared with the model group, the relative melanin activity in the sample group increased by 0.04% and 0.02% at test concentrations of 0.8 nM and 0.4 nM, respectively, but the differences were not statistically significant (p<0.05).

[0139] 3. Results: 3.1 The substance showed a significant decrease in MDA and a significant increase in CAT in step S11, which suggests that it has antioxidant properties.

[0140] 3.2 In step S12, the activity of tyrosinase in this substance increased significantly, and the melanin content increased significantly.

[0141] 3.3 In step S2, the overlap rate of melanin and keratinocytes was not significantly increased in the experiment.

[0142] Bortezomib is known to promote melanin expression, but this ingredient does not promote melanin migration, so there is no need to proceed to step S3 for further verification.

[0143] Using the method described in this application, the hair-darkening effect of the test sample can be accurately determined without further in vitro pig skin testing, which has the advantages of saving costs and improving evaluation timeliness.

[0144] Example 3 1. Except for the test samples, the details of each test are the same as in Example 1. The information of the test samples is as follows: Sample to be tested: receptor-2 for protease activation, amide (SLIGKV-NH2); catalog number: MCE, HY-P0283.

[0145] 2. Results: As shown in Tables 28-33.

[0146] Table 28 Effects of test substances on intracellular SOD levels Note: # indicates a statistically significant difference (p<0.05) compared with the negative control group (NC); * indicates a statistically significant difference (p<0.05) compared with the model control group (M); the above data were processed using SPSS software.

[0147] Compared with the model group, the relative SOD activity in the sample group was reduced by 34.11% at a test concentration of 200 μM, which was statistically significant (p<0.05).

[0148] Table 29 Effects of test substances on intracellular CAT activity Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0149] Compared with the model group, the relative CAT content activity of the sample group was increased by 60.46% and 83.94% at test concentrations of 200 μM and 100 μM, respectively, and the differences were statistically significant (p<0.05).

[0150] Table 30 Effects of test substances on intracellular MDA content Note: # indicates a statistically significant difference (p<0.05) compared with the negative control group (NC); * indicates a statistically significant difference (p<0.05) compared with the model control group (M); the above data were processed using SPSS software.

[0151] Compared with the model group, the relative activity of MDA in the sample group was reduced by 12.75% at a test concentration of 50 μM, which was statistically significant (p<0.05).

[0152] Table 31 Effects of test substances on tyrosinase activity Note: # indicates a statistically significant difference compared to the blank control (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0153] Compared with the model group, the relative tyrosinase activity in the sample group was increased by 62.13%, 55.82%, and 38.82% at test concentrations of 40 nM, 20 nM, and 10 nM, respectively, and the differences were statistically significant (p<0.05).

[0154] Table 32 Effects of the test substances on melanin content Note: # indicates a statistically significant difference (p<0.05) compared with the negative control group (NC); * indicates a statistically significant difference (p<0.05) compared with the model control group (M); the above data were processed using SPSS software.

[0155] Compared with the model group, the relative melanin activity of the sample group increased by 24.41%, 19.90%, and 13.32% at test concentrations of 40 nM, 20 nM, and 10 nM, respectively, and the differences were statistically significant (p<0.05).

[0156] Table 33 Effects of the test substances on melanin migration Note: # indicates a statistically significant difference compared to the negative control group (NC) (p<0.05); * indicates a statistically significant difference compared to the model control group (M) (p<0.05); the above data were processed using SPSS software.

[0157] Compared with the model group, the sample group showed no significant difference in relative melanin content activity at test concentrations of 40 nM, 20 nM, and 10 nM (p<0.05).

[0158] 3. Results: 3.1 The CAT level of this substance increased significantly in step S11, while the other indicators did not change significantly, indicating that it does not have antioxidant properties.

[0159] 3.2 The activity of tyrosinase in this substance increased significantly in step S12, but the increase in melanin content was relatively small.

[0160] 3.3 In step S2, the overlap rate of melanin and keratinocytes was not significantly increased in the experiment.

[0161] Since the raw material has no antioxidant properties and does not promote melanin migration, there is no need to proceed to step S3 for further verification.

[0162] Using the method described in this application, the hair-darkening effect of the test sample can be accurately determined without further in vitro pig skin testing, which has the advantages of saving costs and improving evaluation timeliness.

[0163] This application provides a method for evaluating the efficacy of in vitro hair-darkening raw materials or product combinations and its application. This application employs single-cell culture (an antioxidant model of keratinocytes and a melanin production model of melanocytes), multi-cell co-culture (a melanin migration model based on keratinocyte-melanocyte co-culture), and an in vitro porcine skin evaluation model. The evaluation method of this application is simple, provides intuitive results, and can be widely used for evaluating the efficacy of hair-darkening raw materials or products. It is an ideal method for predicting the efficacy of hair-darkening raw materials or products. Furthermore, the evaluation method of this application can replace live animals and human skin and can be directly used for hair-darkening efficacy testing of chemicals, cosmetics, pharmaceuticals, and other hair-darkening products. The method established based on in vitro animal skin also conforms to the 3R principle of reducing optimization and replacing live animal experiments.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for evaluating the efficacy of an in-vitro hair growth material or product combination, characterized by, The method comprises the following steps: S1, respectively, using the antioxidant model of keratinocytes and the melanin production model of melanocytes to test the sample to be tested; If the test results of the antioxidant model of keratinocytes and the melanin production model of melanocytes are both negative, it can be directly judged that the sample to be tested does not have hair blackening effect, otherwise step S2 is performed; S2, using the melanin migration model of keratinocyte-melanocyte co-culture to test the sample to be tested after step S1; If the test result of the melanin migration model of keratinocyte-melanocyte co-culture is negative, it is directly judged that the sample to be tested does not have hair blackening effect; if the test result is positive, step S3 is performed; S3, using the ex vivo pig skin evaluation model to test the sample to be tested after step S2; If the test result of the ex vivo pig skin evaluation model is negative, it is directly judged that the sample to be tested does not have hair blackening effect; if the test result is positive, the sample to be tested has hair blackening effect.

2. The method for evaluating the in-vitro hair-growing material or the product combination efficacy according to claim 1, wherein, The method for testing the sample to be tested by using the antioxidant model of keratinocytes comprises the following steps: 1) The cultured keratinocytes are divided into a negative control group, a model control group, a positive control group and a test group. The keratinocytes in the negative control group and the model control group are cultured in a maintenance culture medium. The keratinocytes in the positive control group are cultured in a maintenance culture medium added with a Vc derivative. The keratinocytes in the test group are cultured in a maintenance culture medium added with the sample to be tested. Then, the model control group, the positive control group and the test group are respectively cultured in a culture medium containing an oxidizing agent; 2) The keratinocytes in the negative control group, the model control group, the positive control group and the test group after step 1) are broken, and the superoxide dismutase activity, the catalase activity and the malondialdehyde content of the cells in each group are detected; In the case that the superoxide dismutase activity and the catalase activity of the model control group are lower than those of the negative control group, the malondialdehyde content of the model control group is higher than that of the negative control group, the superoxide dismutase activity and the catalase activity of the positive control group are higher than those of the model control group, and the malondialdehyde content of the positive control group is lower than that of the model control group, if the superoxide dismutase activity and the catalase activity of the test group are higher than those of the model control group, and the malondialdehyde content of the test group is lower than that of the model control group, with a significant difference p<0.05, the test result is positive, otherwise it is negative.

3. The in-vitro hair growth promoting material or product combination efficacy evaluation method according to claim 1, wherein The method for testing the sample to be tested by using the melanin production model of melanocytes comprises the following steps: 1) The melanoma cells are divided into a negative control group, a model control group, a positive control group and a test group. The melanoma cells in the negative control group and the model control group are cultured in a maintenance culture medium. The melanoma cells in the positive control group are cultured in a maintenance culture medium added with rapamycin. The melanoma cells in the test group are cultured in a maintenance culture medium added with the sample to be tested. Then, the model control group, the positive control group and the test group are respectively cultured in a culture medium containing an oxidizing agent; 2) co-culturing the melanoma cells of the negative control group, the model control group, the positive control group and the test group treated in step 1) with the culture medium containing the oxidant, then centrifuging to collect the cells, lysing the cells, obtaining the supernatant, and determining the absorbance of the supernatant; In the case that the tyrosinase activity of the model control group is lower than that of the negative control group, and the tyrosinase activity of the positive control group is higher than that of the model control group, if the tyrosinase activity of the test group is higher than that of the model control group with a significant difference p<0.05, the test result is positive, otherwise, it is negative. Or, 2) co-culturing the melanoma cells of the model control group, the positive control group and the test group treated in step 1) with the culture medium containing H2O2, then centrifuging to collect the cells, adding NaOH solution to dissolve the cells, obtaining the cell solution, heating the cell solution to lyse the melanosome, and determining the absorbance value of the cell solution to determine the melanin content; In the case that the melanin content of the model control group is lower than that of the negative control group, and the melanin content of the positive control group is higher than that of the model control group, if the melanin content of the test group is higher than that of the model control group with a significant difference p<0.05, the test result is positive, otherwise, it is negative.

4. The in-vitro hair growth promoting material or product combination efficacy evaluation method according to claim 1, wherein The method for testing the sample by using the melanin migration model of the co-cultured keratinocytes-melanocytes comprises the following steps: 1) digesting the melanoma cells and the keratinocytes to obtain a single cell mixed suspension, and dividing the cells into a negative control group, a model control group, a positive control group and a test group, culturing the cells of the negative control group and the model control group in the maintenance culture medium, culturing the cells of the positive control group in the maintenance culture medium added with rapamycin, culturing the cells of the test group in the maintenance culture medium added with the sample to be tested, and then culturing the cells of the model control group, the positive control group and the test group in the culture medium containing the oxidant; 2) performing immunofluorescence staining on the cells of the negative control group, the model control group, the positive control group and the test group treated in step 1), and determining the melanin migration amount; If the melanin migration amount of the test group is higher than that of the model control group with a significant difference p<0.05, the test result is positive, otherwise, it is negative.

5. The in-vitro hair-growing material or product combination efficacy evaluation method according to claim 2, wherein The molar concentration of the oxidant in the method for testing the sample by using the antioxidant model of the keratinocytes is 0.125-2 mmol / L.

6. The in-vitro hair-growing material or the method for evaluating the efficacy of a product combination according to claim 3, wherein The molar concentration of the oxidant in the method for testing the sample by using the melanin production model of the melanocytes is 0.125-0.5 mmol / L.

7. The in-vitro hair growth promoting material or product combination efficacy evaluation method according to claim 4, wherein The molar concentration of the oxidant in the method for testing the sample by using the melanin migration model of the co-cultured keratinocytes-melanocytes is 0.1-0.5 mmol / L.

8. The in-vitro hair-growing material or the method for evaluating the efficacy of a product combination according to any one of claims 5 to 7, characterized in that, The oxidant comprises H2O2.

9. The in-vitro hair growth promoting material or product combination efficacy evaluation method according to claim 1, wherein The keratinocytes comprise HaCaT; And / or, the melanocytes comprise B16 or A875.

10. The application of the method for evaluating the efficacy of hair-growing raw materials or product combinations in vitro according to any one of claims 1-9 in evaluating hair-growing products.

Images