Method for screening whitening effect of raw materials by utilizing 3D melanin skin model

Abstract

The invention relates to a method for screening a whitening effect of raw materials by utilizing a 3D melanin skin model, which comprises the following steps: S1, selecting the 3D melanin skin model, and respectively setting a blank control group, a positive control group, a negative control group and a sample group; s2, the positive control group and the sample group are subjected to UVB irradiation, kojic acid is added into the negative control group, UVB irradiation is carried out, and the blank control group is not treated in the culture period and is kept away from light; s3, after the irradiation is finished, adding a raw material sample to be detected into the sample group, and continuing to culture; s4, after culture is finished, respectively detecting skin apparent brightness, melanin content, MITF gene expression and TYR gene expression of the sample group, the blank control group, the positive control group and the negative control group; and S5, judging the whitening effect of the raw material sample according to the detection results of the four indexes in the step S4. The screening and evaluation indexes of the invention cover two dimensions of skin appearance and microcosmic, and the result is more accurate.

Description

Technical Field

[0001] The present disclosure relates to the technical field of cosmetic raw material performance screening, and in particular to a method for screening raw materials for whitening efficacy using a 3D melanin skin model. Background Art

[0002] In recent years, whitening cosmetics have been favored by more and more Oriental women. In Western countries, whitening products are also used in the prevention and treatment of irregular hyperpigmentation, such as age spots and freckles. Therefore, many cosmetics companies have conducted extensive research on changing skin pigmentation and developing highly effective whitening products. As a physical barrier against ultraviolet radiation, melanin has a key photoprotective function for the skin. However, excessive melanin production can cause skin spots, uneven skin tone, dullness and other problems, affecting people's skin beauty and self-confidence. Therefore, improving hyperpigmentation and whitening the skin are urgent needs of many consumers.

[0003] In the development of whitening functional skin care products, evaluating the whitening efficacy of its active ingredients is one of the important steps in the early stage of formula design. With the continuous investment in the research and development of whitening ingredients and formulas, a convincing efficacy evaluation method should be established. In vitro reconstruction of human skin models has been widely used in studies such as skin irritation, skin sensitization and skin phototoxicity, and in vitro construction of skin models containing melanocytes can be used for the efficacy evaluation of whitening cosmetics.

[0004] At present, the model used for whitening evaluation at the cellular level is mainly mouse melanoma B16 cells. Due to its advantages such as multiple passaging, fast growth and low culture requirements, it has become the preferred cell for screening whitening active ingredients. However, there are always differences between B16 cells and human melanocytes, and after several generations of culture, its melanin production ability will decrease, which may lead to problems such as missed detection and false detection during the screening process.

[0005] 3D artificial skin models have become good skin replacement models because their tissue structure and physiological functions are very similar to those of normal skin tissues, and have been widely used in the evaluation of the efficacy of cosmetic raw materials and finished products in recent years. Among them, the 3D melanin skin model is constructed by co-culturing normal human epidermal cells and melanocytes using an air-liquid surface culture method. It has a similar structure to normal human epidermal tissues, which helps to improve the accuracy of screening raw materials with whitening activity.

[0006] CN115501156A discloses a cosmetic raw material composition with whitening and soothing effects and its evaluation system, which uses a 3D skin model efficacy evaluation module to evaluate the efficacy of finished cosmetics containing the raw material composition. However, this scheme only analyzes the whitening efficacy of the raw material from the surface level, does not conduct a comprehensive evaluation from the gene level related to melanin, and does not establish a detailed scoring system, so the accuracy of the screening results is not high enough. Summary of the invention

[0007] Based on this, the purpose of the present invention is to evaluate the whitening efficacy of raw materials from multiple aspects and improve the accuracy of the whitening efficacy screening results of raw materials.

[0008] To achieve the above objectives, the present disclosure provides the following technical solutions:

[0009] A method for screening raw materials for whitening efficacy using a 3D melanin skin model comprises the following steps:

[0010] S1. Select a 3D melanin skin model and set up a blank control group, a positive control group, a negative control group and a sample group;

[0011] S2. The positive control group and the sample group were subjected to UVB irradiation, kojic acid was added to the negative control group and subjected to UVB irradiation, and the blank control group was not treated and protected from light during the culture period;

[0012] S3. After the irradiation is completed, the raw material sample to be tested is added to the sample group and the culture is continued;

[0013] S4. After the culture is completed, the skin apparent brightness, melanin content, MITF gene expression level and TYR gene expression level of the sample group, the blank control group, the positive control group and the negative control group are detected respectively;

[0014] S5. Determine the whitening efficacy of the raw material sample according to the detection result in step S4.

[0015] Preferably, in step S5, the ratios of skin apparent brightness, melanin content, MITF gene expression level, and TYR gene expression level of the sample group, the blank control group, and the negative control group to the positive control group are calculated respectively;

[0016] GraphPad Prism was used to draw the graphs, and the results were expressed as Mean±SD. One-way ANOVA was used for comparison among the groups, and all statistical analyses were two-tailed.

[0017] p<0.05 was considered to be significantly different, indicating that the test substance had a certain whitening effect, and the score was 0.3;

[0018] p<0.01 is considered to be a very significant difference, indicating that the test substance has a significant whitening effect, and the score is 0.5;

[0019] p<0.001 is considered to be extremely significant, indicating that the test substance has an extremely significant whitening effect, with a score of 0.7;

[0020] p<0.0001 is considered to be extremely significant, indicating that the test substance has an extremely significant whitening effect, and the score is 1.0;

[0021] The scores of the four detection indicators are summarized to determine the whitening efficacy of the raw material samples in the sample group.

[0022] More preferably, the skin apparent brightness and the melanin content are used as macro-indicators, and the MITF gene expression level and the TYR gene expression level are used as micro-indicators. When the score of at least one of the macro-indicators and at least one of the micro-indicators in the sample group is greater than or equal to 0.3, the raw material sample in the sample group is determined to have a whitening effect.

[0023] Preferably, in step S2, the irradiation intensity is 0.2-0.4 mW / cm 2 The irradiation temperature is 28°C and the irradiation dose is 50-70 mJ / cm 2 .

[0024] More preferably, the number of irradiation days is the ratio of the irradiation dose to the irradiation intensity.

[0025] Preferably, the added amount of the raw material sample is 0.5 to 2 mL, and the concentration of the raw material sample is 5 mg / mL to 10 mg / mL.

[0026] More preferably, step S3 is repeated for 4 consecutive days.

[0027] Preferably, the method further comprises the step of evaluating the solubility of the raw material sample:

[0028] The raw material sample with a solubility in water of ≥1000 μg / mL is directly dissolved in the culture medium;

[0029] The raw material sample with a solubility in water of less than 1000 μg / mL was dissolved in dimethyl sulfoxide solvent.

[0030] Preferably, the culture conditions of the 3D melanin skin model are: 37±0.5°C, (5±1)% CO2 concentration, and saturated humidity.

[0031] Preferably, in step S4, a stereo microscope is used to take an apparent photo of the 3D melanin skin model, and a colorimeter is used to detect the apparent brightness of the skin;

[0032] The melanin content of the 3D melanin skin model was detected by alkaline lysis method, and the expression of the MITF gene and the TYR gene was detected by RNA transcription and qCPR.

[0033] The technical solution claimed in the present disclosure has achieved the following beneficial effects:

[0034] The detection indicators disclosed in the present invention include skin apparent brightness, melanin content and gene expression related to melanin, so that the objects of action of cosmetic raw materials cover both apparent and microscopic dimensions, and a systematic evaluation system is established, which has the advantages of accuracy and efficiency. It provides a fast and reliable evaluation method for the preliminary verification and screening of whitening active raw materials in skin care products, provides a reference basis for the compounding scheme of whitening raw materials, and also provides an effective basis for further research on animal replacement experiments in the skin care industry, skin care product development and efficacy verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are merely embodiments of the present disclosure, and a person of ordinary skill in the art can obtain other drawings based on the provided drawings without paying any creative work.

[0036] Figure 1 Flow chart of the method for screening raw materials for whitening efficacy using a 3D melanin skin model.

[0037] Figure 2 is the relative apparent brightness of raw material samples 1 to 5.

[0038] Figure 3a is the relative melanin content of raw material samples 1 to 5.

[0039] Figure 3b These are the skin appearance pictures of raw material samples 1 to 5.

[0040] Figure 4 The relative gene expression of MITF of samples 1 to 5 is shown.

[0041] Figure 5 is the relative gene expression of TYR of raw samples 1 to 5. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and beneficial effects of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0043] Example

[0044] This embodiment provides a method for screening the whitening efficacy of raw materials using a 3D melanin skin model, combining apparent brightness, melanin content, skin appearance photos and melanin-related gene expression results to screen the whitening activity of the target raw materials, such as Figure 1 As shown, the following steps are included:

[0045] S1. Evaluate the dissolution mode of the raw material sample to be tested and determine the dissolution scheme:

[0046] The raw material samples with solubility ≥1000 μg / mL in water were directly dissolved in the culture medium supporting the 3D melanin skin model for subsequent experiments;

[0047] Raw material samples with solubility in water <1000 μg / mL were dissolved in dimethyl sulfoxide solvent and then subjected to subsequent experiments;

[0048] S2. Cultivate the 3D melanin skin model by first placing it in a six-well plate, adding 1 ml of matching culture medium to each well, and then incubating it in an incubator for 24 hours for subsequent experiments. The 3D melanin skin model needs to be cultured with a matching special culture medium, and all culture conditions are 37±0.5℃, (5±1)% CO2 concentration, and saturated humidity.

[0049] S3. Determine the volume of the raw material sample added and the dissolution scheme of the raw material sample, and prepare the raw material sample solution;

[0050] S4. Set the parameters of the phototoxicity machine and set the UVB intensity to 0.2-0.4 mW / cm 2 The temperature was set at 28°C and the irradiation dose was 50-70 mJ / cm 2 ;

[0051] Among them, according to the formula: irradiation time = irradiation dose / irradiation intensity, the experimental irradiation time is calculated;

[0052] S5. Set up sample group, blank control group, positive control group and negative control group;

[0053] The sample group needs to be irradiated with UVB every day during the culture period; the blank control group does not receive any irradiation during the culture period and takes measures to avoid light; the positive control group, i.e., the UVB irradiation group, needs to be irradiated with UVB every day during the culture period; kojic acid is added to the negative control group, and irradiated with UVB every day during the culture period. Kojic acid is a common whitening ingredient;

[0054] During irradiation, the 3D melanin skin model was transferred from the six-well plate to a 24-well plate, 100 μL of PBS was added to the 24-well plate, and the plate was placed in a phototoxic instrument. The cover of the plate was opened for irradiation.

[0055] S6. After the irradiation is completed, the 3D melanin skin model in the sample group is transferred to a six-well plate, 1 mL of the raw material sample dissolved in the culture medium is added to each well plate, and the 3D melanin skin model is continued to be cultured in the incubator;

[0056] S7. Repeat step S6 for the next 4 days. On the 5th day, take out the 3D melanin skin model from the well plate, wipe the liquid at the bottom with a cotton swab, place it in a new well plate, place the 3D melanin skin model on the stage, and take a picture of the appearance of each 3D melanin skin model with a stereo microscope;

[0057] S8. Place the 3D melanin skin model on a flat and hard white surface, carefully remove the culture medium under the model with a cotton swab, align the detection hole of the colorimeter vertically with the surface of the model for detection, and detect the apparent brightness (L*) of the skin. Each skin model is measured three times;

[0058] S9. After the apparent brightness (L*) was measured, the 3D melanin skin model was cut with a scalpel, placed in a 1.5 mL centrifuge tube, and 800 μL NaOH (containing 10% DMSO) was added. The tube was vortexed for several minutes to facilitate full lysis, and then placed in an 80°C metal bath for 3 hours to fully dissolve the melanin precipitate. The rotation speed was 5000 r / min and centrifuged for 5 minutes. Then, the 96-well plate was taken out, and 200 μL of melanin solution was pipetted with a pipette and added to the 96-well plate. Three replicate wells were set for each sample, and the OD value was measured at 405 nm by an enzyme marker;

[0059] S10. RNA was extracted from each group of 3D melanin skin models, reverse transcribed into cDNA, and the expression of melanin production-related genes MITF and TYR was detected by QPCR;

[0060] S11. Summarize the apparent brightness, melanin content, and gene measurement results in steps S8 to S10, calculate the ratio of the apparent brightness of the test sample group to the apparent brightness of the UVB-induced group (positive control group), the ratio of the melanin content of the sample group to the melanin content of the UVB-induced group, and the ratio of the gene expression of the sample group to the gene expression of the UVB-induced group, and obtain the relative expression of the apparent brightness, the relative expression of the melanin content, and the relative expression of the gene;

[0061] For a more intuitive comparison, the relative expression of apparent brightness, melanin content and gene expression of the blank control group, negative control group and UVB-induced group were also calculated. GraphPad Prism was used for drawing, and the results were expressed as Mean±SD. One-way ANOVA statistical analysis was used for comparison between groups, and all statistical analyses were two-tailed.

[0062] Among them, p<0.05 is considered to be significantly different, indicated by "*", indicating that the test substance has a certain whitening effect, with a score of 0.3;

[0063] p<0.01 is considered to be a very significant difference, indicated by "**", indicating that the test substance has a significant whitening effect, with a score of 0.5;

[0064] p<0.001 is considered to be extremely significant, indicated by "***", indicating that the test substance has an extremely significant whitening effect, with a score of 0.7;

[0065] p<0.0001 is considered to have an extremely significant difference, indicated by "****", indicating that the test substance has an extremely significant whitening effect, with a score of 1.0;

[0066] There are a total of 4 detection indicators in this embodiment, L* value and melanin content as 2 macro indicators, and two genes related to melanin as 2 micro indicators. According to the 4-choice-2 principle, that is, if at least one of the micro and macro indicators has a score of 0.3 or above (including 0.3), the test substance is judged to have a whitening effect.

[0067] The scores of all test indicators are summarized according to the above-mentioned scoring criteria to judge the strength of the whitening effect of the test substance.

[0068] This embodiment uses an in vitro 3D melanin skin model to screen the whitening efficacy of raw material samples. The detection indicators include skin appearance photos, skin appearance brightness, melanin content, and gene expression related to melanin, so that the objects of cosmetic raw materials cover both the appearance and microscopic dimensions, and a systematic evaluation system is established, which has the advantages of accuracy and efficiency, and provides a fast and reliable evaluation method for the preliminary verification and screening of whitening active raw materials for skin care products, provides a reference basis for the compounding scheme of whitening raw materials, and also provides an effective basis for further research on animal replacement experiments, skin care product development, and efficacy verification in the skin care industry. The following is a further clear explanation of the scheme and its technical effects in the above embodiment in combination with specific application examples.

[0069] Application Examples

[0070] 1. Experimental Materials

[0071] 1. Experimental Materials and Instruments

[0072] 1.1 3D Melanin Skin Model

[0073] In this application example, the 3D melanin epidermal model was purchased from Hangzhou GeneFluid Biotechnology Co., Ltd.

[0074] 1.2 Main Reagents

[0075] (1) 3D melanin skin culture related reagents: 3D melanin epidermis model culture medium was purchased from Genentech Biotechnology Co., Ltd., and DPBS was purchased from Gibco, USA;

[0076] (2) Chemical reagents: DMSO, NaOH, and kojic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0077] 1.3 Solution preparation

[0078] (1) Sodium hydroxide DMSO solution: Weigh 1.2 g of sodium hydroxide solid, add 27 mL of deionized water to dissolve, and then add 3 mL of DMSO solution and mix well;

[0079] (2) Kojic acid solution: Weigh 10 mg of kojic acid powder and add 10 mL of culture medium for culturing melanin skin to a concentration of 1 mg / mL. Vortex to dissolve, filter through a 0.22 μm filter membrane to sterilize, and store at 4°C in the dark.

[0080] 1.4 Instruments

[0081] Microplate reader (Thermo, USA), CO2 incubator (Thermo, USA), stereo microscope (Leica, Germany), biological safety cabinet (Thermo, USA), high-speed low-temperature centrifuge (Thermo, USA), UV light exposure system (Melton, USA), QPCR (Roche, Germany), colorimeter (Minolta, CM-26DG).

[0082] 2. Raw material samples to be tested

[0083] The names of raw material samples (test substances) are shown in Table 1:

[0084] Table 1

[0085] serial number Sample name 1 Active ingredient A 2 Active ingredient B 3 Active Ingredients C 4 Active ingredient D 5 Active substance E

[0086] 3. Whitening efficacy screening of raw material samples to be tested

[0087] 1) Determine the volume of raw material sample added and the dissolution scheme of the raw material sample, and prepare the raw material sample solution with a concentration of 5 mg / mL to 10 mg / mL;

[0088] 2) Set the parameters of the phototoxicity machine and set the UVB intensity to 0.2-0.4 mW / cm 2 The temperature was set at 28°C and the irradiation dose was 50-70 mJ / cm 2 , the irradiation time was set to 5 days;

[0089] 3) Taking a normally cultured 3D melanin skin model, setting up a sample group, a blank control group, a positive control group, and a negative control group;

[0090] Among them, the sample group needs to be irradiated with UVB every day during the culture period; the blank control group does not receive any irradiation during the culture period and takes measures to avoid light; the positive control group, i.e., the UVB irradiation group, needs to be irradiated with UVB every day during the culture period; 1 mL of kojic acid is added to the negative control group, and UVB is irradiated every day during the culture period;

[0091] During irradiation, the 3D melanin skin model was transferred from the six-well plate to a 24-well plate, 100 μL of PBS was added to the 24-well plate, and the plate was placed in a phototoxic instrument, the cover of the plate was opened, and irradiation was performed;

[0092] 4) After the irradiation is completed, the 3D melanin skin model in the sample group is transferred to a six-well plate, 1 mL of the raw material sample dissolved in the culture medium is added to each well of the plate, and the 3D melanin skin model is continued to be cultured in the incubator;

[0093] 5) Repeat step S6 for the next 4 days. On the 5th day, take out the 3D melanin skin model from the well plate and wipe the liquid at the bottom with a cotton swab;

[0094] 6) According to the steps in the embodiment, the skin appearance photos of the sample group, the blank control group, the positive control group and the negative control group before and after the experiment are respectively detected and taken, and the skin appearance brightness, melanin content, MITF gene expression level and TYR gene expression level of the sample group, the blank control group, the positive control group and the negative control group are respectively detected, and the ratios of the skin appearance brightness, melanin content, MITF gene expression level and TYR gene expression level of the sample group, the blank control group and the negative control group to the positive control group are respectively calculated.

[0095] 2. Results Analysis

[0096] GraphPad Prism was used for drawing, and the results were expressed as Mean ± SD. One-way ANOVA statistical analysis was used for comparison among the groups. All statistical analyses were two-tailed. P < 0.05 was considered to be different, indicated by "*", p < 0.01 was considered to be significantly different, indicated by "**", p < 0.001 was considered to be very significantly different, indicated by "***", and p < 0.0001 was considered to be extremely significantly different, indicated by "****".

[0097] 2.1 3D melanin epidermal model test results

[0098] 2.1.1 Apparent brightness

[0099] After the experiment, the melanin skin model was placed on a flat and hard white surface, and the colorimeter detection hole was vertically aligned with the model surface for detection. Each model was read 3 times, and the relative expression of the UVB group as the control was calculated. The relative expression of apparent brightness (L* value) is as follows Figure 2 As shown, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 are the comparisons between the sample group and the UVB control group.

[0100] 2.1.2 Melanin content

[0101] The melanin model after the L* value was determined was cut with a scalpel, placed in a 1.5 mL centrifuge tube, and 330 μL of NaOH (containing 10% DMSO) was added. Vortexing was performed to facilitate full lysis. The melanin precipitate was placed in an 80°C metal bath for 2 h to fully dissolve. The melanin precipitate was vortexed and mixed. Then, the 96-well plate was taken out, and 100 μL of melanin solution was added to each well. Three replicate wells were set for each sample. The OD value was measured at 405 nm, and the relative expression of the UVB group as the control was calculated. The relative melanin content was as follows: Figure 3aAs shown, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 are the comparisons between the sample group and the UVB control group. 2.1.3 MITF mRNA detection, TYR mRNA detection

[0102] After the experiment, the 3D melanin skin model was cut off and placed in Trizol. It was vortexed with a vortexer to fully mix the skin and liquid. The reaction lasted for 30 minutes, with interval vortexing in between. Add 200 μl of chloroform to each RNAfree tube, press the upper and lower ends of the RNAfree tube with your hands, oscillate for 15 seconds, and let stand for 3 minutes. Turn on the low-temperature centrifuge in advance, precool to 4°C, adjust the speed to 12000r / min, and centrifuge for 10 minutes. Carefully remove the centrifuged RNAfree tube, use a pipette to draw 350 μl of supernatant and add it to a new RNAfree tube, mark it, and discard the original RNAfree tube.

[0103] Add 350μl isopropanol to a new RNAfree tube, hold the two ends of the RNAfree tube and shake it up and down, and put it in a -20℃ refrigerator for 20min. Take out the RNAfree tube from the 20℃ refrigerator, put it in a low-speed centrifuge, set the speed to 12000r / min, centrifuge for 10min, and you can see some white precipitate at the bottom of the RNAfree tube. After 10min, take out the RNAfree tube, discard the supernatant, add 1ml of 75% ethanol diluted with DEPC water to each tube, and shake it up and down until the white precipitate falls off. Put the RNAfree tube into a low-speed centrifuge, set the speed to 7500r / min, centrifuge for 5min, discard the supernatant, then continue to put it into a low-speed centrifuge and set the speed to 12000r / min, centrifuge for 1min, discard the supernatant, and blow dry in a fume hood, which takes about 5 to 10min. Add 25μL of DEPC water to dissolve.

[0104] Finally, the RNA concentration of 1 μL was detected with the Nanodrop quantifier. The multi-control water bath was turned on in advance, one temperature was set to 85°C, and the other temperature was set to 37°C. Calculate the volume of RNA required to reverse transcribe 50 μL of CDNA, add 10 μL of the mix in the reverse transcription kit, and add the rest to the reverse transcription kit to make the system 50 μL. After spotting the plate, the plate was placed in the QPCR instrument for detection for 1 hour. The results of TYRmRNA detection and MITFmRNA detection are as follows: Figure 4 and Figure 5 shown.

[0105] 2.2 Comprehensive evaluation results and analysis

[0106] All test substances were Figure 1The results of the experimental significant differences in 3D melanin skin levels of the five test substances detected in this application example are summarized in Table 2, and the evaluation and scoring are performed according to the standards in Table 3.

[0107] Table 2

[0108]

[0109] Table 3

[0110]

[0111] Table 4

[0112]

[0113] The results of the whitening efficacy evaluation of the test substances are shown in Table 4. According to the summary score results, active substance A in this batch of test substances does not have whitening efficacy, while the other four active substances all have whitening efficacy. The whitening efficacy intensity is ranked as No. 2 > No. 5 > No. 4 > No. 3. The best whitening efficacy raw material screened out is active substance B.

[0114] In this application example, the whitening activity of the target raw material is screened by combining the macroscopic and microscopic test results. The macroscopic evaluation indicators include apparent brightness, melanin content and skin appearance photos, and the microscopic evaluation indicators include MITF and TYR genes related to melanin. Combining the evaluation indicators of the two to comprehensively judge the whitening effect of the raw material has the advantages of being fast and efficient, and also makes the screening results more convincing and accurate.

[0115] Comparative Example 1

[0116] The difference between this comparative example and the aforementioned application example is that only the macroscopic indexes of the 3D melanin skin level of the five raw material samples to be tested are tested, that is, only the skin apparent brightness (L* value) and melanin content are tested. The significant difference results of the macroscopic experiment are summarized in Table 5, and are evaluated and scored according to the standards in Table 6.

[0117] Table 5

[0118] serial number L* Value Melanin content 1 ns ns 2 **** **** 3 *** ns 4 **** **** 5 *** ****

[0119] Table 6

[0120]

[0121] Table 7

[0122] serial number L* Value Melanin content Summary 1 0 0 0 2 1 1 2.0 3 0.7 0 0.7 4 1 1 2.0 5 0.7 1 1.7

[0123] The results of the whitening efficacy evaluation of the raw material samples to be tested in terms of macro indicators are shown in Table 7. According to the summary score results, active substance A in this batch of test materials does not have whitening efficacy, and the other four active substances all have whitening efficacy. The whitening efficacy intensity is ranked as No. 2 = No. 4 > No. 5 > No. 3, that is, the raw materials with the best whitening efficacy are active substances B and D, but the whitening effects of active substances B and D cannot be compared.

[0124] Comparative Example 2

[0125] The difference between this comparative example and the aforementioned application example is that only the microscopic indicators of 3D melanin skin level of the five raw material samples to be tested are tested, that is, only the skin MITF gene expression and TYR gene expression are tested. The results of the significant difference of the microscopic experiment are summarized in Table 8, and are evaluated and scored according to the standards in Table 7.

[0126] Table 8

[0127]

[0128] Table 9

[0129]

[0130] The results of the whitening efficacy evaluation of the raw material samples to be tested in terms of micro-indicators are shown in Table 9. According to the summary score results, active substance A in this batch of test materials does not have whitening efficacy, and the other four active substances all have whitening efficacy. The whitening efficacy intensity is ranked as No. 2 = No. 5 > No. 4 > No. 3. The raw materials with the best whitening efficacy are active substances B and E, but the whitening effects of active substances B and E cannot be compared.

[0131] It can be seen from the above results that the active substances with the best whitening effect screened out by the detection methods of Comparative Examples 1 and 2 are inconsistent, and it is impossible to judge the strength of the whitening effect between the active substances with the best whitening effect, that is, screening whitening raw materials by a single macroscopic or single microscopic detection result is prone to problems such as insufficient detection accuracy and insufficient detection. The technical solution of the present application combines the detection results of macroscopic indicators and microscopic indicators, and comprehensively judges the whitening effect of the raw materials to be tested based on the evaluation results of the two, and screens out the raw material with the best whitening effect as active substance B, making the screening results more convincing and accurate.

[0132] The embodiments and application examples described above are merely illustrative descriptions of the present disclosure and are not intended to limit the scope of the present disclosure. Without departing from the design spirit of the present disclosure, various modifications and improvements made to the technical solutions of the present disclosure by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present disclosure.

Claims

1. A method for screening raw materials for whitening efficacy using a 3D melanin skin model, characterized in that: The following steps are involved: S1. Select a 3D melanin skin model and set up a blank control group, a positive control group, a negative control group and a sample group; S2. The positive control group and the sample group were subjected to UVB irradiation, kojic acid was added to the negative control group and subjected to UVB irradiation, and the blank control group was not treated and protected from light during the culture period; S3. After the irradiation is completed, the raw material sample to be tested is added to the sample group and the culture is continued; S4. After the culture is completed, the skin apparent brightness, melanin content, MITF gene expression and TYR gene expression of the sample group, the blank control group, the positive control group and the negative control group are detected respectively; S5. Determine the whitening efficacy of the raw material sample according to the detection results of the four indicators in step S4.

2. The method according to claim 1, characterized in that In step S5, the ratios of skin apparent brightness, melanin content, MITF gene expression level, and TYR gene expression level of the sample group, the blank control group, and the negative control group to the positive control group are calculated respectively; GraphPad Prism was used to draw the graphs, and the results were expressed as Mean±SD. One-way ANOVA was used for comparison among the groups, and all statistical analyses were two-tailed. p<0.05 was considered to be significantly different, indicating that the test substance had a certain whitening effect, and the score was 0.3; p<0.01 is considered to be a very significant difference, indicating that the test substance has a significant whitening effect, and the score is 0.5; p<0.001 is considered to be extremely significant, indicating that the test substance has an extremely significant whitening effect, with a score of 0.7; p<0.0001 is considered to be extremely significant, indicating that the test substance has an extremely significant whitening effect, and the score is 1.0; The scores of the four detection indicators are summarized to determine the whitening efficacy of the raw material samples in the sample group.

3. The method according to claim 2, characterized in that The skin apparent brightness and the melanin content are used as macro-indicators, and the MITF gene expression level and the TYR gene expression level are used as micro-indicators. When the score of at least one of the macro-indicators and at least one of the micro-indicators in the sample group is greater than or equal to 0.3, it is determined that the raw material sample in the sample group has a whitening effect.

4. The method according to claim 1, characterized in that: In step S2, the irradiation intensity is 0.2-0.4 mW / cm 2 The irradiation temperature is 28°C and the irradiation dose is 50-70 mJ / cm 2 .

5. The method according to claim 4, characterized in that The number of irradiation days is the ratio of the irradiation dose to the irradiation intensity.

6. The method according to claim 1, characterized in that The added amount of the raw material sample is 0.5 to 2 mL, and the concentration of the raw material sample is 5 mg / mL to 10 mg / mL.

7. The method according to claim 6, characterized in that Repeat step S3 for 4 consecutive days.

8. The method according to claim 1, characterized in that The method further comprises the step of evaluating the solubility of the raw material sample: The raw material sample with a solubility in water of ≥1000 μg / mL is directly dissolved in the culture medium; The raw material sample with a solubility in water of less than 1000 μg / mL was dissolved in dimethyl sulfoxide solvent.

9. The method according to claim 1, characterized in that: The culture conditions of the 3D melanin skin model are: 37±0.5° C., (5±1)% CO2 concentration, and saturated humidity.

10. The method according to claim 1, characterized in that In step S4, a stereo microscope is used to take an apparent photo of the 3D melanin skin model, and a colorimeter is used to detect the apparent brightness of the skin; The melanin content of the 3D melanin skin model was detected by alkaline lysis method, and the expression of the MITF gene and the TYR gene was detected by RNA transcription and qCPR.

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