Screening method for anti-aging raw materials of cosmetics

Abstract

The invention discloses a method for screening anti-aging raw materials of cosmetics, the anti-aging activity of a test substance is screened based on a H2O2 damaged HDFa cell model, detection indexes of the method comprise cell inflammatory factors, metalloproteinase, mitochondrial membrane potential and the like, and the anti-aging activity of the test substance is comprehensively evaluated through a plurality of detection indexes. Compared with a single index, the method is higher in result accuracy, has the characteristics of high efficiency, rapidness, wide application range and the like, provides a new idea for screening anti-aging cosmetic raw materials, and provides a certain theoretical basis for animal substitution experiments in the skin care product industry and development and efficacy verification of skin care products.

Description

Technical Field

[0001] The invention relates to a method for screening cosmetic anti-aging raw materials, and belongs to the technical field of cosmetic raw material performance testing. Technical Background

[0002] Aging is a natural physiological phenomenon of the human body, involving many physiological processes. Aging theories include free radical theory, inflammatory theory, immune theory, etc. The skin is prone to oxidative stress response when stimulated by external factors. Relevant studies have shown that oxidative stress can induce aging of the body. Therefore, it is necessary to develop corresponding anti-aging daily chemicals, which is in line with the current market trend. At present, the core competitiveness of daily chemicals mainly comes from raw materials. Screening effective anti-aging raw materials is an effective guarantee for product development. Therefore, this patent provides a method for screening cosmetic anti-aging raw materials, which provides a certain theoretical basis for the screening of anti-aging raw materials.

[0003] This method complies with the 3R principle, making up for the shortcomings of the traditional method of long test cycle and high detection cost, and is conducive to the rapid screening of anti-aging raw materials. The cause of skin aging is the dermis, which is mainly composed of fibroblasts; H2O2 is a classic oxidative stress inducer that can cause the body to produce a series of oxidative stress reactions, such as aging, wrinkles, etc. Therefore, this method uses H2O2 to stimulate HDFa cells to establish an aging model.

[0004] The inflammatory factors detected by this method are IL-6 and IL-8, which are consistent with the evaluation of inflammatory aging; changes in mitochondrial membrane potential are one of the signs of early cell aging. Detecting this indicator can effectively screen out raw materials that have the ability to resist early cell aging; MMP-1 plays an important role in the aging process and is the most important enzyme for degrading type I and type III collagen. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, destroys the normal structure of collagen fibers and elastic fibers, and leads to the appearance of wrinkles and other aging manifestations on the skin. It is an essential evaluation indicator for screening anti-aging raw materials.

[0005] This method uses the above typical indicators and a comprehensive scoring system to screen out the best anti-aging raw materials, making up for the singleness of the current screening of cosmetic anti-aging raw materials. This comprehensive evaluation system provides an effective method for the screening of anti-aging raw materials and provides stronger support for the development of anti-aging cosmetics. There are currently no relevant literature or patent reports on this evaluation system. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a method for screening cosmetic anti-aging raw materials. The method selects typical inflammatory factors as inflammatory aging indicators; selects changes in mitochondrial membrane potential as early aging indicators; and then selects the important aging indicator MMP-1. Through a scoring system, the cosmetic anti-aging raw materials are comprehensively screened, which has the advantages of being efficient, rapid and comprehensive.

[0007] The technical solution to achieve the above purpose is: a method for screening cosmetic anti-aging raw materials, screening the anti-aging activity of the target raw materials, specifically comprising the following steps:

[0008] S1, evaluate the solubility of the test substance and determine the dissolution scheme;

[0009] S2, cultured HDFa cells;

[0010] S3, HDFa cell activity detection, determination of test substance detection concentration, including the following steps:

[0011] S31, inoculating and culturing HDFa cells in the logarithmic growth phase in step S2 for 16 h to 24 h, adding a test substance, wherein the test substance detection includes multiple concentration gradients, and a blank group is a culture medium solution without any sample added, culturing for 16 h to 24 h, adding MTT to detect absorbance, and calculating cell survival rate;

[0012] S32, screening according to step S31 to obtain the test substance concentration corresponding to the cell survival rate of the HDFa cell model of 90% (CV90), and setting this concentration as the drug administration concentration for subsequent anti-aging tests;

[0013] S4, take the HDFa cells in the logarithmic growth phase in step S2, culture for 16 to 24 hours, and set up a blank group, a model group, and a sample group respectively. Add complete culture medium to the blank group, add H2O2 solution diluted with complete culture medium to the model group and the sample group, culture for 1 to 3 hours, discard the culture medium, add 1 to 2 mL of buffer to each well for 2 to 4 minutes, wash 1 to 3 times, add complete culture medium to the blank group and the model group, add the test substance concentration corresponding to CV90 in step S32 diluted with complete culture medium to the sample group, culture for 16 to 24 hours, and collect the supernatant and HDFa cells respectively.

[0014] S5, taking the supernatant in step S4 to detect the content of inflammatory factors IL-6, IL-8 and metalloproteinase MMP-1.

[0015] S6, taking the HDFa cells collected in step S4, detecting the mitochondrial membrane potential, and recording the distribution of high potential and low potential cells.

[0016] S7, summarize the IL-6, IL-8 content determination results and MMP-1 content determination results in step S5, calculate the content ratio of the sample group and the model group, and obtain their relative expression levels; summarize the distribution of high and low potential cells in step S6; GraphPad Prism was used for the above graphs, and the results were expressed as Mean±SD; One-way ANOVA statistical analysis was used for comparison between groups.

[0017] p<0.05 is considered to be significantly different, indicated by "*", indicating that the test substance has a certain inhibitory effect, with a score of 0.33;

[0018] p<0.01 is considered to be a very significant difference, indicated by "**", indicating that the test substance has a significant inhibitory effect, with a score of 0.67;

[0019] p<0.001 is considered to be extremely significant, indicated by "***", indicating that the test substance has an extremely significant inhibitory effect, with a score of 1.0;

[0020] According to the above scoring criteria, the scores of inflammatory aging indicators IL-6, IL-8, typical aging indicator mmp-1 and early aging indicator mitochondrial membrane potential distribution were obtained respectively. The scores were added and divided by 4 to calculate the average value. If the average value is ≥ 0.5, the test substance is considered to have good anti-aging activity.

[0021] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S1, the water-soluble sample is directly dissolved in the complete culture medium; the water-insoluble sample is first prepared to a high concentration with dimethyl sulfoxide, and then diluted to the effective concentration with the complete culture medium; during the dissolution process, high temperature, ultrasound and other auxiliary dissolution methods can be used according to the characteristics of the test substance.

[0022] In the above-mentioned method for screening cosmetic anti-aging raw materials, when dimethyl sulfoxide is used as a solvent, the volume fraction of the solvent shall not exceed 0.1% of the total volume of the sample.

[0023] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S2, the complete culture medium used for HDFa cell culture is configured as 90% Fibroblast Basal Medium + 10% FBS, and the culture conditions are: temperature of 37±0.5°C, CO2 concentration of (5±1)%.

[0024] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S31, HDFa cells are inoculated in a 96-well plate, and the density of the inoculated cell suspension is 0.5-2.0×10 4 / mL, 100uL was inoculated in each well, 5 to 8 concentration gradients of the test substance were set, and 3 or more replicate wells were set for each concentration.

[0025] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S4, HDFa cells are cultured at a concentration of 3 to 5×10 4 Cells / mL were inoculated into 6-well plates, 2 mL of cell suspension was added to each well, and cultured for 24 h.

[0026] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S4, the concentration of H2O2 is 50-200umol / L, and the buffer solution is PBS solution.

[0027] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S5, the contents of inflammatory factors IL-6, IL-8 and metalloproteinase MMP-1 are detected using an ELISA kit.

[0028] In the above-mentioned method for screening cosmetic anti-aging raw materials, in step S6, mitochondrial membrane potential is detected by flow cytometry, and the staining agent is JC-1.

[0029] The screening method for cosmetic anti-aging raw materials of the present invention comprises a plurality of typical indicators of aging, and each indicator is incorporated into a comprehensive scoring system, thereby providing a comprehensive, rapid and efficient anti-aging screening method, making up for the deficiency of the traditional single indicator, providing a relatively comprehensive evaluation method for the early verification, development and screening of anti-aging active raw materials of daily chemical products, providing a corresponding basis for the compounding of anti-aging raw materials, and providing a strong guarantee for the development of better skin care products with anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the survival rate of HDFa human primary fibroblasts of test substances A to D;

[0031] Figure 2 The relative secretion of IL-6 in the supernatant of different groups of HDFa human primary fibroblasts induced by H2O2;

[0032] Figure 3 The relative secretion of IL-8 in the supernatant of different groups of HDFa human primary fibroblasts induced by H2O2;

[0033] Figure 4 The relative secretion of MMP-1 in the supernatant of different groups of HDFa human primary fibroblasts induced by H2O2;

[0034] Figure 5 The distribution of mitochondrial membrane potential in different groups of HDFa human primary fibroblasts induced by H2O2;

[0035] Figure 6 The present invention is a screening flow chart of a method for screening cosmetic anti-aging raw materials. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods thereof are described in detail below in conjunction with the accompanying drawings:

[0037] Unless otherwise specified, the reagents and materials used in the examples can be purchased from the market.

[0038] 1. Experimental Materials

[0039] 1. Experimental Materials and Instruments

[0040] 1.1 Cells

[0041] HDFa human primary fibroblasts are derived from ATCC, Chinese Academy of Sciences Cell Bank or China Type Culture Collection Center Cell Bank and other registered member units of the World Federation of Culture Collections. In this embodiment, HDFa human primary fibroblasts were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0042] 1.2 Main Reagents

[0043] (1) Cell culture related reagents: Fetal bovine serum and PBS (1x) were purchased from Gibco, USA; Fibroblast Basal Medium was purchased from ATCC.

[0044] (2) Chemical reagents: MTT was purchased from Sigma, USA; DMSO and hydrogen peroxide (H2O2) were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0045] (3) ELISA test: IL-6 ELISA kit, IL-8 ELISA kit, and MMP-1 ELISA kit were purchased from Xinbosheng Biotechnology Co., Ltd.;

[0046] (4) Mitochondrial membrane potential detection: JC-1 test kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0047] 1.3 Solution preparation

[0048] (1) Fibroblast Basal Medium complete medium for HDFa human primary fibroblasts: Take 45 mL of Fibroblast Basal Medium medium, add 5 mL of fetal bovine serum (FBS), mix thoroughly and store at 4°C. Preheat in a 37°C water bath before use;

[0049] (2) MTT solution: weigh 100 mg of MTT powder, add 20 mL of PBS, vortex to dissolve, filter through a 0.22 μm filter membrane to sterilize, store at -20°C away from light, and thaw at 4°C before use;

[0050] (3) MTT detection solution: MTT solution was diluted to 0.5 mg / mL in complete culture medium;

[0051] (4) H2O2 modeling agent: Take 20uL of H2O2 solution, add 10mL of complete culture medium to prepare a 2mmol / L H2O2 solution, and then dilute it to 50umol / L with complete culture medium.

[0052] 1.4 Instruments

[0053] CO2 incubator (Thermo, USA), inverted microscope (Olympus, Japan), biological safety cabinet (Thermo, USA), automatic cell counter (Nexcelom, USA), full-wavelength microplate reader (Thermo, USA), centrifuge (Thermo, USA)

[0054] 2. Raw material samples to be tested

[0055] The names of raw material samples (test substances) are shown in Table 1.

[0056] Table 1, raw material sample table:

[0057] serial number Sample name 1 Test substance A 2 Test substance B 3 Test substance C 4 Test substance D

[0058] 2. H2O2-induced HDFa human primary fibroblast senescence model to evaluate the anti-aging activity of raw materials

[0059] 1. Cell Culture

[0060] HDFa human primary fibroblasts were cultured with complete medium in an incubator at 37°C and 5% CO2. The cells were subcultured when the cell proliferation rate reached 70-80%. The medium was discarded and 2 mL of trypsin was added. The cells were placed in a CO2 incubator for digestion for 3 minutes. 2 times the medium was added to terminate the digestion. The cells were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and fresh medium was added to resuspend the cells. The cells were subcultured at a ratio of 1:3.

[0061] 2. MTT cytotoxicity test and analysis of the test substance

[0062] 2.1 Cytotoxicity assay

[0063] HDFa human primary fibroblasts with good cell morphology and in logarithmic growth phase were prepared with complete culture medium at 1×10 4100uL of cell suspension at a concentration of cells / mL was inoculated in a 96-well plate. No cells were inoculated in the outer circle of the 96-well plate. 200uL of PBS solution was added and the plate was cultured in an incubator at 37°C and 5% CO2. The original culture medium was discarded after 24h, and 100uL of test substances of different concentrations prepared with fresh complete culture medium was added to each well. Six concentration gradients were set for each test substance. The concentrations of liquid raw materials (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, and 0.0316%, respectively. The concentrations of solid raw materials were 1000μg / mL, 316μg / mL, 100μg / mL, 31.6μg / mL, 10μg / mL, and 3.16μg / mL, respectively. Three replicate wells were set for each concentration. Fresh complete culture medium was used instead of blank group. The plate was cultured at 37°C and 5% CO2. Culture in a CO2 incubator, discard the original culture medium after 24 hours, wash twice with PBS solution, add 100uL of MTT detection solution to each well, incubate in a 37°C, 5% CO2 incubator for 4 hours, remove the supernatant, wash twice with PBS, add 100uL of DMSO solution to each well, mix well, measure the absorbance at a wavelength of 570nm, use 630nm as the reference wavelength, and record the results.

[0064] 2.2 Analysis of cytotoxicity results

[0065] Calibration OD = OD570-OD630

[0066] The cell viability calculation formula is as follows:

[0067] Cell survival rate = [(As-Ab) / (Ac-Ab)] x 100%

[0068] As: Experimental well (containing cell culture medium, MTT, and test substance)

[0069] Ac: control well (containing cell culture medium, MTT, no test substance)

[0070] Ab: Blank well (medium without cells and test substance, MTT)

[0071] Calculation of 90% cell viability (CV90)

[0072]

[0073] Where:

[0074] a: The cell viability exceeds the minimum value of 90%;

[0075] c: cell viability is less than 90% of the maximum value;

[0076] b and d indicate the concentrations corresponding to the cell activities in a and c.

[0077] The cell viability of the test substance was calculated according to the above formula, and the test substance concentration corresponding to CV90 was selected as the dosage concentration for anti-aging activity determination.

[0078] 3. Determination of the anti-aging efficacy of the test substances in the H2O2-induced HDFa human primary fibroblast model

[0079] 3.1 Detection of inflammatory factors IL-6 and IL-8 secretion in the H2O2-induced HDFa human primary fibroblast model

[0080] HDFa human primary fibroblasts with good cell morphology and in logarithmic growth phase were cultured in complete medium at 4 × 10 4 The cell suspension with a concentration of 100 μg / mL was inoculated in 6-well plates with 2 mL per well, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The supernatant was discarded, and blank group, model group and sample group were set up respectively. 2 mL of H2O2 solution with a concentration of 50 umol / L was added to each well of the model group and sample group, and the blank group was replaced with complete culture medium, and incubated in an incubator at 37°C and 5% CO2 for 1 hour, the supernatant was discarded, and the cells were washed twice with PBS solution. 2 mL of fresh complete culture medium was added to the blank group and model group, and 2 mL of the test substance with the corresponding concentration of CV90 prepared with complete culture medium was added to the sample group, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The supernatant was collected, and the content of IL-6 and IL-8 in the collected supernatant was detected using an ELISA kit. The operation steps were strictly carried out in accordance with the instructions of the kit.

[0081] The standard curve was fitted, and the OD values ​​corresponding to different groups were substituted into the standard curve to calculate the contents of IL-6 and IL-8. The H2O2-induced model group was taken as 100% expression, and the relative expression of IL-6 and IL-8 in the blank group and sample group was calculated. The calculation formula is as follows:

[0082] Relative expression level = sample group content / induced control group content

[0083] 3.2 Detection of MMP-1 secretion in H2O2-induced HDFa human primary fibroblast model

[0084] HDFa human primary fibroblasts with good cell morphology and in logarithmic growth phase were cultured in complete medium at 4 × 10 4Cell suspension with a concentration of 100 μg / mL was added to each well of a 6-well plate, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The supernatant was discarded, and blank group, model group and sample group were set up respectively. 2 mL of H2O2 solution with a concentration of 50 umol / L was added to each well of the model group and sample group, and the blank group was replaced with complete culture medium. The cells were incubated in an incubator at 37°C and 5% CO2 for 1 hour, the supernatant was discarded, and the cells were washed twice with PBS solution. 2 mL of fresh complete culture medium was added to the blank group and model group, and 2 mL of the test substance with the corresponding concentration of CV90 prepared with complete culture medium was added to the sample group. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The supernatant was collected, and the content of mmp-1 in the collected supernatant was detected using an ELISA kit. The operation steps were strictly carried out in accordance with the instructions of the kit.

[0085] The standard curve was fitted, and the OD values ​​corresponding to different groups were substituted into the standard curve to calculate the content of mmp-1. The H2O2-induced model group was taken as 100% expression, and the relative expression of mmp-1 in the blank group and the sample group was calculated. The calculation formula is as follows:

[0086] Relative expression level = sample group content / induced control group content

[0087] 3.3 Detection of cell membrane potential distribution in H2O2-induced HDFa human primary fibroblast model

[0088] HDFa human primary fibroblasts with good cell morphology and in logarithmic growth phase were cultured in complete medium at 4 × 10 4 The cell suspension with a concentration of 100 μg / mL was inoculated in 6-well plates with 2 mL per well. The plates were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The supernatant was discarded and blank, model and sample groups were set up respectively. 2 mL of H2O2 solution with a concentration of 50 umol / L was added to each well of the model group and sample group, and the blank group was replaced with complete culture medium. The plates were incubated in an incubator at 37°C and 5% CO2 for 1 hour. The supernatant was discarded and the plates were washed twice with PBS solution. 2 mL of fresh complete culture medium was added to the blank group and model group, and 2 mL of the test substance with the corresponding concentration of CV90 prepared with complete culture medium was added to the sample group. The plates were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The supernatant was discarded, 500 uL of trypsin was added to each well and placed in the incubator for 3 minutes. 1 mL of complete culture medium was added to each well to terminate digestion, and the cells were collected by centrifugation. The mitochondrial membrane potential distribution of the collected cells was detected by flow cytometry using the JC-1 kit. The operation steps were strictly carried out in accordance with the instructions of the kit.

[0089] 4. Results Analysis

[0090] 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. 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 "***".

[0091] 4.1 Cytotoxicity test of the test substance

[0092] See also Figure 1 The cell viability MTT test results of the test substances (raw material samples) A ​​to D are as follows: Figure 1 As shown, CV90 is calculated based on the result, and the CV90 values ​​of the samples to be tested are shown in Table 2.

[0093] Table 2, CV90 values ​​of the samples to be tested:

[0094] serial number CV90 A 0.73％ B 1％ C 5％ D 3mg / mL

[0095] 4.2 Results of H2O2-induced senescence model of human primary HDFa cells

[0096] 4.2.1 IL-6 and IL-8 secretion in cell supernatant

[0097] See also Figure 2 and Figure 3 , collect the supernatant of HDFa cells, detect the secretion of IL-6 and IL-8 according to the ELISA kit, and strictly follow the instructions to detect and calculate the secretion of inflammatory factors IL-6 and IL-8 in the supernatant, and calculate the relative secretion of the H2O2 model group as the control group.

[0098] The relative secretion of IL-6 Figure 2 As shown in Figure 2, the relative secretion of IL-6 is as follows Figure 3 As shown, ###P<0.001 is the comparison between H2O2 induced control group and blank control group, *P<0.05, **P<0.01, ***P<0.001 is the comparison between sample group and H2O2 induced control group.

[0099] 4.2.2 MMP-1 secretion in cell supernatant

[0100] See also Figure 4 , collect the supernatant of HDFa cells, detect the secretion of mmp-1 according to the ELISA kit, and strictly follow the instructions to detect and calculate the secretion of mmp-1 in the supernatant, and calculate the relative secretion of the H2O2 model group as the control group.

[0101] The relative secretion of mmp-1 Figure 4As shown, ###P<0.001 is the comparison between H2O2 induced control group and blank control group, *P<0.05, **P<0.01, ***P<0.001 is the comparison between sample group and H2O2 induced control group.

[0102] 4.2.3 Distribution of mitochondrial membrane potential in HDFa primary human fibroblasts

[0103] See also Figure 5 , collect cells, stain the cells using the JC-1 staining kit, and detect the distribution of mitochondrial membrane potential by flow cytometry. Figure 5 and Table 3.###P<0.001 for the comparison between H2O2 induced control group and blank control group, *P<0.05, **P<0.01, ***P<0.001 for the comparison between sample group and H2O2 induced control group.

[0104] Table 3, mitochondrial membrane potential distribution of the samples to be tested:

[0105] serial number High Potential Low potential A 62.4％ 37.6％ B 49.1％ 50.9％ C 76.1％ 23.9％ D 53.8％ 46.2％

[0106] 4.3 Comprehensive evaluation results and analysis

[0107] All tested substances were Figure 6 The evaluation was performed according to the process shown in the figure, and the scoring criteria were evaluated according to Table 4. The test results of the four samples tested in this embodiment are shown in Table 5. According to the scoring criteria, the scoring results are shown in Table 6. It can be seen from Table 6 that the comprehensive anti-aging effect of the test objects in this embodiment after the scores are ranked is: C>A=B>D, and the test objects with a score greater than 0.5 are: C, which has a good anti-aging effect.

[0108] Table 4, scoring criteria table:

[0109]

[0110] Table 5, test results of the samples to be tested:

[0111] serial number Human IL-6 Human IL-8 mmp-1 Mitochondrial membrane potential A ns. ns. ns. ** B ns. ** ns. ns. C *** * ** *** D ns. ns. * ns.

[0112] Table 6, scoring results of the tested samples:

[0113] serial number Human IL-6 Human IL-8 mmp-1 Mitochondrial membrane potential average value A 0 0 0 0.67 0.17 B 0 0.67 0 0 0.17 C 1.0 0.33 0.67 1.0 0.75 D 0 0 0.33 0 0.08

[0114] See also Figure 6 The present invention provides a method for screening anti-aging raw materials, which comprehensively evaluates and screens anti-aging active raw materials by combining different anti-aging evaluation indicators, and includes the following steps:

[0115] S1, evaluate the solubility of the test substance and determine the dissolution scheme;

[0116] Water-soluble samples are directly dissolved in complete culture medium; water-insoluble samples are first prepared with dimethyl sulfoxide to a high concentration, and then diluted with complete culture medium to the effective concentration; during the dissolution process, high temperature, ultrasound and other auxiliary dissolution methods can be used according to the characteristics of the test substance. When using dimethyl sulfoxide as a solvent, the volume fraction of the solvent shall not exceed 0.1% of the total volume of the sample.

[0117] S2, cultured HDFa cells;

[0118] S3, HDFa cell activity assay to determine the test concentration of the test substance, including the following steps:

[0119] S31, inoculating and culturing the logarithmically growing HDFa cells in step S2 for 16 h to 24 h, adding the test substance, wherein the test substance detection includes multiple concentration gradients, and the blank group is a culture medium solution without adding any sample, culturing for 16 h to 24 h, adding MTT to detect the absorbance, and calculating the cell survival rate;

[0120] S32, screening according to step S31 to obtain the test substance concentration corresponding to the cell survival rate of the HDFa cell model of 90% (CV90), which is the drug administration concentration for subsequent efficacy testing;

[0121] S4, take the HDFa cells in the logarithmic growth phase in step S2, culture for 16 to 24 hours, and set up a blank group, a model group, and a sample group respectively. Add complete culture medium to the blank group, add H2O2 solution diluted with complete culture medium to the model group and the sample group, culture for 1 to 3 hours, discard the culture medium, add 1 to 2 mL of buffer to each well for 2 to 4 minutes, wash 1 to 3 times, add complete culture medium to the blank group and the model group, add the test substance concentration corresponding to CV90 in step S32 diluted with complete culture medium to the sample group, culture for 16 to 24 hours, and collect the supernatant and HDFa cells respectively.

[0122] S5, taking the supernatant in step S4 to detect the content of inflammatory factors IL-6, IL-8 and metalloproteinase MMP-1. This detection step is strictly carried out according to the instructions of the kit.

[0123] S6, taking the HDFa cells collected in step S4, detecting the mitochondrial membrane potential, and recording the proportion of high potential and low potential cells in the total cell population. This staining step is strictly carried out according to the JC-1 staining kit.

[0124] S7, summarize the IL-6, IL-8 content determination results and MMP-1 content determination results in step S5, calculate the content ratio of the sample group and the model group, and obtain their relative expression levels; summarize the proportion of high- and low-potential cells in the total cell population in step S6; GraphPad Prism was used for the above graphs, and the results were expressed as Mean±SD; One-wayANOVA statistical analysis was used for comparison between groups.

[0125] p<0.05 is considered to be significantly different, indicated by "*", indicating that the test substance has a certain inhibitory effect, with a score of 0.33;

[0126] p<0.01 is considered to be a very significant difference, indicated by "**", indicating that the test substance has a significant inhibitory effect, with a score of 0.67;

[0127] p<0.001 is considered to be extremely significant, indicated by "***", indicating that the test substance has an extremely significant inhibitory effect, with a score of 1.0;

[0128] According to the above scoring criteria, the scores of IL-6, IL-8, mmp-1 and mitochondrial membrane potential distribution were obtained respectively. The scores were added and divided by 4 to calculate the average value. If the average value was ≥ 0.5, the test substance was considered to have good anti-aging activity.

[0129] The inflammatory factors detected by this method are IL-6 and IL-8, which are consistent with the evaluation of inflammatory aging; changes in mitochondrial membrane potential are one of the signs of early cell aging. Detecting this indicator can effectively screen out raw materials that have the ability to resist early cell aging; MMP-1 plays an important role in the aging process and is the most important enzyme for degrading type I and type III collagen. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, destroys the normal structure of collagen fibers and elastic fibers, and leads to the appearance of wrinkles and other aging manifestations on the skin. It is an essential evaluation indicator for screening anti-aging raw materials.

[0130] This method uses the above typical indicators and a comprehensive scoring system to score, screen out the best anti-aging raw materials, and comprehensively evaluate the anti-aging activity of the test substances through multiple detection indicators, which makes up for the current singleness of the screening of cosmetic anti-aging raw materials. The results are more accurate than those of a single indicator. At the same time, it has the characteristics of high efficiency, rapidity, and a wide range of applications. It provides new ideas for screening anti-aging cosmetic raw materials and provides a certain theoretical basis for animal replacement experiments in the skin care industry, the development of skin care products, and efficacy verification.

[0131] Those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. As long as they are within the spirit of the present invention, any changes or modifications to the above embodiments will fall within the scope of the claims of the present invention.

Claims

1. A method for screening cosmetic anti-aging raw materials, characterized in that: Based on the results of the H2O2-damaged HDFa cell aging model test, the anti-aging activity of the target raw materials was screened, including the following steps: S1, evaluate the solubility of the test substance and determine the dissolution scheme; S2, cultured HDFa cells; S3, HDFa cell activity assay to determine the test concentration of the test substance, including the following steps: S31, taking the HDFa cells in the logarithmic growth phase in step S2 and inoculating them for 16h to 24h, adding the test substance, wherein the test substance detection includes multiple concentration gradients, and the blank group is a culture medium solution without adding any sample, culturing for 16h to 24h, adding MTT to detect the absorbance, and calculating the cell survival rate; S32, screening according to step S31 to obtain the test substance concentration corresponding to the cell survival rate of the HDFa cell model of 90% (CV90), and setting the concentration as the drug administration concentration for subsequent efficacy testing; S4, take the HDFa cells in the logarithmic growth phase in step S2, culture for 16 to 24 hours, and set up a blank group, a model group, and a sample group respectively. Add complete culture medium to the blank group, add H2O2 solution diluted with complete culture medium to the model group and the sample group, culture for 1 to 3 hours, discard the culture medium, add 1 to 2 mL of buffer to each well for 2 to 4 minutes, wash 1 to 3 times, add complete culture medium to the blank group and the model group, add the test substance concentration corresponding to CV90 in step S32 diluted with complete culture medium to the sample group, culture for 16 to 24 hours, and collect the supernatant and HDFa cells respectively. S5, taking the supernatant collected in step S4 to detect the content of inflammatory factors IL-6, IL-8 and metalloproteinase MMP-1. S6, taking the HDFa cells collected in step S4, detecting the mitochondrial membrane potential, and recording the proportion of high potential and low potential cells in the total cell population. S7, summarize the IL-6, IL-8 content determination results and MMP-1 content determination results in step S5, calculate the content ratio of the sample group and the model group, and obtain their relative expression levels; summarize the proportion of high- and low-potential cells in the total cell population in step S6; GraphPad Prism was used for the above graphs, and the results were expressed as Mean±SD; One-way ANOVA statistical analysis was used for comparison between groups. p<0.05 is considered to be significantly different, indicated by "*", indicating that the test substance has a certain inhibitory effect, with a score of 0.33 p<0.01 is considered to be a very significant difference, indicated by "**", indicating that the test substance has a significant inhibitory effect, with a score of 0.67; p<0.001 is considered to be extremely significant, indicated by "***", indicating that the test substance has an extremely significant inhibitory effect, with a score of 1.0; According to the above scoring criteria, the scores of inflammatory aging indicators IL-6, IL-8, typical aging indicator mmp-1 and early aging indicator mitochondrial membrane potential distribution were obtained respectively. The scores were added and divided by 4 to calculate the average value. If the average value is ≥ 0.5, the test substance is considered to have good anti-aging activity.

2. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S1, the water-soluble sample is directly dissolved in the complete culture medium; the water-insoluble sample is first prepared with dimethyl sulfoxide to a high concentration, and then diluted with the complete culture medium to the final effective concentration; during the dissolution process, high temperature, ultrasound and other auxiliary dissolution methods can be used according to the characteristics of the test substance.

3. The method for screening cosmetic anti-aging raw materials according to claim 3, characterized in that: When using dimethyl sulfoxide as a solvent, the volume fraction of the solvent shall not exceed 0.1% of the total volume of the sample.

4. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S2, the complete culture medium used for HDFa cell culture is configured as 90% Fibroblast Basal Medium + 10% FBS, and the culture conditions are: temperature of 37±0.5°C and CO2 concentration of (5±1)%.

5. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S31, HDFa cells were inoculated in a 96-well plate, and the density of the inoculated cell suspension was 0.5-2.0×10 4 / mL, 100uL was inoculated in each well, 5 to 8 concentration gradients of the test substance were set, and 3 or more replicate wells were set for each concentration.

6. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S4, HDFa cells were cultured at a rate of 3 to 5 × 10 4 Cells / mL were inoculated into 6-well plates, 2 mL of cell suspension was added to each well, and cultured for 24 h.

7. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S4, the concentration of H2O2 is 50-200umol / L, and the buffer solution is PBS solution.

8. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S5, the contents of inflammatory factors IL-6, IL-8 and metalloproteinase MMP-1 are detected using an ELISA kit.

9. The method for screening cosmetic anti-aging raw materials according to claim 1, characterized in that: In step S6, flow cytometry is used to detect mitochondrial membrane potential, and the dye is JC-1.

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