Establishing method of saccharification damaged cell model and anti-sugar efficacy testing method
By culturing HFF-1 cells in vitro in the laboratory and inducing a glycation damage model, and detecting CML content in combination with ELISA method, the blank points in the evaluation of anti-sugar efficacy of cosmetics were solved, and a rapid, economical and accurate anti-sugar efficacy test was achieved.
Patent Information
- Application Number
- CN202510201113.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-11-28
- Filing Date
- 2025-02-24
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art lacks effective methods for evaluating the anti-sugar efficacy of cosmetics, especially in laboratory conditions, with the lack of standardized testing methods.
HFF-1 cells were cultured in vitro, and a glycated damage cell model was established using glyoxal induction, and the content of N-ε-carboxymethyl lysine in cells was detected by ELISA method, and the effects of the samples to be tested were compared with the positive control group.
This method can quickly and economically evaluate the anti-sugar effects of cosmetics and their raw materials. By detecting changes in CML content, it objectively reflects the anti-sugar effects of the sample to be tested, and has high accuracy and repeatability.
Smart Images
Figure CN120173869A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of cosmetic testing, and specifically relates to a method for establishing a glycation-damaged cell model and a method for testing anti-glycation efficacy. Background Art
[0002] Skin aging is an irresistible natural law of humans and cannot be avoided, while skin glycation will accelerate skin aging. In addition to endogenous factors such as the polyol pathway, there are also some exogenous factors that accelerate the skin glycation process, such as tobacco, ultraviolet rays, air pollution, etc. The glycation process is accompanied by the generation of free radicals (ROS), and AGEs are one of the important influencing factors accelerating skin glycation. Pentosidine, CML and MGO derivatives are commonly used AGEs (advanced glycation end-products) biomarkers. Anti-glycation has attracted wide attention from the public. Different from the screening of raw materials such as whitening and moisturizing, which have industry-recognized efficacy evaluation criteria and evaluation indicators, there are still blank points in the evaluation of anti-glycation efficacy.
[0003] The evaluation tests for cosmetic efficacy claims include human efficacy evaluation tests, consumer use tests and laboratory tests. Laboratory tests have the advantages of short cycle, low cost, clear targeting, etc. Among them, the cell model method can simply simulate the normal physiological environment of body cells to a certain extent. It can not only screen substances that inhibit glycation formation, but also screen substances that inhibit glycation damage and rapidly remove glycoproteins (or intermediate products), and has the advantages of low cost, short experimental cycle, strong repeatability, and the ability to determine the dose of cosmetic raw materials that cause biochemical reactions. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for establishing a glycation-damaged cell model and a method for testing anti-glycation efficacy.
[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0006] A method for establishing a glycation-damaged cell model, characterized in that a glycation-damaged model is established by culturing HFF-1 cells in vitro and inducing HFF-1 with glyoxal.
[0007] Specifically, it includes the following steps: 1) Culturing human skin fibroblasts HFF-1 in vitro; performing the next test with a sample concentration without obvious cytotoxicity; 2) Setting a normal control group and a model group to detect the content of N-ε-carboxymethyllysine CML in HFF-1 cells; 3) Analyzing the data by statistical methods. Compared with the normal control group, the CML content in the model group decreases and there is a statistical difference, indicating that the induction of the in vitro cell glycation-damaged model is successful.
[0008] In step 1), the test method for cytotoxicity is the CCK-8 assay. The sample concentration with a cell viability ≥ 90% is selected as the sample concentration without obvious cytotoxicity.
[0009] The statistical method described is to use GraphPad Prism 9 statistical analysis software. One-way ANOVA is used to analyze and compare the data differences between groups. When the variances are homogeneous, the LSD test is used; when the variances are not homogeneous, the Dunnnett T3 test is used. A p < 0.05 is considered to have statistical significance.
[0010] The present invention also includes a method for testing the anti-glycation efficacy of a cosmetic and its raw materials. According to the established method, a glycation-damaged cell model is established, and a normal control group, a model group, a positive control group, and a test sample group are set up. The content of N-ε-carboxymethyllysine (CML) in HFF-1 cells is detected, and the CML contents of the model group, the test sample group, and the positive control group are compared to judge the anti-glycation efficacy of the test sample.
[0011] The detection method for the content of N-ε-carboxymethyllysine (CML) is the ELISA method; preferably, an ELISA kit is used for the operation, and an enzyme-linked immunosorbent assay reader is used to measure the OD value, and the CML content of each group of cells is calculated according to the OD value.
[0012] Compared with the model group, the CML contents in the test sample group and the positive control group are significantly reduced, both with p < 0.01, which proves that the test sample and the positive control group have the effect of significantly reducing intracellular CML, and it can be considered that the test sample has anti-glycation efficacy.
[0013] In step 3), aminoguanidine is used as a positive control to establish a positive control group.
[0014] The specific steps of step 3) are as follows:
[0015] Adjust the concentration of HFF-1 cells, inoculate them in a well plate, and place them in a cell culture incubator overnight. Set up a normal control group, a model group, a test sample group, and a positive control group;
[0016] Dilute the test sample and aminoguanidine with complete medium respectively, and add them to the well plates of the test sample group and the positive control group. Add complete medium to the remaining wells to make the final concentration of the test sample the non-toxic concentration of the CCK-8 assay, and place them in a cell culture incubator for culture;
[0017] Add complete medium to the normal control group, and add glyoxal solution to the remaining wells, and place them in a cell culture incubator for induction.
[0018] Compared with the prior art, the beneficial effects of the present invention are:
[0019] The present invention provides a method for testing the antiglycation efficacy of a cosmetic and its raw materials. The content of CML in cells is determined by ELISA method. By analyzing the CML content of images in the normal control group, model group, test sample group and positive control group, whether there are significant differences among groups is compared, the establishment of the glycation-damaged cell model is judged, and whether the test product has antiglycation efficacy is obtained. This method is different from human detection, is convenient and simple to operate, inexpensive, and has high repeatability. And it can directly reflect the antiglycation efficacy of the test sample through objective scientific data, and the measurement result has high accuracy.
[0020] Glyoxal belongs to reactive carbonyl species (RCS), which is a by-product of cell metabolism. It can damage important cell components such as proteins, nucleic acids, and lipids, and has potential toxicity, playing a role in aging and various chronic diseases. Glyoxal is considered an important glycating agent and precursor of AGEs, mainly reacting with protein residues to cause the formation of AGEs. CML is the most common type of AGEs in organisms and is often used as a representative substance of AGEs to explore the physiological toxicity, formation mode, and inhibition mechanism of endogenous AGEs, etc. CML can crosslink with macromolecular substances such as proteins, nucleic acids, and lipids in almost all skin cells. In addition, it can directly trap and crosslink extracellular matrix proteins (such as elastin and collagen), changing the elasticity and function of tissues, resulting in a decrease in skin elasticity and an increase in hardness, and ultimately leading to skin collapse and wrinkle formation.
[0021] Aminoguanidine is considered a typical antiglycation agent, mainly acting in the middle stage of the glycation reaction. It captures α-dicarbonyl intermediates through nucleophilic reaction (carbonyl scavenging activity), reduces the content of CML in cells, and inhibits collagen degradation, thereby exerting antiglycation efficacy.
[0022] In the present invention, aminoguanidine is used as a positive control substance to establish a positive control group, and the antiglycation efficacy can be tested by inducing glycation damage of HFF-1 cells in vitro with glyoxal.
[0023] Enzyme-Linked Immunosorbent Assay (ELISA) is a laboratory technique widely used in immunology and molecular biology. The ELISA method used in this invention is the sandwich method, specifically manifested in coating a microplate with a purified antibody to make a solid-phase carrier, and then sequentially adding a specimen or a standard, a biotinylated anti-CML antibody, and HRP-labeled avidin to the microplate coated with the anti-CML antibody. After thorough washing, the substrate TMB is used for color development. TMB is converted into blue under the catalysis of peroxidase and then into the final yellow under the action of an acid. The intensity of the color is positively correlated with CML in the sample. The absorbance (OD value) is measured at a wavelength of 450 nm using an enzyme-labeled instrument, and the sample concentration is calculated. Description of the Drawings
[0024] Figure 1 This is a bar chart of the experimental results of the induced glycation damage model of the present invention. Detailed Embodiments
[0025] In order to enable those skilled in the art of this technical field to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the drawings and the best embodiments.
[0026] A method for testing the anti-glycation efficacy of a cosmetic and its raw materials belongs to a laboratory test in the evaluation of cosmetic efficacy tests. By culturing HFF-1 cells in vitro, a glycation damage cell model is established, and the change in the content of CML in the cells is compared to evaluate the anti-glycation efficacy of the cosmetic.
[0027] The specific operation steps are as follows.
[0028] 1) Cell resuscitation: Take out a cryopreserved human skin fibroblast cell line from -80°C or a liquid nitrogen tank, quickly thaw it in a 37°C water bath within 2 minutes, prepare about 10 mL of high-glucose DMEM cell culture medium containing 10% fetal bovine serum in a centrifuge tube, quickly transfer the cryopreservation solution containing the cells into the centrifuge tube, mix well by pipetting, centrifuge at a low speed of 800 rpm for 5 minutes, aspirate the liquid in the tube, add 1 mL of fresh medium and mix well by pipetting again, evenly divide it into 10 cm cell culture dishes, add 8 - 10 mL of fresh medium to each dish, and each cryopreservation tube can be thawed into 2 - 3 dishes. Place the cell culture dishes in a cell culture incubator and culture them for 24 - 72 h in an environment of 37°C and 5% CO2. ， When the cells grow to 80% confluence under a microscope, subculture can be carried out.
[0029] Cell subculture: Rinse the cells twice with 1 mL of PBS buffer, add 2 mL of 0.25% trypsin, place it in the cell culture incubator for 3 - 5 min, observe the cells under the microscope. When the cells become round, it means the cell digestion is completed. At this time, gently blow down the cells, collect them with a centrifuge tube and add 4 mL of cell culture medium. After pipetting and mixing evenly, centrifuge at a low speed of 800 rpm for 5 min, resuspend again, and evenly divide them into cell culture dishes containing 8 - 10 mL of medium. Place the cell culture dishes in the cell culture incubator for continued culture.
[0030] 2) Cytotoxicity test of the test sample (CCK-8 test): Digest and collect HFF-1 cells in the cell culture dish with 0.25% trypsin, adjust the cell concentration to 6×10 4 cells / mL, inoculate 100 μL / well into a 96-well plate. Add 100 μL / well of PBS buffer to the surrounding wells of the 96-well plate. Place the 96-well plate in the cell culture incubator and culture overnight. Dilute the test sample with complete medium to different concentrations, add 100 μL / well to the 96-well plate. At the same time, set up a normal control group and a blank control group, and place them in the cell culture incubator for continued culture for 24 ± 2 h. Take out the 96-well plate, add 10 μL of CCK-8 solution to each well, place it in the cell culture incubator for continued culture for 1 - 2 h, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at 450 nm for each group.
[0031] According to the OD value measurement results, calculate the cell survival rate at each concentration. The cell survival rate = (test well - blank well) / (normal well - blank well). Select the sample concentration with a cell survival rate ≥ 90% as the sample concentration without obvious cytotoxicity for the next experiment.
[0032] 3) CML determination test: Digest and collect HFF-1 cells in the cell culture dish with 0.25% trypsin, adjust the cell concentration to 1×10 5 cells / mL, inoculate 500 μL / well into a 24-well plate. Place the 24-well plate in the cell culture incubator and culture for 24 h. Set up a normal control group, a model group, a test sample group, and a positive control group, with 4 wells in each group. Dilute glyoxal, the test sample, and aminoguanidine with cell culture medium and add them to the corresponding wells. Add complete cell culture medium to the remaining wells, with 250 μL / well in each well, so that the final concentration of the test sample in the 24-well plate is the non-toxic concentration, the concentration of glyoxal is 0.5 mmol·L -1 and the concentration of aminoguanidine is 1.5 mmol·L -1 . Place the 24-well plate with the added drugs in the cell culture incubator and culture for 48 h.
[0033] Extract cell protein: prepare an ice box in advance, pre-cool PBS, protein lysis buffer, PMSF, Eppendorf tubes and other reagents and consumables. After 24 hours of culture, add PMSF to the protein lysis buffer to make the final concentration of PMSF 1mM, take out the 24-well plate, discard the supernatant, add 1ml pre-cooled PBS to each well and wash twice, discard the PBS, add 200μL lysis buffer to each well, and gently shake the culture dish to fully lyse it; scrape the cells with a cell scraper, aspirate the cells and lysis buffer into a 1.5ml Eppendorf tube, and mark it; centrifuge the lysed cells at 4℃ and 12000rpm for 5min, take the supernatant and transfer it to a new 1.5ml Eppendorf tube, wrap it with sealing film, freeze it with liquid nitrogen, and store it at -80℃.
[0034] ELISA method for determination of CML content: 1) Before measuring ELISA, move the sample and various reagents in the ELISA kit to room temperature (18-25℃) for at least 30 minutes, and centrifuge the sample at 12000rpm for 3 minutes, and aspirate the supernatant into a new Eppendorf tube for ELISA detection; 2) Prepare the required reagents according to the method described in the kit instructions and set aside; 3) Sample addition: add 100μL of standard or sample to be tested to each well, attach an ELISA plate sticker, and incubate at 37℃ for 2h; 4) Discard the liquid in the well and spin dry; 5) Add 100μL of biotin-labeled antibody working solution to each well, attach a new ELISA plate sticker, and incubate at 37℃ for 1h; 6) Discard the liquid in the well, spin dry, add 200μL of washing solution to each well, soak for 2min, and wash the plate 3 times; 7) Add 100μL of 8) Discard the liquid in the wells, spin dry, add 200μL of washing solution to each well, soak for 2min, and wash the plate 5 times; 9) Add 90μL of TMB to each well, incubate at 37℃ in the dark for 15-30min; 10) Add 50μL of stop solution to each well in sequence, and measure the OD450 value with a microplate reader within 5min.
[0035] Data statistical analysis: GraphPad Prism 9 statistical analysis software was used to compare the data differences among the groups. LSD test was used when the variance was equal, and Dunnutt T3 test was used when the variance was unequal. p < 0.05 was considered to be statistically significant.
[0036] The results of the cytotoxicity test of the tested samples are shown in Table 1;
[0037] Table 1
[0038]
[0039] The bar graph of the experimental results of the induced glycation damage model is as follows Figure 1 The experimental results of the induced glycation damage model - Density (mean) and IOD values are shown in Table 2.
[0040] Table 2
[0041]
[0042]
[0043] Note: Compared with the normal control group, * represents P < 0.05, ** represents P < 0.01. Compared with the model group, # represents P < 0.05, ## represents P < 0.01.
[0044] Result analysis: Compared with the normal control group, the CML content in the cells of the model group increased significantly (p < 0.01), indicating that the establishment of the glycation damage model was successful. Compared with the model group, the CML content in the test sample group and the positive control group decreased significantly (both p < 0.01), indicating that the test sample and aminoguanidine had a significant effect on reducing intracellular CML, and it can be considered that the test sample has an anti-glycation effect.
[0045] In summary, the present invention provides a method for testing the anti-glycation effect of a cosmetic and its raw materials. By establishing an in vitro glycation damage cell model to simulate the increase in skin CML content, using aminoguanidine as a positive control, and detecting the ability of the test sample to reduce CML, the anti-glycation effect of the test sample is evaluated. This method has the advantages of clear indicators, short test period, and low cost, and can target and verify the anti-glycation effect of cosmetics.
[0046] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. A method for establishing a glycated cell damage model, characterized in that: The method comprises the following steps: culturing HFF-1 cells in vitro and inducing HFF-1 with glyoxal to establish a glycation damage model.
2. The method for establishing a glycated damaged cell model according to claim 1, characterized in that: Specifically, the following steps are included: 1) culturing human skin fibroblasts HFF-1 in vitro; conducting the next test at a sample concentration without obvious cytotoxicity; 2) setting up a normal control group and a model group to detect the N-ε-carboxymethyllysine CML content in HFF-1 cells; 3) using statistical methods to analyze the data. Compared with the normal control group, the CML content in the model group was reduced and there was a statistical difference, indicating that the induction of the in vitro cell glycation damage model was successful.
3. The method for establishing a glycated damaged cell model according to claim 2, characterized in that: The cytotoxicity test method in step 1) is the CCK-8 test method, and a sample concentration with a cell survival rate of ≥ 90% is selected as the sample concentration without obvious cytotoxicity.
4. The method for establishing a glycated damaged cell model according to claim 1, characterized in that: The statistical method was to use GraphPad Prism 9 statistical analysis software, and one-way ANOVA analysis was used to compare the data differences among the groups. When the variances were equal, the LSD test was used, and when the variances were unequal, the Dunnutt T3 test was used. p < 0.05 was considered to be statistically significant.
5. A method for testing the anti-sugar efficacy of cosmetics and their raw materials, characterized in that: A glycated damaged cell model is established according to the establishment method according to any one of claims 1 to 4, a normal control group, a model group, a positive control group and a test sample group are set, the N-ε-carboxymethyllysine CML content of HFF-1 cells is detected, the CML content of the model group, the test sample group and the positive control group is compared, and the anti-glycemic efficacy of the test sample is determined.
6. The method for testing the anti-sugar efficacy of cosmetics and their raw materials according to claim 4, characterized in that: The method for detecting the CML content of N-ε-carboxymethyllysine is ELISA; preferably, an ELISA kit is used for the operation, an OD value is measured using an enzyme marker, and the CML content of each group of cells is calculated according to the OD value.
7. The method for testing the anti-sugar efficacy of cosmetics and their raw materials according to claim 4, characterized in that: Compared with the model group, the CML content in the test sample group and the positive control group was significantly reduced, both p < 0.01, proving that the test sample and the positive control group have the effect of significantly reducing intracellular CML, and it can be considered that the test sample has anti-sugar effect.
8. The method for testing the anti-sugar efficacy of cosmetics and their raw materials according to claim 4, characterized in that: In step 3), aminoguanidine is used as a positive control to establish a positive control group.
9. The method for testing the anti-sugar efficacy of cosmetics and their raw materials according to claim 4, characterized in that: The specific steps of step 3) are: Adjust the concentration of HFF-1 cells, inoculate them into well plates, place them in a cell culture incubator overnight, and set up a normal control group, a model group, a test sample group, and a positive control group; Dilute the test sample and aminoguanidine with complete culture medium, add them to the wells of the test sample group and the positive control group, add complete culture medium to the remaining wells, make the final concentration of the test sample to be the non-toxic concentration of the CCK-8 test, and place in a cell culture incubator for culture; Complete culture medium was added to the normal control group, and glyoxal solution was added to the remaining wells, and the cells were placed in a cell culture incubator for induction.