Evaluation method of freckle-removing and whitening effects and application thereof

Abstract

The invention relates to a freckle-removing and whitening effect evaluation method and application thereof. The evaluation method comprises the following steps: (1) culturing MNT-1 cells by using a culture medium containing a to-be-detected product and a culture medium not containing the to-be-detected product respectively, cracking the MNT-1 cells by using a cell lysis solution, centrifugally collecting melanin precipitates, dissolving the collected melanin precipitates by using an alkali solution, and collecting the dissolved melanin precipitates; respectively measuring the content I of melanin in the dissolving solution containing the to-be-detected product and the content II of melanin in the dissolving solution without the to-be-detected product; and (2) analyzing the significant difference between the melanin content I and the melanin content II to obtain a P value, and if the P value is less than 0.05, determining that the to-be-detected product has the freckle-removing and whitening effects. The culture medium in the step (1) contains a basic culture medium, bFGF, IBMX and alpha-MSH; the basic culture medium is prepared from DMEM (Dulbecco Modified Eagle Medium), AIM-V (Alcohol Immunodeficiency Virus), fetal calf serum, NEAA (Neotropium Acetic The evaluation method is high in accuracy, high in stability and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a method for evaluating freckle-removing and whitening efficacy and application thereof. Background Art

[0002] With the growing demand for freckle-removing and whitening products, the evaluation methods for their efficacy are gaining increasing attention. Currently, methods for evaluating freckle-removing and whitening efficacy primarily include in vitro cell experiments, animal experiments, and human trials. In vitro cell experiments, due to their ease of use, low cost, and high reproducibility, are widely used for preliminary screening and evaluation of freckle-removing and whitening efficacy.

[0003] Tumor cells, due to their advantages of multiple passages, rapid growth, and ease of culture, are preferred cell models for screening whitening agents, such as mouse melanoma B16F10 cells, human melanoma A375 cells, and human melanoma MNT-1 cells. MNT-1 cells are a fibroblast-like cell line isolated from the lymph nodes of melanoma patients. They are highly pigmented melanocytes with active melanin production and are commonly used in pigmentation research, melanosome characterization, melanosome biogenesis, effects on human pigmentation, and membrane trafficking.

[0004] In the prior art, there are various methods for evaluating the efficacy of freckle removal and whitening using cells. CN114574541A discloses a method for accurately detecting the effect of whitening products on melanin content in mouse B16F10 cells. This method uses cell counting to make the cell number of the sample treatment group, positive control group, and blank control group consistent. Then, the cells are treated with a lysate and the melanin content of the cell population with the same cell number is directly measured. Although this method can eliminate the error in melanin content measurement caused by different cell numbers in different samples, it requires cell counting during the experiment, which increases the number of steps and experimental errors.

[0005] CN101532950A discloses an experimental method for toxicity and efficacy analysis of whitening chemicals using primary cultured human skin melanocytes. The method includes culturing and identifying primary human skin melanocytes, performing toxicity analysis of whitening chemicals using primary human melanocytes, and analyzing the efficacy of whitening chemicals using primary human melanocytes. Although this method can replace live animals and human skin and be directly used for toxicity and efficacy testing of whitening substances in products such as chemicals, cosmetics, and pharmaceuticals, obtaining and culturing primary cells is relatively difficult, and batch-to-batch variability can affect the stability of experimental results.

[0006] On May 28, 2024, the National Medical Products Administration (NMPA) announced that, effective November 24, 2024, bioanalytical methods must undergo methodological validation in accordance with the requirements of the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Technical Guidelines for Validation of Analytical Procedures Q2(R2) and Technical Guidelines for Development of Analytical Procedures Q14. The 2025 edition of the Chinese Pharmacopoeia, Part IV, stipulates that bioanalytical methods must also undergo analytical validation in accordance with the requirements. A search of Chinese and foreign literature on methods for inhibiting melanin production in melanocytes revealed no mention of analytical method validation. Data generated using unvalidated methods pose risks to specificity, accuracy, and reproducibility. Konrad Kleszczyński et al. used thiazolium blue tetrazolium bromide (MTT) reagent to detect the cell viability of melanocytes (Konrad Kleszczyński1 et al. Melatonin exerts oncostatic capacity and decreasesmelanogenesis in human MNT-1 melanoma cells. J Pineal Res. 2019 November; 67(4): e12610. doi: 10.1111 / jpi.12610.); while Shilpi Goenka used Alamar Blue dye or 3-(4,5-dimethyl-2-yl)-5-(3-methoxyphenyl)-2-(4-thiophenyl)-2H tetrazolium solution to determine the toxicity of MNT-1 cells (Shilpi Goenka et al. Depigmenting effect of Xanthohumol from hop extract in MNT-1 human melanoma cells and normal human melanocytes.https: / / doi.org / 10.1016 / j.bbrep.2021.100955); Chi-Hyun Park et al. treated human melanoma MNT-1 cells with EZ-CYTOX reagent after incubation of the test substance with dehydroepiandrosterone or chemical inhibitors for a specified time. The culture medium was collected and the absorbance value was measured at 450 nm or 650 nm (Chi-Hyun Park et al. A natural compound harmine decreases melanin synthesis through regulation of the DYRK1A / NFATC3 pathway.https: / / doi.org / 10.1016 / j.jdermsci.2021.05.003).These methods are complicated to operate and require measuring the absorbance value twice, which leads to large errors in the results and low accuracy.

[0007] In view of the shortcomings of the existing technology, developing an evaluation method for the freckle removal and whitening efficacy with high accuracy, high detection efficiency, good stability and simple operation has become one of the technical problems that need to be solved urgently. Summary of the Invention

[0008] In response to the shortcomings of the prior art, the present invention aims to provide a method for evaluating the freckle-removing and whitening efficacy of a product and its application. Specifically, the present invention provides a method for evaluating the freckle-removing and whitening efficacy of a product and its application. This method is economical, fast, highly accurate, highly specific, highly stable, and reproducible.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for evaluating freckle removal and whitening efficacy, the evaluation method comprising:

[0011] (1) MNT-1 cells are cultured with a culture medium containing the test substance, the MNT-1 cells are lysed with a cell lysis solution, and the melanin precipitate is collected by centrifugation. The collected melanin precipitate is dissolved with an alkaline solution, and the melanin content in the dissolved solution is measured I.

[0012] MNT-1 cells were cultured in a culture medium without the test substance, lysed with a cell lysis solution, and melanin precipitates were collected by centrifugation. The collected melanin precipitates were dissolved with an alkaline solution, and the melanin content in the dissolved solution was measured.

[0013] (2) Analyze the significant difference between melanin content I and melanin content II to obtain the P value. If the P value is less than 0.05, it is determined that the product to be tested has the effect of removing spots and whitening.

[0014] The culture medium in step (1) contains a basal culture medium and bFGF, IBMX and α-MSH. The basal culture medium contains: DMEM, AIM-V, fetal bovine serum, NEAA and penicillin / streptomycin double antibody.

[0015] Here, less than 0.05 may be less than 0.05, less than 0.04, less than 0.03, less than 0.02, less than 0.01, etc.

[0016] MNT-1 cells are a fibroblast-like cell line isolated from the lymph nodes of melanoma patients. They are highly pigmented melanocytes with active melanin production and are commonly used in pigment research, melanosome properties, melanosome biogenesis, effects on human pigmentation, and membrane transport. This innovative method uses MNT-1 cells as a model cell for in vitro cell evaluation. The melanin content in MNT-1 cells is used to assess the freckle-removing and whitening efficacy of the test product. This method enables rapid, accurate, and stable evaluation of freckle-removing and whitening efficacy. Compared to other common melanoma cells, such as human melanoma A375 cells and mouse melanoma B16F10 cells, MNT-1 cells exhibit superior evaluation stability, providing guidance for evaluating the efficacy of cosmetics.

[0017] Furthermore, the present inventors have inventively discovered that adding bFGF to the culture medium can further enhance the growth stability of MNT-1 cells, while adding IBMX can further accelerate the growth and differentiation of MNT-1 cells. Adding α-MSH can promote the activation of MNT-1 cells and increase melanin production. The present invention evaluates the freckle-removing and whitening efficacy by assessing the relative amount of melanin produced in cells. The addition of these additives can further enhance the accuracy, stability, and repeatability of the evaluation method.

[0018] Preferably, in the culture medium of step (1), the amount of bFGF added is 10-20 ng / mL, the amount of IBMX added is 33-55 μg / mL, and the amount of α-MSH added is 83-333 ng / mL.

[0019] Among them, the specific point values ​​in 10-20ng / mL can be selected as 10ng / mL, 12ng / mL, 14ng / mL, 16ng / mL, 18ng / mL, 20ng / mL, etc., the specific point values ​​in 33-55μg / mL can be selected as 33μg / mL, 40μg / mL, 45μg / mL, 50μg / mL, 55μg / mL, etc., and the specific point values ​​in 83-333ng / mL can be selected as 83ng / mL, 100ng / mL, 150ng / mL, 200ng / mL, 250ng / mL, 300ng / mL, 333ng / mL, etc.

[0020] Preferably, the basic culture medium comprises, by volume percentage, 65-75% DMEM, 6-10% AIM-V, 15-25% fetal bovine serum, 0.5-1.5% NEAA, and 0.5-1.5% penicillin / streptomycin dual antibody.

[0021] Among them, the specific point values ​​in 65-75% can be selected from 65%, 67%, 69%, 71%, 73%, 75%, etc., the specific point values ​​in 6-10% can be selected from 6%, 7%, 8%, 9%, 10%, etc., the specific point values ​​in 15-25% can be selected from 15%, 17%, 19%, 21%, 23%, 25%, etc., and the specific point values ​​in 0.5-1.5% can be selected from 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, etc.

[0022] Preferably, in step (1), the MNT-1 cell seeding density of culturing the MNT-1 cells with the medium containing the test article and culturing the MNT-1 cells with the medium without the test article is independently selected from 9×10 4 -9.5×10 4 / mL, for example, it can be 9×10 4 / mL, 9.1×10 4 / mL, 9.2×10 4 / mL, 9.3×10 4 / mL, 9.4×10 4 / mL, 9.5×10 4 pieces / mL, etc.

[0023] Preferably, the MNT-1 cell seeding density in step (1) of culturing the MNT-1 cells in the medium containing the test article and culturing the MNT-1 cells in the medium without the test article is the same.

[0024] The present invention controls the seeding density of MNT-1 cells to 9-9.5×10 4 cells / mL, which can improve the growth stability of MNT-1 cells, thereby further improving the stability and repeatability of the freckle removal and whitening evaluation method based on the MNT-1 cell model.

[0025] Preferably, the culture time of culturing MNT-1 cells with the medium containing the test article and the culture time of culturing MNT-1 cells with the medium without the test article are each independently selected from 48-72 hours, for example, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, etc.

[0026] Preferably, the culturing time of the MNT-1 cells in the culture medium containing the test article and the culturing time of the MNT-1 cells in the culture medium without the test article in step (1) are the same.

[0027] Preferably, the cell lysis solution in step (1) comprises a non-denaturing cell lysis solution.

[0028] Native cell lysis buffer contains a nonionic detergent that lyses cells and releases cytoplasmic proteins, soluble membrane proteins, and nuclear proteins under native conditions. Protein lysis products obtained under native conditions maximize the preservation of protein properties and functions, such as antigen-antibody binding and enzymatic activity. Since melanin is a protein present on the MNT-1 cell membrane, using native cell lysis buffer allows for maximum extraction without damaging the melanin.

[0029] Preferably, the cell lysate further contains additives.

[0030] Preferably, the mass percentage of the additive in the cell lysate is 2-7%, for example, 2%, 3%, 4%, 5%, 6%, 7%, etc.

[0031] Preferably, the additives in the cell lysate include phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium edetate.

[0032] Phenylmethylsulfonyl fluoride is a serine protease inhibitor, imidazole is a metal ion chelator that inhibits the activity of metalloproteinases, sodium fluoride is a phosphatase inhibitor, and sodium EDTA is a metal ion chelator. The present invention creatively discovered that the simultaneous addition of phenylmethylsulfonyl fluoride, imidazole, sodium fluoride, and sodium EDTA to a cell lysate can fully lyse MNT-1 cells while ensuring the stability of melanin, improving the accuracy of melanin content detection, and thereby enhancing the repeatability and stability of the freckle removal and whitening assessment method.

[0033] Preferably, the mass ratio of phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium edetate is (0.5-2):(0.5-2):(0.5-2):(0.1-1).

[0034] Among them, the specific point values ​​in 0.5-2 can be selected from 0.5, 0.8, 1.1, 1.4, 1.7, 2, etc., and the specific point values ​​in 0.1-1 can be selected from 0.1, 0.3, 0.5, 0.7, 0.9, 1, etc.

[0035] Preferably, the cracking temperature in step (1) is 3-5°C, and the cracking time is 15-25 minutes.

[0036] Among them, the specific point values ​​in 3-5℃ can be selected from 3℃, 4℃, 5℃, etc., and the specific point values ​​in 15-25min can be selected from 15min, 18min, 21min, 25min, etc.

[0037] Preferably, the alkaline solution in step (1) comprises a DMSO solution of an alkali.

[0038] In the present invention, DMSO is used as a solvent to dissolve the melanin, which can further improve the dissolution efficiency and dissolution stability of the melanin.

[0039] Preferably, the base comprises NaOH and / or KOH.

[0040] Preferably, the pH value of the alkaline solution is 12.5-13.5, for example, it can be 12.5, 12.7, 12.9, 13.1, 13.3, 13.5, etc.

[0041] Controlling the pH value of the alkaline solution at 12.5-13.5 can improve the dissolution efficiency and dissolution stability of melanin, thereby improving the repeatability and stability of the freckle removal and whitening evaluation method.

[0042] Preferably, in the culture medium containing the test article in step (1), the concentration of the test article is below the maximum safe concentration of the test article for MNT-1 cells.

[0043] Preferably, the method for testing the maximum safe concentration of the test substance on MNT-1 cells is:

[0044] MNT-1 cells were cultured with culture media containing different concentrations of the test substance. The test substance concentration at which the survival rate of MNT-1 cells was above 90-100% was the maximum safe concentration of the test substance for MNT-1 cells.

[0045] Preferably, the detection reagent for the survival rate of MNT-1 cells includes CCK-8 solution.

[0046] Preferably, the test product includes any one or a combination of at least two of raw materials with freckle-removing and whitening effects, cosmetic products, drugs for treating chloasma, drugs for treating age spots, or drugs for treating post-inflammatory pigmentation.

[0047] In a second aspect, the present invention provides an application of the freckle-removing and whitening efficacy evaluation method as described in the first aspect in evaluating the freckle-removing and whitening efficacy of cosmetics or raw materials having freckle-removing and whitening efficacy.

[0048] In a third aspect, the present invention provides an application of the freckle-removing and whitening efficacy evaluation method as described in the first aspect in evaluating the efficacy of drugs for treating chloasma, senile plaques or post-inflammatory pigmentation.

[0049] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention provides a method for evaluating freckle-removing and whitening efficacy using MNT-1 cells. MNT-1 cells are used as model cells for an in vitro cell evaluation method, and the freckle-removing and whitening efficacy of a test product is evaluated based on the melanin content in the MNT-1 cells. The method is economical and fast, has high accuracy, strong specificity, high stability, and good repeatability.

[0052] Furthermore, adding bFGF, IBMX, and α-MSH to the culture medium of MNT-1 cells can further enhance the growth stability of MNT-1 cells, promote their growth and differentiation, and increase melanin production. This method evaluates the freckle-removing and whitening efficacy by assessing the relative amount of melanin produced in cells. The addition of these additives significantly improves the stability and repeatability of the evaluation method.

[0053] Furthermore, the seeding density of MNT-1 cells was controlled at 9-9.5×10 4 cells / mL, which can improve the growth stability of MNT-1 cells, thereby further improving the stability and repeatability of the freckle removal and whitening evaluation method based on the MNT-1 cell model.

[0054] Furthermore, in the process of extracting melanin from cells, the MNT-1 cells are lysed by using a non-denaturing cell lysis solution, which can extract the melanin to the greatest extent without destroying it.

[0055] Furthermore, by simultaneously adding phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium edetate to the cell lysate, the stability of melanin can be ensured while fully lysing the MNT-1 cells, thereby improving the accuracy of melanin content detection and thereby improving the repeatability and stability of the freckle removal and whitening evaluation method.

[0056] Furthermore, in the process of dissolving melanin, using an alkaline solution with a pH value of 12.5-13.5 to dissolve the melanin can further improve the dissolution efficiency and dissolution stability of the melanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the regression curve of the melanin production inhibition rate of Thiamidol in the first repeated test of Example 1 in Test Example 1.

[0058] Figure 2 This is the regression curve of the melanin production inhibition rate of Thiamidol in the second repeated test of Example 1 in Test Example 1.

[0059] Figure 3 This is the regression curve of the melanin production inhibition rate of Thiamidol in the third repeated test of Example 1 in Test Example 1.

[0060] Figure 4 This is the regression curve of the melanin production inhibition rate of Thiamidol in the fourth repeated test of Example 1 in Test Example 1.

[0061] Figure 5 This is the regression curve of the melanin production inhibition rate of Thiamidol in the fifth repeated test of Example 1 in Test Example 1.

[0062] Figure 6 This is the regression curve of the melanin production inhibition rate of Thiamidol in the sixth repeated test of Example 1 in Test Example 1. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] The sources of the raw materials in the following embodiments are shown in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] The following MNT-1 cell resuscitation method is as follows: Add 10 mL of culture medium to a 100 mm cell culture dish. Remove the cryovial containing MNT-1 cells (cell number CVCL5624) from a -80°C freezer and quickly place it in a preheated 37°C water bath with constant shaking to thaw the cell suspension within 1 minute. Add the thawed cell suspension to the aforementioned cell culture dish and agitate thoroughly. Incubate the cells at 37°C with 5% CO2. Once the cells have attached, replace with fresh medium and continue culturing. Subculture the cells when they are approximately 80% grown.

[0069] Example 1

[0070] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells:

[0071] (1) Determination of the maximum safe concentration of Thiamidol for MNT-1 cells:

[0072] MNT-1 cells in good condition and in the exponential growth phase were obtained, digested with trypsin for 30 seconds, counted using a cell counter, and seeded into 96-well plates at a seeding density of 9.5 × 10 4Cells were cultured for 32 hours at 37°C and 5% CO2. Culture media containing different concentrations of the test article (thiamidol concentrations in the culture media were 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, and 200 μmol / L) and culture media without the test article (i.e., the control group) were then added and cultured for 24 hours. 10 μL of CCK-8 solution was then added to each well, incubated at 37°C for 2 hours, shaken on a shaker for 5 minutes, and the OD value of each well was measured at a wavelength of 450 nm to calculate relative cell viability.

[0073] Relative cell viability = OD value of the test group / OD value of the control group × 100%.

[0074] Table 2 shows the relative cell viability test results for different Thiamidol concentrations. It shows that at a concentration of 25 μmol / L, the survival rate of MNT-1 cells was above 103.5%, with no significant cytotoxicity. This indicates that 25 μmol / L is the maximum safe concentration for the tested Thiamidol.

[0075] Table 2

[0076] Concentration (μmol / L) 6.25 12.5 25 50 100 200 Cell survival rate / % 119.2 117.6 103.5 85.2 52.8 27.7 SD 0.052 0.042 0.031 0.033 0.006 0.0004

[0077] (2) Determination of relative melanin content:

[0078] MNT-1 cells were seeded in 6-well plates at a density of 9.2 × 10 4 / mL, 2mL / well. After adhesion, the culture medium containing different concentrations of the test article (the concentration of Thiamidol in the culture medium was 25μmol / L, 12.5μmol / L, 6.25μmol / L, 3.125μmol / L, and 1.5625μmol / L) and the culture medium without the test article (control group) were replaced and treated for 60h (2.0mL / well). After 60h of culture, the cultured cells were discarded from the culture medium and washed 3 times with PBS. The cell culture dish was placed on an ice plate, and 400μL of non-denaturing cell lysis buffer containing 5% additives (phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium ethylenediaminetetraacetic acid in a mass ratio of 1:1:1:0.5) was added to each dish and lysed at 4℃ for 20min. The lysate was centrifuged at 4℃ and 13000r / min for 10min, and the melanin precipitate was collected. Add 330 μL of NaOH in DMSO (pH 13) to the melanin precipitate and vortex to dissolve it completely. Place it in an 80°C metal bath for 2 hours to dissolve the melanin precipitate completely. Vortex to mix thoroughly, then centrifuge at 12,000 rpm for 3 minutes. Collect the supernatant to obtain the melanin solution.

[0079] 96-well plate was added 200 μL of the above melanin solution, and three replicates were set up. A blank group was set up (200 μL of PBS solution was added to the 96-well plate), and OD values were measured at 405 nm. The relative melanin production was calculated, and the P value was calculated.

[0080] The relative melanin production = (OD value of the test sample group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) x 100%.

[0081] The above medium was prepared by adding 15 ng / mL of bFGF, 40 μg / mL of IBMX, and 200 ng / mL of α-MSH to a basal medium (DMEM 70%, AIM-V 8%, fetal bovine serum 20%, NEAA 1%, and penicillin / streptomycin double antibiotic 1% by volume percentage).

[0082] The test results of the relative melanin production are shown in Table 3. It can be seen that when the sample concentration is 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 3.125 μmol / L, and 1.5625 μmol / L, there is a significant difference, and it is determined that the test product has a depigmentation and whitening effect.

[0083] Table 3

[0084] Group Relative melanin content mean (%) P-value Significance control group 100 / / Thiamidol-25μmol / L 34.0 ＜0.001 *** Thiamidol-12.5μmol / L 49.2 ＜0.001 *** Thiamidol-6.25μmol / L 58.7 ＜0.001 *** Thiamidol-3.125μmol / L 65.1 ＜0.001 *** Thiamidol-1.5625μmol / L 69.5 ＜0.001 ***

[0085] Example 2

[0086] This example provides a method for evaluating the whitening efficacy of the raw material α-arbutin using MNT-1 cells:

[0087] (1) Determining the maximum safe concentration of α-arbutin on MNT-1 cells:

[0088] According to the method of Example 1, it was determined that the maximum safe concentration of α-arbutin was 200 μmol / L.

[0089] (2) Determining the relative content of melanin:

[0090] MNT-1 cells were inoculated in a 6-well plate at a density of 9 x 10 4 / mL, 2mL / well. After adhesion, the culture medium containing different concentrations of the test article (the concentration of α-arbutin in the culture medium was 200μmol / L, 100μmol / L, 50μmol / L, 25μmol / L, and 12.5μmol / L) and the culture medium without the test article (control group) were replaced and treated for 48h (2.0mL / well). After 48h of culture, the cultured cells were discarded from the culture medium and washed 3 times with PBS. The cell culture dish was placed on an ice plate, and 400μL of non-denaturing cell lysis buffer containing 3% additives (phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium ethylenediaminetetraacetic acid in a mass ratio of 0.5:2:2:0.1) was added to each dish and lysed at 3℃ for 25min. The lysate was centrifuged at 4℃ and 12000r / min for 12min, and the melanin precipitate was collected. Add 350 μL of KOH in DMSO (pH 12.5) to the melanin precipitate and vortex to dissolve it completely. Place it in a 75°C metal bath for 2.5 hours to dissolve the melanin precipitate completely. Vortex to mix thoroughly, then centrifuge at 13,000 rpm for 3 minutes. Collect the supernatant to obtain the melanin solution.

[0091] Add 200 μL of the above melanin solution to each well of a 96-well plate, set up 4 replicate wells, set up a blank well (add 200 μL of PBS solution to the 96-well plate), measure the OD value at 405 nm, calculate the relative melanin production, and calculate the P value.

[0092] Relative melanin production = (OD value of the test group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0093] The culture medium was formulated as follows: 20 ng / mL of bFGF, 33 μg / mL of IBMX, and 333 ng / mL of α-MSH were added to a basal culture medium (DMEM 65%, AIM-V 9%, fetal bovine serum 23%, NEAA 1.5%, and penicillin / streptomycin 1.5%, by volume).

[0094] The test results of relative melanin production are shown in Table 4. It can be seen that when the sample concentrations are 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, and 12.5 μmol / L, there are significant differences, which indicates that α-arbutin has the effect of removing spots and whitening.

[0095] Table 4

[0096]

[0097]

[0098] Example 3

[0099] The present embodiment provides a method for evaluating artemisinic acid by using MNT-1 cells:

[0100] (1) Determining the maximum safe concentration of artemisinic acid to MNT-1 cells:

[0101] Referring to the method of Example 1, it is determined that 300 μmol / L is the maximum safe concentration of the test product artemisinic acid.

[0102] (2) Determining the relative content of melanin:

[0103] MNT-1 cells are inoculated in a 6-well plate with a seeding density of 9.5 x 10 4 After adhesion, the medium containing different concentrations of the test product (the concentration of artemisinic acid in the medium is 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, and 12.5 μmol / L, respectively) and the medium without the test product (control group) are replaced, respectively, and treated for 72 h (2.0 mL / well). After 72 h of culture, the cells are discarded, washed with PBS for 3 times, and the cell culture dish is placed on an ice plate. 400 μL of non-denaturing cell lysate containing 7% additives (benzylsulfonyl fluoride, imidazole, sodium fluoride, and sodium ethylenediaminetetraacetate with a mass ratio of 2:0.5:0.5:1) are added to each dish, and the lysate is lysed at 5°C for 15 min. The lysate is centrifuged at 4°C and 14000 r / min for 8 min, and the melanin precipitate is collected. 320 μL of KOH DMSO solution (pH 13.5) is added to the melanin precipitate and vortexed to facilitate complete dissolution. The mixture is dissolved in a 85°C metal bath for 1.5 h to completely dissolve the melanin precipitate. After vortex mixing, the mixture is centrifuged at 11000 r / min for 6 min, and the supernatant is collected to obtain the melanin lysate;

[0104] 200 μL of the above melanin lysate is added to each well of a 96-well plate, 5 replicates are set, and a blank well (200 μL of PBS solution is added to the 96-well plate) is set. The OD value is measured at 405 nm, the relative melanin production is calculated, and the P value is calculated.

[0105] The formula of the above-mentioned medium is: 10 ng / mL of bFGF, 55 μg / mL of IBMX, and 83 ng / mL of α-MSH are added to the basic medium (DMEM 75%, AIM-V 7%, fetal bovine serum 17%, NEAA 0.5%, and penicillin / streptomycin double antibody 0.5% by volume percentage).

[0106] The test results of the relative melanin production are shown in Table 5. It can be seen that when the sample concentration is 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, and 12.5 μmol / L, there is a significant difference, and it is determined that artemisinic acid has a depigmentation and whitening effect.

[0107] Table 5

[0108] Group Relative melanin content mean (%) P-value Significance control group 100 / / Artemisinic acid -200 μmol / L 50.67 ＜0.001 *** Artemisinic acid -100 μmol / L 62.33 ＜0.001 *** Artemisinic acid -50 μmol / L 71.00 ＜0.001 *** Artemisinic acid -25 μmol / L 82.37 ＜0.001 *** Artemisinic acid -12.5μmol / L 86.55 ＜0.01 **

[0109] Example 4

[0110] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that the total amount of the additive in the native cell lysate is maintained at 5%, and the additive is replaced with phenylmethylsulfonyl fluoride, imidazole, and sodium fluoride in a mass ratio of 1:1:1. The remaining steps remain the same as in Example 1.

[0111] Example 5

[0112] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that the total amount of the additive in the native cell lysate is maintained at 5%, and the additive is replaced with phenylmethylsulfonyl fluoride, imidazole, and sodium ethylenediaminetetraacetic acid in a mass ratio of 1:1:0.5. The remaining steps remain the same as in Example 1.

[0113] Example 6

[0114] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that the total amount of the additive in the native cell lysate is maintained at 5%, and the additive is replaced with phenylmethylsulfonyl fluoride, sodium fluoride, and sodium edetate in a mass ratio of 1:1:0.5. The remaining steps remain the same as in Example 1.

[0115] Example 7

[0116] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that the total amount of the additive in the native cell lysate is maintained at 5%, and the additive is replaced with imidazole, sodium fluoride, and sodium EDTA in a mass ratio of 1:1:0.5. The remaining steps remain the same as in Example 1.

[0117] Example 8

[0118] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that, during the dissolution of the melanin precipitate, 330 μL of the NaOH-DMSO solution is maintained unchanged, and the pH value of the NaOH-DMSO solution is adjusted to 12. The remaining steps remain the same as in Example 1.

[0119] Example 9

[0120] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that, during the dissolution of the melanin precipitate, 330 μL of the NaOH-DMSO solution is maintained unchanged, and the pH value of the NaOH-DMSO solution is adjusted to 14. The remaining steps remain the same as in Example 1.

[0121] Example 10

[0122] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The difference from Example 1 is that in step (2), the seeding density of MNT-1 cells is adjusted to 8.5×10 4 The remaining steps were the same as those in Example 1.

[0123] Example 11

[0124] This example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The difference from Example 1 is that in step (2), the seeding density of MNT-1 cells is adjusted to 1×10 5 The remaining steps were the same as those in Example 1.

[0125] Comparative Example 1

[0126] This comparative example provides a method for evaluating Thiamidol, a whitening ingredient, using A375 cells. The method differs from Example 1 only in that MNT-1 cells are replaced with an equal inoculum of A375 cells, and the remaining steps remain the same as Example 1.

[0127] Comparative Example 2

[0128] This comparative example provides a method for evaluating Thiamidol, a whitening ingredient, using B16F10 cells. The method differs from Example 1 only in that MNT-1 cells are replaced with an equal inoculation amount of mouse melanoma B16F10 cells.

[0129] Comparative Example 3

[0130] This comparative example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that bFGF is not added to the culture medium. That is, the culture medium formula is as follows: 40 μg / mL IBMX and 200 ng / mL α-MSH are added to a basal culture medium (by volume percentage, DMEM 70%, AIM-V 8%, fetal bovine serum 20%, NEAA 1%, and penicillin / streptomycin 1%). The remaining steps are consistent with Example 1.

[0131] Comparative Example 4

[0132] This comparative example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that IBMX is not added to the culture medium. Specifically, the culture medium formula is as follows: 15 ng / mL of bFGF and 200 ng / mL of α-MSH are added to a basal culture medium (by volume percentage, DMEM 70%, AIM-V 8%, fetal bovine serum 20%, NEAA 1%, and penicillin / streptomycin 1%). The remaining steps are consistent with Example 1.

[0133] Comparative Example 5

[0134] This comparative example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that α-MSH is not added to the culture medium. That is, the culture medium formula is as follows: 15 ng / mL of bFGF and 40 μg / mL of IBMX are added to a basal culture medium (by volume percentage, DMEM 70%, AIM-V 8%, fetal bovine serum 20%, NEAA 1%, and penicillin / streptomycin 1%). The remaining steps are consistent with Example 1.

[0135] Comparative Example 6

[0136] This comparative example provides a method for evaluating Thiamidol, a whitening ingredient, using MNT-1 cells. The method differs from Example 1 only in that bFGF, IBMX, and α-MSH are not added to the culture medium. That is, the culture medium formula, calculated by volume percentage, is DMEM 70%, AIM-V 8%, fetal bovine serum 20%, NEAA 1%, and penicillin / streptomycin dual antibody 1%. The remaining steps remain the same as in Example 1.

[0137] Test Example 1

[0138] This test example verifies the repeatability and stability of the evaluation methods of the above embodiments and comparative examples.

[0139] The evaluation method of each example and comparative example was repeated 6 times, and the repeatability of each evaluation index was evaluated.

[0140] (1) Melanin production inhibition rate:

[0141] Melanin production inhibition rate = (relative melanin content of the control group - relative melanin content of the test product group) / relative melanin content of the control group x 100%.

[0142] According to the calculation results of 6 melanin production inhibition rates, the SD value was calculated.

[0143] (2) Linear regression equation r value:

[0144] The melanin production inhibition rate was taken as the ordinate, and the concentration of the test product in the culture medium was taken as the abscissa, a regression curve was drawn, and the r value was calculated.

[0145] Taking Example 1 as an example, the melanin production inhibition rate regression curves of thiamidol in Example 1 of 6 experiments are shown in Figure 1-6 , and the r values are 0.9911, 0.9970, 0.9953, 0.9975, 0.9912 and 0.9942, respectively. It shows that the detection method of Example 1 has good stability and repeatability, and the error is small.

[0146] The SD value test results of 6 melanin production inhibition rates of each example and comparative example test method are shown in Table 6 and Table 7, and the SD value test results of the linear regression equation r value are shown in Table 8.

[0147] Table 6

[0148]

[0149]

[0150] Table 7

[0151]

[0152] Table 8

[0153]

[0154]

[0155] From the test data in Tables 6-8, it can be seen that the evaluation method of the present application related to the whitening and freckle-removing effect has high stability and good repeatability. And under the safe concentration of the test product, the melanin production inhibition rate and the concentration of the test product have a good linear relationship.

[0156] Comparison of the data in Example 1 with Comparative Examples 1-2 demonstrates that MNT-1 cells exhibit superior evaluation stability compared to other melanoma cells. Comparison of the data in Example 1 with Comparative Examples 3-6 demonstrates that the addition of bFGF, IBMX, and α-MSH to the culture medium of MNT-1 cells enhances the growth stability of MNT-1 cells, accelerates the growth and differentiation process of MNT-1 cells, and improves the stability and reproducibility of the evaluation method.

[0157] Comparison of the data in Example 1 with those in Examples 4-7 shows that by simultaneously adding phenylmethylsulfonyl fluoride, imidazole, sodium fluoride, and sodium EDTA to the non-denaturing cell lysate, the four components cooperate with each other to synergistically enhance the detection accuracy of the melanin content, thereby improving the repeatability and stability of the freckle removal and whitening evaluation method.

[0158] From the comparison of the data of Example 1 and Examples 8-9, it can be seen that in the process of dissolving melanin, controlling the pH value of the alkaline solution at 12.5-13.5 can improve the dissolution efficiency and dissolution stability of melanin, thereby improving the stability of the freckle removal and whitening evaluation method.

[0159] From the comparison of the data of Example 1 and Examples 10-11, it can be seen that the seeding density of MNT-1 cells is controlled at 9-9.5×10 4 cells / mL, which can improve the growth stability of MNT-1 cells, thereby further improving the stability and repeatability of the freckle removal and whitening evaluation method based on the MNT-1 cell model.

[0160] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0161] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0162] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for evaluating freckle removal and whitening efficacy, characterized in that: The evaluation method includes: (1) Cultivating MNT-1 cells with a culture medium containing a test substance, lysing the MNT-1 cells with a cell lysis solution, collecting a melanin precipitate by centrifugation, dissolving the collected melanin precipitate with an alkaline solution, and measuring the melanin content in the dissolved solution I; MNT-1 cells are cultured in a medium without the test substance, the MNT-1 cells are lysed with a cell lysis buffer, melanin precipitates are collected by centrifugation, the collected melanin precipitates are dissolved with an alkaline solution, and the melanin content in the dissolved solution is measured. (2) Analyze the significant difference between melanin content I and melanin content II to obtain the P value. If the P value is less than 0.05, it is determined that the product to be tested has the effect of removing spots and whitening. The culture medium in step (1) contains basal culture medium and bFGF, IBMX and α-MSH; The basic culture medium contains: DMEM, AIM-V, fetal bovine serum, NEAA and penicillin / streptomycin double antibodies.

2. The evaluation method according to claim 1, wherein: In the culture medium of step (1), the amount of bFGF added is 10-20 ng / mL, the amount of IBMX added is 33-55 μg / mL, and the amount of α-MSH added is 83-333 ng / mL; Preferably, the basic culture medium comprises, by volume percentage, 65-75% DMEM, 6-10% AIM-V, 15-25% fetal bovine serum, 0.5-1.5% NEAA, and 0.5-1.5% penicillin / streptomycin dual antibody.

3. The evaluation method according to claim 1 or 2, characterized in that In step (1), the MNT-1 cell seeding density of culturing the MNT-1 cells with the medium containing the test substance and culturing the MNT-1 cells with the medium without the test substance is independently selected from 9×10 4 -9.5×10 4 / mL; Preferably, the culture time of culturing the MNT-1 cells with the medium containing the test article and the culture time of culturing the MNT-1 cells with the medium not containing the test article are each independently selected from 48-72 hours.

4. The evaluation method according to any one of claims 1 to 3, characterized in that The cell lysate in step (1) comprises a non-denaturing cell lysate; Preferably, the cell lysate further contains additives; Preferably, the mass percentage of the additive in the cell lysate is 2-7%; Preferably, the additives in the cell lysate include phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium edetate; Preferably, the mass ratio of phenylmethylsulfonyl fluoride, imidazole, sodium fluoride and sodium edetate is (0.5-2):(0.5-2):(0.5-2):(0.1-1); Preferably, the cracking temperature in step (1) is 3-5°C, and the cracking time is 15-25 minutes.

5. The evaluation method according to any one of claims 1 to 4, characterized in that: The alkaline solution in step (1) comprises a DMSO solution of an alkali; Preferably, the base comprises NaOH and / or KOH; Preferably, the pH value of the alkaline solution is 12.5-13.

5.

6. The evaluation method according to any one of claims 1 to 5, characterized in that: In the culture medium containing the test article in step (1), the concentration of the test article is below the maximum safe concentration of the test article for MNT-1 cells; Preferably, the method for testing the maximum safe concentration of the test substance on MNT-1 cells is: MNT-1 cells were cultured with culture media containing different concentrations of the test substance. The test substance concentration at which the survival rate of MNT-1 cells was above 90-100% was the maximum safe concentration of the test substance for MNT-1 cells.

7. The evaluation method according to claim 6, wherein: The detection reagent for the survival rate of MNT-1 cells includes CCK-8 solution.

8. The evaluation method according to any one of claims 1 to 7, characterized in that: The test products include any one or a combination of at least two of raw materials with freckle removal and whitening effects, cosmetic products, drugs for treating chloasma, drugs for treating age spots, or drugs for treating post-inflammatory pigmentation.

9. Use of the freckle-removing and whitening efficacy evaluation method according to any one of claims 1 to 8 in evaluating the freckle-removing and whitening efficacy of cosmetics or raw materials having freckle-removing and whitening efficacy.

10. Use of the method for evaluating the freckle-removing and whitening efficacy of any one of claims 1 to 8 in evaluating the efficacy of a drug for treating chloasma, senile plaques or post-inflammatory pigmentation.

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