Method for screening antioxidant effects of cosmetic raw materials

Abstract

The invention relates to a cosmetic raw material anti-oxidation effect screening method which comprises the following steps: S1, taking a normally growing 3D skin model, and respectively setting a blank control group, an induction control group and a sample group; s2, respectively adding a complete culture medium into the blank control group and the induction control group for culturing, and adding a raw material sample with corresponding administration concentration prepared by the complete culture medium into the sample group for culturing; s3, H2O2 is added into the induction control group and the sample group for continuous culture; s4, respectively detecting the contents of inflammatory factors MDA and SOD in the culture media of the induction control group and the sample group, and respectively detecting the immunofluorescence intensity and nuclear translocation condition of NRF2 protein of the 3D skin models of the induction control group and the sample group; and S5, respectively calculating secretion volume ratios of MDA, SOD and NRF2 proteins in the group and the induction control group, and judging the antioxidant efficacy of the raw material sample according to the three relative ratios. The screening method provided by the invention is visual, comprehensive and higher in accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic raw material performance testing, and in particular to a method for screening the antioxidant efficacy of cosmetic raw materials. Background Art

[0002] In the development of antioxidant and anti-aging skin care products, the antioxidant efficacy evaluation of active raw materials is one of the important steps in the early stage of formula design. Traditional methods of evaluating the antioxidant efficacy of active raw materials mainly rely on keratinocyte or 3D skin reactive oxygen detection experiments. The experimental results are usually not intuitive and not convenient for high-throughput raw material screening. With the introduction of the circular economy concept of the 3R principle, the 3R principle has become a generally followed criterion for biomedical scientific research and related regulatory management. The European Cosmetics Regulations have also issued a ban on animal experiments in cosmetic products. These regulatory changes have promoted the development and application of in vitro alternative verification methods. The construction of in vitro alternative efficacy verification methods will help to achieve the development and screening of antioxidant cosmetic raw materials more economically and efficiently.

[0003] Under normal and oxidative stress conditions, redox balance is maintained at the transcriptional level primarily through Nrf2 / Keap1 / ARE / HO-1 signaling. The Nrf2 / Keap1 / ARE / HO-1 signaling pathway has been established as a major cellular defense mechanism against oxidative stress both under physiological conditions and in disease models of various systems. Nrf2 is maintained in its inactive form within the cell by Keap1 unless redox homeostasis is disturbed. Activated Nrf2 accumulates in the cell nucleus and binds to ARE on DNA, upregulating the transcription of related genes. A series of genes including glutamate cysteine ​​ligase (GCLC), thioredoxin reductase 1 (Txnrd1), NAD(P)H-quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) are regulated by Nrf2 binding to ARE sequences, mainly regulating the transcription of detoxification and antioxidant enzyme-related genes. These enzymes act as ROS scavengers and electrophilic neutralizers (such as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), catalase), and indirectly induce glutathione synthetase.

[0004] Reducing the production of ROS or inducing the elimination of ROS ultimately affects the oxidative state of cells and provides strong antioxidant protection. The Nrf2 pathway has been widely recognized by the academic community as a key factor in endogenous antioxidant activity. Researchers screened related lead compounds by observing changes in Nrf2 expression at the cellular level (see Man S, Bi J, Liu F, Xie W, Ma L. Vitamin C Inhibited Pulmonary Metastasis through Activating Nrf2 / HO-1Pathway; Mol Nutr Food Res. 2024Mar; 68(6): e2300706. doi: 10.1002 / mnfr.202300706. Epub 2024Feb 28. PMID: 38419398., etc.).

[0005] 3D skin models have similar physiology and structure to normal human skin, and commercially available 3D skin is produced in a unified manner, with relatively stable structural characteristics. When using 3D skin models to detect antioxidant active substances, H2O2 chemical induction methods can be used to promote the secretion of ROS, construct an oxidative damage model, and simulate the oxidative damage state of real skin. Human skin oxidative damage occurs in the epidermis of the skin. Most 3D skin models are mainly composed of keratinocytes and fibroblasts. The antioxidant efficacy of cosmetic raw materials is evaluated by observing the expression of the antioxidant protein NRF2 on the epidermis and detecting the content of antioxidant-related factors in the culture medium. However, the current screening of antioxidant raw materials on 3D skin focuses on the generation of ROS, and there are few studies on the expression of upstream related proteins and genes, and the detection structure is not comprehensive and accurate enough. Summary of the invention

[0006] Based on this, the purpose of the present disclosure is to provide a more intuitive and comprehensive method for screening the antioxidant efficacy of cosmetic raw materials with higher accuracy of test results.

[0007] To achieve the above objectives, the present disclosure provides the following technical solutions:

[0008] A method for screening the antioxidant efficacy of cosmetic raw materials, comprising the following steps:

[0009] S1. Take a normally grown 3D skin model and set up a blank control group, an induced control group and a sample group respectively;

[0010] S2. adding complete medium to the blank control group and the induced control group for culturing, and adding raw material samples of corresponding dosing concentrations prepared by complete medium to the sample group for culturing;

[0011] S3. adding H2O2 to the induced control group and the sample group in step S2 and continuing to culture;

[0012] S4. respectively detecting the contents of inflammatory factors MDA and SOD in the culture medium of the induced control group and the sample group in step S3, and

[0013] Respectively detecting the immunofluorescence intensity and nuclear translocation of the NRF2 protein in the induced control group and the sample group in step S3;

[0014] S5. Calculate the secretion ratios of MDA, SOD and NRF2 proteins in the sample group and the induced control group respectively, and determine the antioxidant efficacy of the raw material sample based on the three relative ratios.

[0015] Preferably, in step S5, GraphPad Prism is used to draw a graph, and the results are expressed as Mean ± SEM; One-way ANOVA statistical analysis is used for comparison between groups, and all statistical analyses are two-tailed; wherein

[0016] p<0.05 is considered to be significantly different, indicated by "*", indicating that the raw material sample has a certain inhibitory effect, with a score of 0.3;

[0017] p<0.01 is considered to be very significantly different, indicated by "**", indicating that the raw material sample has a significant inhibitory effect, with a score of 0.7;

[0018] p<0.001 is considered to have an extremely significant difference, represented by "***", indicating that the raw material sample has an extremely significant inhibitory effect, with a score of 1.0;

[0019] Determine whether the NRF2 protein undergoes nuclear translocation and activates the antioxidant pathway, and compare the final scores of each of the raw material samples obtained by adding the scores of the three detection indicators. If the score is greater than or equal to 0.3, it is judged to have antioxidant effect. The higher the score, the better the antioxidant effect. In this way, the raw material sample with the best antioxidant effect is screened out.

[0020] Preferably, the method further comprises a solubility evaluation step before performing step S1:

[0021] Dissolving the raw material sample having a solubility in water of ≥1000 μg / mL directly in the complete culture medium;

[0022] The raw material sample with a solubility in water of less than 1000 μg / mL is dissolved in dimethyl sulfoxide or ethanol and then added to the complete culture medium.

[0023] More preferably, when the dimethyl sulfoxide or the ethanol is used as the solvent, the volume percentage concentration of the solvent does not exceed 1% of the total volume, and the volume ratio of the solvent in the blank control group, the induced control group and the sample group is kept the same.

[0024] Preferably, in step S2, the concentration of the raw material sample is 10 μM-50 μM, the addition amount is 10 μL-30 μL, and the incubation time is 16 to 24 hours.

[0025] More preferably, the concentration of the raw material sample is 30 μM, the addition amount is 20 μL, and the culture time is 18 to 22 hours.

[0026] Preferably, it is characterized in that, in step S3, the concentration of H2O2 is 500μM-800μM, the addition amount is 10μL-30μL, and the incubation time is 16-24h.

[0027] More preferably, in step S3, the amount of H2O2 added is 20 μL, and the culture time is 18 to 22 h.

[0028] Preferably, it is characterized in that before performing step S1, the 3D skin model is cultured in gas-liquid two-phase conditions at 37±0.5°C, (5±1)% CO2 concentration and saturated humidity for 10 to 15 hours.

[0029] Preferably, in step S4, the 3D skin model is fixed, dehydrated, paraffin-embedded and paraffin-sectioned before immunofluorescence intensity analysis of the NRF2 protein is performed.

[0030] The technical solution claimed in the present disclosure has achieved the following beneficial effects:

[0031] The MDA inflammatory factor, SOD inflammatory factor and NRF2 protein were used as antioxidant efficacy detection and evaluation indicators, focusing on the key factors regulating the endogenous antioxidant capacity of the body, and comprehensively verifying the antioxidant efficacy of the raw material samples. The expression and localization of the Nrf2 protein were observed on an in vitro artificial epidermis model, providing visual test results, which can intuitively judge the antioxidant capacity of the raw material samples and detect the activity of related antioxidant enzymes, ensuring the accuracy of the test results. It provides a fast and reliable evaluation method for the preliminary verification and screening of antioxidant active raw materials for skin care products, provides a reference basis for the compounding scheme of antioxidant 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 This is a screening flow chart for the antioxidant efficacy screening method of cosmetic raw materials.

[0034] Figure 2 is the relative expression of SOD of the active substance AD.

[0035] Figure 3 is the relative expression of MDA of active substance AD.

[0036] Figure 4a The NRF2 nuclear translocation status of active substance AD.

[0037] Figure 4b NRF2 immunofluorescence images of active substance AD.

[0038] Figure 5 The relative expression intensity of NRF2 immunofluorescence of active substance AD.

[0039] Figure 6 This is the ROS staining fluorescence image of the active substance AD.

[0040] Figure 7 is the relative expression intensity of ROS fluorescence of active substance AD. DETAILED DESCRIPTION

[0041] 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.

[0042] Example

[0043] This embodiment provides a method for screening the antioxidant efficacy of cosmetic raw materials, and screening the antioxidant activity of target raw materials according to the detection results of the 3D skin model, comprising the following steps:

[0044] S1. Evaluate the solubility of raw material samples and determine the dissolution scheme;

[0045] S2. Cultivating 3D skin;

[0046] S3. Take the normally grown 3D skin model in step S2, set up a blank control group, an induced control group and a sample group. The blank control group and the induced control group are added with complete culture medium, and the sample group is added with raw material samples of corresponding drug concentrations prepared by complete culture medium. After culturing for 16h to 24h, H2O2 is added to the induced control group and the sample group, and the culture is continued for 16h to 24h. The contents of antioxidant-related factors MDA and SOD in the culture fluid of the induced control group and the sample group are respectively detected;

[0047] S4. Take the 3D skin model processed in step S4, fix, dehydrate, embed in paraffin, and slice the skin model, then perform immunofluorescence staining of the antioxidant-related protein NRF2, observe the nuclear translocation of the protein, and quantify the fluorescence intensity.

[0048] S5. Summarize the results of antioxidant factor content determination in steps S4 to S5, calculate the ratio of MDA and SOD secretion in the test raw material sample group to that in the model group, and obtain the relative expression of inflammatory factors; use Image J software to calculate the immunofluorescence intensity and calculate the ratio of NRF2 protein secretion in the test raw material sample group to that in the induced model group, and observe the nuclear translocation.

[0049] GraphPad Prism was used for graphing, and the results were expressed as Mean ± SEM. One-way ANOVA was used for comparison among groups. All statistical analyses were two-tailed.

[0050] p<0.05 is considered to be significantly different, indicated by "*", indicating that the raw material sample has a certain inhibitory effect, with a score of 0.3;

[0051] p<0.01 is considered to be a very significant difference, indicated by "**", indicating that the raw material sample has a significant inhibitory effect, with a score of 0.7;

[0052] p<0.001 is considered to be extremely significant, indicated by "***", indicating that the raw material sample has an extremely significant inhibitory effect, with a score of 1.0.

[0053] By observing the changes in protein expression levels and whether the NRF2 protein undergoes nuclear translocation to activate the antioxidant pathway, the above-mentioned scoring criteria are summarized and the scores of the three detection indicators are added together to obtain the final score of each raw material sample for comparison. If the score is greater than or equal to 0.3, it is judged to have antioxidant effect. The higher the score, the better the antioxidant effect. The best antioxidant raw materials are thus screened out.

[0054] In a preferred embodiment, in step S1, the raw material sample with a solubility in water ≥1000 μg / mL is directly dissolved in the culture medium for subsequent experiments, and the raw material sample with a solubility in water <1000 μg / mL is dissolved in dimethyl sulfoxide or ethanol solvent for subsequent experiments.

[0055] In a more preferred embodiment, when dimethyl sulfoxide or ethanol is used as the solvent, the volume percentage concentration of the solvent shall not exceed 1% of the total volume, and the volume ratio of the solvent in the blank control group, the induced control group and the sample group is the same.

[0056] In a preferred embodiment, in step S2, 3D skin purchases a matching special culture medium for gas-liquid two-phase culture, and all culture conditions are 37±0.5°C, (5±1)% CO2 concentration and saturated humidity.

[0057] In a preferred embodiment, in step S3, the concentration of the raw material sample is 10 μM-50 μM (preferably 30 μM), the amount added is 10 μL-30 μL (preferably 20 μlL), and the incubation time is 16-24 h (preferably 18-22 h). The concentration of H2O2 treatment is 500 μM-800 μM, the amount added is 10 μL-30 μL (preferably 20 μL), the incubation time is 16-24 h (preferably 18-22 h), and the buffer is DPBS buffer.

[0058] In a preferred embodiment, in step S3, the contents of antioxidant related factors MDA and SOD are detected using relevant detection kits.

[0059] Application Examples

[0060] The solution in the above embodiment is explained in more detail below in conjunction with specific application examples.

[0061] 1. Experimental Materials

[0062] 1. Experimental Materials and Instruments

[0063] 1.1 3D Skin

[0064] The 3D skin is derived from a commercially available, functionally stable human reconstructed 3D epidermal model.

[0065] In this application example, the EpiSkin 3D skin model was selected and purchased from Shanghai Sianfunuo Biotechnology Co., Ltd.

[0066] 1.2 Main Reagents

[0067] (1) 3D skin culture related reagents: EpiSkin maintenance medium and EpiSkin detection medium were purchased from Shanghai Sianfuno Biotechnology Co., Ltd., and DPBS was purchased from Gibco, USA;

[0068] (2) Chemical reagents: Hydrogen peroxide (H2O2) was purchased from Sigma, USA; DMSO, dewaxing solution, isopropanol, and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0069] (3) Kit detection: Total SOD activity detection kit and malondialdehyde (MDA) detection kit were purchased from Nanjing Jiancheng Bioengineering Institute;

[0070] (5) Immunofluorescence detection: The primary antibody Anti-NRF2 and the secondary antibody goat anti-rabbit IgG H&L (FITC) were purchased from Abcam.

[0071] 1.3 Solution preparation

[0072] (1) Different concentrations of ethanol were used for dehydration of paraffin sections, and anhydrous ethanol and ultrapure water were used for embedding;

[0073] (2) Blocking solution: Prepare the blocking solution using 1.5% BSA dissolved in PBS and add 0.025% Triton-X-100;

[0074] (3) Dilute the primary and secondary antibodies according to the recommended ratio using blocking buffer.

[0075] 1.4 Instruments

[0076] Microplate reader (Thermo, USA), CO2 incubator (Thermo, USA), upright fluorescence microscope (Carl Zeiss, Germany), biological safety cabinet (Thermo, USA), high-speed low-temperature centrifuge (Thermo, USA), paraffin embedding system (Leica, Germany), paraffin sectioner (Leica, Germany).

[0077] 2. Raw material samples to be tested

[0078] The names of raw material samples (test substances) are shown in Table 1:

[0079] Table 1

[0080] serial number Sample name 1 Active ingredient A 2 Active ingredient B 3 Active Ingredients C 4 Active ingredient D

[0081] 2. H2O2-induced 3D skin oxidative damage model to evaluate the antioxidant activity of raw material samples

[0082] 1.3D skin culture

[0083] After transportation, the 3D epidermal model was placed in a 6-well plate for stable culture, with 1 mL of maintenance culture medium in each well. The culture conditions were 37°C, 5% CO2 and saturated humidity. It was used after stable culture for 12 hours.

[0084] 2. Construction of H2O2-induced 3D skin oxidative damage model and raw material detection

[0085] Sample group and induced control group: 20 μL of the raw material sample with a concentration of 30 μM and DPBS were evenly applied on the surface of the 3D epidermal model. Incubate in a CO2 incubator at 37°C, 5% CO2, and 95% relative humidity for 16-24 hours. Rinse with DPBS buffer, gently absorb the residual liquid on the surface of each 3D epidermal model with a cotton swab, and move the 3D epidermal models one by one into the well plate with maintenance culture medium;

[0086] Take out the culture plate with the 3D epidermal model and put it into the biosafety cabinet. Add 20 μL of 500 μM-800 μM H2O2 (H2O2 is diluted with culture medium) to the two 3D epidermal models respectively. Culture them for 16-24 hours at 37°C, 5% CO2 and saturated humidity. Rinse thoroughly with DPBS buffer to remove irritants on the surface of the 3D epidermal model. Gently absorb the residual liquid on the surface of each 3D epidermal model with a cotton swab, and move the epidermal models one by one into the well plate with maintenance culture medium.

[0087] Blank control group: 20 μL of the sample to be tested and DPBS were evenly applied on the surface of the 3D epidermal model. Incubate in a CO2 incubator at 37°C, 5% CO2 and 95% relative humidity for 24 hours. Rinse with DPBS buffer and gently absorb the residual liquid on the surface of each epidermal model with a cotton swab.

[0088] 3. Detection of factors related to oxidative damage

[0089] Collect the maintenance culture medium in the well plate in step 2 and use the kit to detect the content of SOD and MDA. The operation steps are consistent with the instructions of the kit.

[0090] 4. Immunofluorescence Detection

[0091] The 3D skin model material was cut with a hole puncher, fixed in 4% tissue fixative for 1 hour, and washed with PBS for 5 minutes. Dehydration with gradient ethanol: 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100% ethanol were soaked for 2 minutes each for dehydration, and then soaked in 50% xylene-ethanol and pure xylene twice for 3 minutes each. Finally, the skin material was soaked in liquid paraffin and placed in the paraffin embedding system overnight at 65°C.

[0092] Embed the skin material using the paraffin embedding system. During operation, place the forceps into the heating hole, and the embedding frame and embedding cassette into the left and right heat slots respectively. On the hot stage, open the dispenser and inject the wax liquid into the embedding cassette. Take the sample out of the embedding frame, put it into the embedding cassette, and adjust the position. Place the embedding cassette in the cooling point to pre-fix the tissue sample. Transfer the sample and embedding cassette to the cold stage for rapid cooling into blocks. Take the tissue embedding block out of the embedding cassette and store it.

[0093] Use a paraffin slicer to slice the skin material embedded block. Install the carrier with the wax block on the clamping part of the slicer. Check whether the blade is sharp before installing the knife, and install the slicer on the knife holder with the blade facing up. Loosen the knife holder and move the knife holder so that the blade is close to the paraffin block. Tighten the knife holder and fix the machine head. Adjust the angle of the slicer so that the line on the slicer holder is aligned with the line in the middle of the base, and finally make the slicer and the paraffin block form an angle of about 15 degrees. Adjust the thickness gauge as needed, and the thickness of 5 to 12μm is appropriate.

[0094] After everything is adjusted, you can start slicing. At this time, hold the handle of the rotating wheel with your right hand, open the safety button on the wheel with your thumb, and shake it clockwise for one circle to cut off a piece. Continuous shaking can cut off a continuous wax strip. At this time, hold the wax strip with your left hand. When the wax strip is about 20 cm long, stop shaking and turn off the safety button. Use the brush to pick up the wax strip and put it on the spreader. Use a sticky slide to fish out the wax strip and place it on the baking machine for patch processing. The baking temperature is 42-55℃.

[0095] Then, the sections were soaked in 100% tissue dewaxing solution for 2 times, 8 minutes each time, and 100%, 95%, 85%, and 75% ethanol were used for 3 minutes each. Then, the sections were soaked in distilled water for 5 minutes, and then added to the pre-prepared antigen repair solution and boiled at 97°C for 20 minutes for antigen repair. After cooling to room temperature naturally, the sections were added to the blocking solution for 2 hours, and then the diluted primary antibody was added and placed in a wet box in a refrigerator at 4°C overnight. The second antibody was hybridized the next day. The type of secondary antibody used was determined according to the species of the primary antibody. The secondary antibody was incubated at room temperature for 1 hour, and then the sections were washed three times with PBS for 3 minutes each time. Finally, the cell nuclei were counterstained with DAPI, and the sections were washed three times with PBS for 3 minutes each time. The sections were sealed with anti-fluorescence quenching sealing agent, and then the fluorescence brightness was observed using an upright fluorescence microscope to observe the nuclear translocation of NRF2 protein.

[0096] Select the data software to calculate the standard curve, substitute the OD value of the sample to be tested into the standard curve to calculate the content of SOD and MDA. Calculate the relative expression with the control group as 100%, and the calculation formula is as follows:

[0097] Relative expression level = sample group content / control group content.

[0098] 4. Results Analysis

[0099] GraphPad Prism was used for drawing, and the results were expressed as Mean ± SEM. One-way ANOVA was used for statistical analysis among the groups. All statistical analyses were two-tailed. p<0.05 was considered to be significantly different, indicated by "*", p<0.01 was considered to be very significantly different, indicated by "**", and p<0.001 was considered to be extremely significantly different, indicated by "***".

[0100] 4.1 Detection of SOD and MDA secretion in the culture medium of H2O2-induced 3D skin oxidative damage model

[0101] The 3D epidermal culture medium was collected and the secretion of oxidative damage factors SOD and MDA in the culture medium was detected and calculated according to the steps in the kit instructions. The relative expression of SOD and MDA was calculated with the control group (induced control group) as the control. Figure 2 and Figure 3 As shown, *P<0.05, **P<0.01, ***P<0.001 are the comparisons between the sample group and the H2O2-induced control group.

[0102] 4.2 Immunofluorescence detection of NRF2 in 3D skin oxidative damage model induced by H2O2

[0103] The 3D epidermis after treatment was collected and fixed with paraformaldehyde, then dehydrated, paraffin-embedded, and sliced ​​for immunofluorescence staining. The target protein NRF2 was subjected to nuclear translocation analysis and immunofluorescence intensity detection, and the relative expression level was calculated with the Control group (induced control group) as the control. The nuclear translocation of NRF2 and the immunofluorescence intensity are shown in Figures 4 and Figure 5 As shown, *P<0.05, **P<0.01, ***P<0.001 are the comparisons between the sample group and the H2O2-induced control group.

[0104] 4.3 Comprehensive evaluation results and analysis

[0105] All raw material samples are in accordance with Figure 1 The results of the experimental significant differences of the four raw material samples tested in this application example at the 3D skin level are summarized in Table 2. The evaluation was performed according to the standards in Table 3, and the evaluation results of the antioxidant efficacy of the test samples are shown in Table 4.

[0106] Table 2

[0107] serial number SOD MDA NRF2 A ns ns *** B ** *** *** C ** *** *** D ns ns ns

[0108] Table 3

[0109]

[0110] Table 4

[0111] serial number SOD MDA NRF2 Score summary A 0 0 1 1 B 0.7 1 1 2.7 C 0.7 1 1 2.7 D 0 0 0 0

[0112] According to the summary score results, the raw material samples in this application example scored greater than 0.3 points except for the active substance D, and were judged to have antioxidant effect. The active substance D did not have antioxidant effect. Among them, the active substances B and C scored the highest with 2.7 points, and had the best antioxidant effect.

[0113] The present invention uses MDA inflammatory factor, SOD inflammatory factor and NRF2 protein as antioxidant efficacy detection and evaluation indicators, focusing on the key factors regulating the endogenous antioxidant capacity of the body, and comprehensively verifying the antioxidant efficacy of the raw material samples. The expression and localization of Nrf2 protein were observed on an in vitro artificial epidermis model, providing visual test results, which can intuitively judge the antioxidant capacity of the raw material samples and detect the activity of related antioxidant enzymes, ensuring the accuracy of the test results. It provides a quick and reliable evaluation method for the preliminary verification and screening of antioxidant active raw materials for skin care products, provides a reference basis for the compounding scheme of antioxidant 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.

[0114] Comparative Example

[0115] The difference between this comparative example and the above application example is that the antioxidant effects of active substances A, B, C and D are screened only by ROS (Reactive Oxygen Species) staining results, and the other conditions remain the same as those of the application example.

[0116] Specifically, the treated 3D skin model was embedded in vivo, sliced ​​with a cryostat, and then ROS staining was performed. The fluorescence signal was observed by fluorescence microscopy and the average fluorescence value was calculated. The lower the fluorescence intensity, the better the antioxidant capacity of the active substance to be tested. The analysis results are as follows: Figure 6 and 7 shown.

[0117] Depend on Figure 6 and Figure 7 The results show that, using the screening method of this comparative example, only active substance B has antioxidant effect, and the other active substances do not have antioxidant effect. Using the screening method of the above application example, the screening results show that both active substance B and active substance C have antioxidant effect, indicating that the screening method used can verify the antioxidant effect of the sample more comprehensively and intuitively, making the results more accurate, and can provide a reliable reference for the compounding scheme of antioxidant raw materials.

[0118] 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 the antioxidant efficacy of cosmetic raw materials, characterized in that: The following steps are involved: S1. Take a normally grown 3D skin model and set up a blank control group, an induced control group and a sample group respectively; S2. adding complete medium to the blank control group and the induced control group for culturing, and adding raw material samples of corresponding dosing concentrations prepared by complete medium to the sample group for culturing; S3. adding H2O2 to the induced control group and the sample group in step S2 and continuing to culture; S4. respectively detecting the contents of inflammatory factors MDA and SOD in the culture medium of the induced control group and the sample group in step S3, and Respectively detecting the immunofluorescence intensity and nuclear translocation of the NRF2 protein in the induced control group and the sample group in step S3; S5. Calculate the secretion ratios of MDA, SOD and NRF2 proteins in the sample group and the induced control group respectively, and determine the antioxidant efficacy of the raw material sample based on the three relative ratios.

2. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 1, characterized in that: In step S5, GraphPad Prism was used to draw graphs, and the results were expressed as Mean ± SEM. One-way ANOVA statistical analysis was used for comparisons among groups, and all statistical analyses were two-tailed. p<0.05 is considered to be significantly different, indicated by "*", indicating that the raw material sample has a certain inhibitory effect, with a score of 0.3; p<0.01 is considered to be a very significant difference, indicated by "**", indicating that the raw material sample has a significant inhibitory effect, with a score of 0.7; p<0.001 is considered to have an extremely significant difference, indicated by "***", indicating that the raw material sample has an extremely significant inhibitory effect, with a score of 1.0; Determine whether the NRF2 protein undergoes nuclear translocation and activates the antioxidant pathway, and compare the final scores of each of the raw material samples obtained by adding the scores of the three detection indicators. If the score is greater than or equal to 0.3, it is judged to have antioxidant effect. The higher the score, the better the antioxidant effect. In this way, the raw material sample with the best antioxidant effect is screened out.

3. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 1 or 2, characterized in that: Also included is a solubility assessment step before performing step S1: Dissolving the raw material sample having a solubility in water of ≥1000 μg / mL directly in the complete culture medium; The raw material sample with a solubility in water of less than 1000 μg / mL is dissolved in dimethyl sulfoxide or ethanol and then added to the complete culture medium.

4. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 3, characterized in that: When the dimethyl sulfoxide or the ethanol is used as the solvent, the volume percentage concentration of the solvent does not exceed 1% of the total volume, and the volume ratio of the solvent in the blank control group, the induced control group and the sample group is kept the same.

5. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 1 or 2, characterized in that: In step S2, the concentration of the raw material sample is 10 μM-50 μM, the addition amount is 10 μL-30 μL, and the incubation time is 16 to 24 hours.

6. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 5, characterized in that: The concentration of the raw material sample is 30 μM, the addition amount is 20 μL, and the culture time is 18 to 22 hours.

7. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 1 or 2, characterized in that: In step S3, the concentration of H2O2 is 500 μM to 800 μM, the amount added is 10 μL to 30 μL, and the culture time is 16 to 24 hours.

8. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 7, characterized in that: In step S3, the amount of H2O2 added is 20 μL, and the culture time is 18 to 22 hours.

9. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 1 or 2, characterized in that: Before performing step S1, the 3D skin model is cultured in gas-liquid two-phase conditions at 37±0.5°C, (5±1)% CO2 concentration and saturated humidity for 10 to 15 hours.

10. The method for screening the antioxidant efficacy of cosmetic raw materials according to claim 1 or 2, characterized in that: In step S4, the 3D skin model is fixed, dehydrated, paraffin-embedded and paraffin-sectioned, and then the immunofluorescence intensity of the NRF2 protein is analyzed.

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