Method for evaluating in-vitro anti-saccharification effect and application thereof

Abstract

The invention provides an in-vitro anti-saccharification effect evaluation method and application thereof, and belongs to the technical field of skin saccharification resistance. A photoaging fibroblast model is adopted, after induction of an inducer, anti-saccharification evaluation is carried out by detecting the contents of collagen and waveform protein after application of a substance to be detected, and it is proved that the anti-saccharification effect of the anti-saccharification substance in the cell model is consistent with the result in human body effect evaluation; the evaluation method can be applied to screening and evaluation of anti-skin saccharification substances.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-skin glycation, and relates to an evaluation method, in particular to an evaluation method for anti-glycation effect in vitro and application thereof. BACKGROUND

[0002] Skin glycation is one of the factors affecting skin aging. Glycation reaction refers to the reaction of free amino groups in proteins, nucleic acids or lipids in the body and free carbonyl groups of glucose or other reducing sugars under non-enzyme conditions to produce a series of stable compounds. Skin glycation can cause denaturation of collagen, yellowing and brittleness, and inhibit skin repair and cell renewal, resulting in dull, inelastic skin and apparent muscle age.

[0003] After the reaction of carbohydrates and proteins, some primary glycation products are formed reversibly, and then irreversible advanced glycation end products (AGEs) are formed. AGEs are a kind of brown reducing sugar covalent adduct formed in the later stage of glycation reaction, and the generation, accumulation and subsequent biological effects of AGEs are the main causes of skin glycation.

[0004] In order to screen anti-glycation active substances, the current scheme in the prior art includes using biochemical experiment method, cell model method, 3D skin model method and human test method to verify the efficacy. The biochemical experiment method is simple in operation and low in cost, and can be used as an effective tool for large-scale screening of anti-glycation raw materials, but due to the limitation of in vitro experiment, it cannot well present the state of real skin. The 3D skin model can highly simulate the state of human skin, but has high cost and low success rate. The human test method is mainly used for efficacy evaluation test of cosmetic finished products, and can directly reflect the effect of anti-glycation cosmetics, but it is time-consuming and not suitable for early active substance screening.

[0005] The cell model method can simulate the normal physiological environment of body cells to a certain extent, and can also detect the content of glycation intermediates in combination with biochemical experiment method, so as to screen anti-glycation substances from multiple dimensions.

[0006] At present, in the evaluation of anti-glycation effect by cell model method, the following glycation cell models are used:

[0007] 1. High glucose (HG) culture method is used to induce glycation model. HG can promote the activation of RAGE (high glycation end product receptor) and lead to the accumulation of AGEs.

[0008] Literature source: Wu C H, Wu C F, Huang H W, et al. Naturally occurring flavonoids attenuate high glucose-induced expression of proinflammatory cytokines in human monocytic THP-1 cells

[0009] [J]. Molecular Nutrition and Food Research, 2009, 53(8): 984-995.

[0010] 2. Exposure of cells to highly reactive dicarbonyl compounds such as GO (glyoxal) or MGO (methylglyoxal) induces glycation model. MGO / GO is often used as an inducer in the study, and aminoguanidine (AG) is selected as a positive control for this cell model. AG is a typical synthetic AGEs inhibitor, which plays a role by removing dicarbonyl intermediates in the glycation reaction.

[0011] Literature source: Lin H, Lin T Y, Lin J A, et al. Effect of Pholidota namiko polysaccharides inhibiting methylglyoxal-induced glycation damage in vitro [J]. Antioxidants, 2021, 10(10): 1589.

[0012] 3. AGE-BSA induction: AGE-BSA is the glycation product of bovine serum albumin (BSA) in a sterile condition with reducing sugar (or reducing sugar derivative) and the like. Using AGE-BSA as a stimulant can induce a glycation cell model.

[0013] Literature source: Sukjamnong S, Chen H, Saad S, et al. Fimbristylis ovata and Artemisia vulgaris extracts inhibited AGE-mediated RAGE expression, ROS generation, and inflammation in THP-1 cells [J]. Toxicological Research, 2022, 38(3): 331-343.

[0014] The above methods all use the method of inducing the accumulation of AGEs to construct the model. Since AGEs are easily combined and cross-linked with collagen after being generated, the glycation degree of the cells can be verified by detecting the content of AGEs-collagen cross-linking products in the cell model. After the active substance is applied, the generation inhibition rate of AGEs-collagen cross-linking products is detected, which can be used to evaluate the anti-glycation effect of the active substance.

[0015] The efficacy evaluation method used in the prior art has fewer dimensions, especially in the evaluation using the cell model, the synchronous evaluation of the intracellular and extracellular matrix detection endpoints is ignored, and only the collagen in the extracellular matrix is used as the evaluation standard, the detection endpoint is single, and the real situation of the human skin microenvironment cannot be approached. AGEs can directly target multiple key functional proteins related to cells, leading to changes in the skin, and the principle of its action is multidimensional. In the action area, it includes the AGEs action target in the extracellular matrix and intracellular, and in the action pathway, AGEs also participates in promoting the activation of nuclear factor NF-κB and inducing the production of various intracellular cytokines. The evaluation method or in vitro evaluation model of the prior art only evaluates the efficacy of anti-glycation substances from individual dimensions, and the accuracy of the anti-glycation effect evaluation is low, such as the degree of AGEs-collagen cross-linking. SUMMARY

[0016] The present application provides an in vitro anti-glycation effect evaluation method and its application to solve the problem of low accuracy of anti-glycation effect evaluation in the prior art. The present application uses a photo-aged fibroblast cell model, which is induced by methylglyoxal. The anti-glycation effect of the test substance is evaluated by detecting the content of extracellular collagen and intracellular vimentin after the test substance is applied. The results are consistent with the human efficacy evaluation, and the method can be applied to the screening and evaluation of anti-skin glycation substances.

[0017] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0018] The present application provides an in vitro anti-glycation effect evaluation method, which uses a photo-aged fibroblast cell model, which is induced by an inducing agent. The anti-glycation effect of the test substance is evaluated by detecting the content of extracellular protein and intracellular protein after the test substance is applied. The inducing agent includes high-concentration glucose, active carbonyl substances and AGE-BSA. The extracellular protein includes collagen, and the intracellular protein includes vimentin.

[0019] Preferably, the inducing agent is an active carbonyl substance, the active carbonyl substance is methylglyoxal, the extracellular protein is type I collagen, and the intracellular protein is vimentin.

[0020] The evaluation method of the in-vitro anti-glycation efficacy provided by the application provides more evaluation dimensions, especially the action targets of extracellular matrix and intracellular AGEs are simultaneously added to the evaluation standard of the efficacy of anti-glycation substances, an effective evaluation scheme is constructed, and the scheme is closer to the real skin microenvironment.

[0021] The application is based on the use of extracellular protein-glycation reaction model and intracellular protein-glycation reaction model to construct an intracellular and extracellular protein detection model, and a multi-layer linkage and multi-dimensional in-vitro anti-glycation effect evaluation method is established, wherein the content of non-crosslinked collagen and vimentin after the application of anti-glycation substances is detected in the cell-induced glycation model, and the efficacy is evaluated from the perspective of the action targets of intracellular and extracellular AGEs.

[0022] The application also uses a collagen contraction model on the basis of the above-mentioned evaluation scheme, detects the improvement effect of the anti-glycation substances on the inhibition of collagen contraction in the MGO-induced collagen contraction model, and more intuitively evaluates the anti-glycation effect from the visual angle.

[0023] In addition, the application also uses a cell carbonylation model on the basis of the above-mentioned evaluation scheme, and evaluates the anti-glycation effect of the active substances through the anti-stimulation effect, anti-carbonylation effect and glycation marker level detection.

[0024] The inhibition capacity of the anti-glycation substances on CML and RAGE and other glycation markers (flow cytometry detection) is detected in the cell-induced glycation model, and the efficacy is evaluated from the perspective of the glycation cell markers.

[0025] The inhibition capacity of the anti-glycation substances on cell carbonylation and the protein expression of NF-κB is detected in the cell-induced glycation model, and the efficacy is evaluated from the perspective of the inhibition of carbonylation.

[0026] The change of the levels of IL6, IL8 and other cytokines after the application of the anti-glycation substances is detected in the cell-induced glycation model, and the efficacy of the anti-glycation substances is evaluated from the perspective of the anti-stimulation.

[0027] In addition, in addition to the above-mentioned cell models, the application also links a human efficacy model, detects the change of the clinical glycation index after the application of the anti-glycation active substances, further verifies and evaluates the efficacy of the anti-glycation active substances, and provides multi-dimensional evaluation indexes and results from the perspective of the clinic.

[0028] On the other hand, the application provides the application of the above-mentioned evaluation method in the screening and / or evaluation of anti-skin glycation substances.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] (1) The application provides a multi-layer linkage evaluation method of in-vitro anti-glycation effect, which adds the action target of extracellular matrix and intracellular AGEs into the standard of anti-glycation substance efficacy evaluation, and is used for screening of efficacy raw materials, and the result is more reliable and the screening efficiency is higher.

[0031] (2) The evaluation method provided by the application can replace the clinical efficacy evaluation method, and a large amount of time and cost can be saved. The application adds the action target of extracellular matrix and intracellular AGEs into the standard of anti-glycation substance efficacy evaluation from the action principle and action target of AGEs, constructs an effective evaluation scheme, and makes it more close to the real skin microenvironment. The method can screen and verify effective anti-glycation active substances, and among them, the anti-glycation ability of Antuoyou in human efficacy evaluation is significantly better than that of other anti-glycation active substances. The clinical results are consistent with the results of cell model evaluation, which shows that the cell model evaluation method provided by the application can provide meaningful reference results for molecular screening and function verification for clinical use.

[0032] (3) The application uses photoaging fibroblasts to construct a model. Since the expression level of RAGE in the photoaging model is up-regulated, more targets are provided for AGEs. At the same time, more active oxygen is generated during the construction of the photoaging model, which can accelerate cell glycation and improve the construction efficiency of the glycation cell model. Glycation in human skin is a complex process, which not only includes endogenous free active carbonyl substances, but also includes factors such as ultraviolet induction from the outside world. Therefore, using photoaging fibroblasts to construct a glycation model is more close to the real skin cell state, which can be used for evaluating anti-glycation active ingredients and screening more suitable efficacy ingredients for human skin. The glycation model constructed by the photoaging cells of the application has an advantage in efficacy evaluation at the P7 stage, that is, the glycation reaction is weak at this stage. The glycation cell model constructed by the photoaging fibroblasts of the application can screen and verify effective anti-glycation active substances, and the clinical results are consistent with the results of cell model evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0033] In all the drawings, ns represents no significant difference, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001; BLK represents a blank group that is not induced by an inducer and is not subjected to sample treatment, and Ctrl represents a control group without adding a detected substance. The numerical value on the graph represents the change rate, wherein the change rate = sample group detection value / MGO group detection value.

[0034] Figure 1 The comparison results of different generations of cells; A is the comparison of CML fluorescence intensity quantitative results, B is the comparison of COL1 fluorescence intensity quantitative results, and C is the comparison of protein carbonylation level quantitative results.

[0035] Figure 2 Cell viability after 7 days of treatment with different concentrations of MGO.

[0036] Figure 3 SA-β-GAL level after 7 days of treatment with different concentrations of MGO.

[0037] Figure 4 Fluorescence images of CML, COL1 and their fusion after treatment with different concentrations of MGO.

[0038] Figure 5 Quantitative results of fluorescence after treatment with different concentrations of MGO; A corresponds to CML and B corresponds to COL1.

[0039] Figure 6 NF-kB level after treatment with different concentrations of MGO; the left panel is the western blot detection image and the right panel is the quantitative result obtained from the western blot detection image.

[0040] Figure 7 Results comparison of different indicators after treatment with different concentrations of MGO; A is P16 protein expression, B is RAGE protein expression, C is protein carbonylation level, and D is IL-8 level.

[0041] Figure 8 Cell viability comparison after treatment with different concentrations of different test substances.

[0042] Figure 9 Cell viability comparison after treatment with different concentrations of Angiozyme.

[0043] Figure 10 CML level comparison after treatment with different concentrations of different test substances.

[0044] Figure 11 CML level comparison after treatment with different concentrations of Angiozyme.

[0045] Figure 12 RAGE level comparison after treatment with different concentrations of different test substances.

[0046] Figure 13 RAGE level comparison after treatment with different concentrations of Angiozyme.

[0047] Figure 14 Collagen level comparison after treatment with different concentrations of different test substances.

[0048] Figure 15 Collagen level comparison after treatment with different concentrations of Angiozyme.

[0049] Figure 16Collagen fluorescence images after treatment with different test substances at different concentrations.

[0050] Figure 17 Comparison of vimentin levels after treatment with different test substances at different concentrations.

[0051] Figure 18 Vimentin fluorescence images after treatment with different test substances at different concentrations.

[0052] Figure 19 Comparison of IL-8 levels after treatment with different test substances at different concentrations.

[0053] Figure 20 Comparison of protein carbonylation levels after treatment with different test substances at different concentrations.

[0054] Figure 21 Comparison of NF-kB protein levels after treatment with different test substances at different concentrations, based on the results of western blot.

[0055] Figure 22 Comparison of the inhibitory effects of different test substances on collagen contraction at different times.

[0056] Figure 23 Comparison of the detection results of AGEs values, ITA values (cell skin color evaluation index), and b values (skin yellow-blue color phase) for different treatment groups.

[0057] Figure 24 Comparison of the detection results of high-density echo values, skin melanin values, and rebound time for different treatment groups.

[0058] Figure 25 Comparison of AGEs values and high-density echo values after treatment with different test substances.

[0059] Figure 26 Comparison of b values and rebound time after treatment with different test substances.

[0060] Figure 27 Comparison of CML and RAGE levels after treatment with different test substances in Comparative Example 1; the left graph is a comparison of CML levels, and the right graph is a comparison of RAGE levels.

[0061] Figure 28 Comparison of CML and RAGE levels after treatment with different test substances in Comparative Example 2; the upper graph is a comparison of CML levels, and the lower graph is a comparison of RAGE levels. DETAILED DESCRIPTION

[0062] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to examples. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturers are adopted. If no manufacturers of all reagents or instruments are specified, the conventional products available in the market are adopted. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application.

[0063] Data analysis and statistical analysis were performed using data processing software, and single factor ANOVA test was used for significance analysis. P<0.05 represents significant difference.

[0064] The main materials involved in the present application are described as follows:

[0065] In the specific embodiments of the present application, the human primary fibroblasts used are foreskin cells from a 23-year-old male. The cells are identified according to the functional manifestations of fibroblasts collagen / MMP-1 / MMP-3 and transcriptome gene alignment. The cell number is G2020001FF.

[0066] Anguoyou is produced by Shanghai Gaoke Biological Co., Ltd. The dry flowers of Eurasian Inula are extracted by ethanol. The extract is concentrated, filtered, adsorbed by resin, eluted by ethanol, and then decolorized by activated carbon. After that, it is mixed with hydroxypropyl cyclodextrin at a ratio of 1:9, dissolved, and then spray dried to obtain.

[0067] Salvia miltiorrhiza extract: dry root and rhizome extract of Salvia miltiorrhiza of Labiatae, CAS No.: 79483-68-4.

[0068] Example 1: Construction of a glycation reaction model of photoaged fibroblasts

[0069] The construction method of photoaged cells is as follows:

[0070] 1) The human primary fibroblasts were inoculated at a density of 8×10 5The P1 generation cells are cultured in 10 cm cell culture dishes, and are cultured at 37 DEG C and 5% CO2, and are irradiated with UVA and changed on the second day and the fourth day of culture, and are cultured to the seventh day to obtain P1 generation cells, which are recorded as P1-UVA. The culture medium used in the culture is fibroblast complete culture medium, which comprises 10% newborn bovine serum, 1% penicillin / streptomycin / ambomycin mixed solution, and the balance is DMEM base medium. The specific operation of irradiating with UVA and changing the medium is as follows: the old culture medium in the culture dish is discarded, washed once with PBS, then fresh PBS is added, the UVA irradiation instrument is closely attached to the cell culture dish, and irradiation is performed for 10 min with a dose of 14.4 J; after irradiation, the PBS is discarded, fresh complete culture medium is added, and the culture is continued.

[0071] 2) The P1 generation cells are subcultured and subjected to the same treatment as in step 1), and the foregoing steps are repeated to finally obtain P2-P18 generation cells, which are recorded as UVA-P2-UVA-P18. The expression levels of glycation (CML), protein carbonylation and collagen in P4, P7 and P16 cells of the photoaging UVA-FF are detected, and the results are shown in Table 1. Figure 1 As shown in Table 1, in the photoaging fibroblasts, COL1 is down-regulated, and the levels of CML and protein carbonylation are significantly up-regulated, indicating that the photoaging fibroblasts themselves have a certain glycation basis, and have a higher construction efficiency when constructing a cell glycation model.

[0072] The present application uses UVA-FF cells (photoaging fibroblasts), which are cultured to P7 (a period with weak glycation reaction), and the inducer is MGO (methylglyoxal). The cell activity (CCK8), carbonylated protein (biochemical method), CML, RAGE (cell flow cytometry), type I collagen (WB or ELISA), SASP (IL6 or IL8 measured by ELISA method), NF-kB (WB or immunofluorescence) and other indicators are detected at different treatment times, and the results of the index detection have verified that the photoaging fibroblast glycation reaction model is successfully constructed.

[0073] (1) The CCK8 detection method is as follows:

[0074] S1. The primary P7-UVA cells are plated into 96-well plates at 8000 cells per well.

[0075] S2. Different final concentrations of MGO inducer compounds are treated, which are 0.5 mM, 0.6 mM, 0.8 mM and 1 mM. After dilution of the culture solution by 1000 times, the cells are added, and the samples are collected on the seventh day of treatment for CCK8 detection.

[0076] (2) The SA-β-GAL detection method is as follows:

[0077] S1. Plate primary P7-UVA cells into 12-well plates at 8000 cells per well;

[0078] S2. Treat with MGO at different final concentrations, 0.5 mM, 0.6 mM, 0.8 mM and 1 mM, respectively. After dilution 1000 times, add to cells. Collect samples on the 7th day of treatment for SA-β-GAL detection.

[0079] (3) The detection method of AGEs-RAGE pathway related indicators is as follows:

[0080] S1. Plate primary P7-UVA cells into 96-well plates at 8000 cells per well;

[0081] S2. Treat with MGO at different final concentrations, 0.5 mM, 0.6 mM, 0.8 mM and 1 mM, respectively. After dilution 1000 times, add to cells. Collect samples on the 7th day of treatment for CML and collagen immunofluorescence staining detection.

[0082] and

[0083] S1. Plate primary P7-UVA cells into 12-well plates at 10000 cells per well;

[0084] S2. Treat with MGO at different final concentrations, 0.5 mM, 0.6 mM, 0.8 mM and 1 mM, respectively. After dilution 1000 times, add to cells. Collect RNA samples on the 7th day of treatment for IL6, RAGE and IL8 qPCR detection.

[0085] (4) The detection method of protein carbonylation is as follows:

[0086] S1. Plate primary P7-UVA cells into 6-cm dishes at 40000 cells per well;

[0087] S2. Treat with compounds as above. Collect samples on the 7th day of treatment for protein carbonylation detection

[0088] and

[0089] S1. Plate primary P7-UVA cells into 6-cm dishes at 40000 cells per well;

[0090] S2. Treat with compounds as above. Collect protein samples on the 7th day of treatment for protein carbonylation and NFKB detection

[0091] The detection results show that Figures 2-7), after 7 days of MGO treatment, the expression levels of IL6, RAGE and the protein carbonylation level and the IL8 content were increased. In conclusion, the model of glycation reaction of photoaging fibroblasts was successfully constructed from multiple dimensions.

[0092] Application of the glycation reaction model of photoaging fibroblasts in the evaluation of anti-glycation effect

[0093] In the present application, UVA-FF cells (photoaging fibroblasts) are used, and the cells are cultured to P7 (a period of weak glycation reaction), the inducer is 0.8 mM MGO, the treatment time is 7 days, and the cell activity, carbonylated protein (biochemical method), CML, RAGE (cell flow), type I collagen (WB or ELISA), vimentin (immunofluorescence), SASP (ELISA, IL6, IL8), and NF-kB (WB) are detected.

[0094] (1) Cell activity detection

[0095] S1. Plate the primary P7-UVA cells into a 12-well plate at 10,000 cells per well;

[0096] S2. Compound treatment:

[0097] Prepare 0.8 mM MGO culture solution, and treat according to the samples shown in Table 1 for 7 days, and change the solution once after 3 days of treatment;

[0098] Table 1 Cell activity detection

[0099]

[0100] Figure 8 and Figure 9 It is shown that the addition of anti-glycation experimental substances can alleviate the decrease in cell activity caused by MGO induction to a certain extent, and aminoguanidine and Angu Optimum have the best alleviating effect.

[0101] (2) Detection of AGEs-RAGE pathway related indicators

[0102] S1. Plate the primary P7-UVA cells into a 6-well plate at 2x10 5 cells per well;

[0103] S2. Compound treatment: Prepare 0.8 mM MGO culture solution, and treat according to the samples shown in Table 2 for 7 days, and change the solution once after 3 days of treatment; digest the cells with 0.05% trypsin, wash with PBS for 3 times, centrifuge, fix with 4% PFA for 20 minutes, and then perform CML and RAGE flow cytometry detection.

[0104] Table 2 Detection of AGEs-RAGE pathway related indicators

[0105]

[0106] Figures 10-13 It is shown that the addition of anti-glycation experimental substances can alleviate the increase of CML and RAGE content caused by glycation to a certain extent, and shows a dose-dependent trend, and the best alleviating effect is that of Antuoyou.

[0107] (3) Collagen and vimentin content detection

[0108] S1. Plate the primary P7-UVA cells into a 96-well plate at 8000 cells per well;

[0109] S2. Compound treatment: prepare 0.8 mM MGO culture solution, and treat according to the samples shown in Table 3 for seven days, and change the solution once in the middle after three days of treatment; after washing with PBS for three times, centrifuge, and fix with 4% PFA for 20 minutes before collagen and vimentin staining.

[0110] Table 3 Collagen and vimentin content detection

[0111] SEQ ID NO: Sample Sample Formulation Formulation Solvent Position Test Concentration 1 Aminoguanidine Solid Water Positive Control 0.032％、0.016％ 2 Carnosine Solid Water Experimental Group 0.125％、0.032％、0.008％ 3 Salvia miltiorrhiza extract Liquid Water Experimental Group 0.125％、0.032％、0.016％、0.008％ 4 An Tang You Solid Water Experimental Group 0.016％、0.008％、0.004％、0.002％ 5 Hydroxypropyl cyclodextrin Solid Water Excipient Control 0.015％、0.0072％、0.0036％、0.0018％、

[0112] Figures 14-16 It is shown that the addition of anti-glycation experimental substances of different concentrations can increase the content of cell collagen and alleviate AGEs-collagen crosslinking caused by glycation, and the best alleviating effect is that of Antuoyou.

[0113] Figure 17 and Figure 18 It is shown that the addition of anti-glycation experimental substances of different concentrations can increase the content of cell collagen and alleviate AGEs-collagen crosslinking caused by glycation, and the best alleviating effect is that of Antuoyou.

[0114] (4) Cytokine content detection

[0115] S1. Plate the primary P7-UVA cells into a 96-well plate at 8000 cells per well;

[0116] S2. Compound treatment: prepare 0.8 mM MGO culture solution, and treat according to the samples shown in Table 4 for seven days, and change the solution once in the middle after three days of treatment; collect the supernatant for ELISA detection of IL8

[0117] Table 4 Cytokine content detection

[0118] SEQ ID NO: Sample Sample Formulation Formulation Solvent Position Test Concentration 1 Aminoguanidine Solid Water Positive Control 0.032％、0.016％ 2 Carnosine Solid Water Experimental Group 0.125％、0.032％、0.008％ 3 Salvia miltiorrhiza extract Liquid Water Experimental Group 0.125％、0.032％、0.016％、0.008％ 4 An Tang You Solid Water Experimental Group 0.016％、0.008％、0.004％、0.002％ 5 Hydroxypropyl cyclodextrin Solid Water Excipient Control 0.015％、0.0072％、0.0036％、0.0018％

[0119] Figure 19It is shown that the addition of anti-glycation experimental substances can inhibit the accumulation of cytokine IL8, and among them, the cleaning efficiency of anti-glycation substances on cytokines is best.

[0120] (5) Protein carbonylation detection

[0121] S1. Plate the primary P7-UVA cells into 6 cm plates at 4x10 5 cells per well;

[0122] S2. Compound treatment: prepare 0.8mM MGO culture solution, and treat according to the sample shown in Table 5 for seven days, and change the solution once after three days of treatment; collect the cell protein sample, and perform protein carbonylation and NF-κB protein immunoblotting detection.

[0123] Table 5 Protein carbonylation detection

[0124] SEQ ID NO: Sample Sample Formulation Formulation Solvent Position Test Concentration 1 Aminoguanidine Solid Water Positive Control 0.016％ 2 Carnosine Solid Water Experimental Group 0.125％、0.032％、0.008％ 3 Salvia miltiorrhiza extract Liquid Water Experimental Group 0.125％、0.032％、0.008％ 4 An Tang You Solid Water Experimental Group 0.016％、0.008％、0.004％、0.002％ 5 Hydroxypropyl cyclodextrin Solid Water Excipient Control 0.015％、0.0072％、0.0036％、0.0018％

[0125] Figure 20 and Figure 21 It is shown that the addition of anti-glycation experimental substances can reduce the level of protein carbonylation and NF-kB protein, and alleviate the cell carbonylation caused by glycation.

[0126] Example 3 Construction and efficacy evaluation of collagen contraction model

[0127] Test object: 1.5% collagen solution

[0128] Inducing agent: MGO (0.8mM, prepared with sterile water)

[0129] Test scheme:

[0130] 1. Add fibroblasts suspended in 1mL DMEM to the collagen solution, and transfer 2mL of the obtained fibroblast collagen solution to each tissue culture dish (diameter 35mm) to obtain 1.5% collagen solution and 1.5x10 5 / mL fibroblasts.

[0131] 2. After placing in a 37°C incubator for 1h, use FPCL (fibroblast-filled collagen lattice) to solidify.

[0132] 3. Then, add anti-glycation substances to the solidified collagen in the efficacy experimental group, and use 2mL DMEM (10% NBCS) in the model control group.

[0133] 4. Add 2mL DMEM containing 800μM MGO to the collagen contraction model, that is, the experimental control group.

[0134] 5. Efficacy experiment group, add anti-glycation substances to DMEM together with MGO to make the final concentration of MGO 800 μM. Anti-glycation substances include: 0.032% aminoguanidine, 0.032% carnosine, 1% salvia miltiorrhiza extract, 0.05% and 0.025% An Tang You.

[0135] 6. On the second day, the collagen gel was separated from the cell culture dish to produce floating FPCL, and the median diameter was measured.

[0136] 7. Every other day, 1 mL of medium was replaced with 1 mL of fresh medium with or without test substances. From the first day, photographs were taken every day for 4 consecutive days, and once on the 6th day, and incubation was stopped after 6 days.

[0137] From Figure 22 It can be seen that various anti-glycation substances have an improvement effect on the MGO-induced collagen contraction inhibition model, and An Tang You has a significant improvement effect on the MGO-induced collagen contraction inhibition.

[0138] Example 4 Anti-glycation human efficacy evaluation

[0139] The reference standard of this scheme is: T / ZHCA 026-2023 Cosmetics Anti-glycation Human Test Method.

[0140] The test scheme includes:

[0141] 1. Matrix group (containing 0.45% hydroxypropyl cyclodextrin) or 0.5% An Tang You sample group, half-face control;

[0142] 2. 0.1% An Tang You group or 0.1% carnosine group, or half-face control;

[0143] The efficacy indicators were detected on the 0th, 14th, 28th, and 56th days of the test, and the subjects were 18-60 years old, healthy men or women.

[0144] Figures 23-26 The results of skin glycation indicators (including AGEs value), melanin value, skin elasticity detection, and skin yellowness detection are shown, wherein An Tang You can effectively reduce the content of AGEs, improve the brightness of the skin, and promote the recovery of skin elasticity, and the effect is proportional to the application time. An Tang You has more significant advantages in improving skin glycation, reducing skin yellowness, and promoting the recovery of skin elasticity compared with carnosine.

[0145] Based on the above results, it is found that An Tang You has more significant anti-glycation effects in cell models, collagen models, and clinical evaluations, i.e., the evaluation results of the cell model are consistent with the human efficacy evaluation results, proving that the cell model evaluation method provided by the present application can predict the human efficacy of anti-glycation active molecules, and replace the clinical evaluation method.

[0146] Comparative Example 1

[0147] According to the method of Examples 1-3, the photoaging fibroblast model is replaced by normally cultured fibroblast model, and after being cultured to P7, the same procedure is followed, the sugarization is induced, the fibroblast sugarization model is constructed, and the carnosine, An Tang You and Danshen extract are added for treatment, and the CML, RAGE (cell flow) are detected, and according to the results of Figure 27 , it is found that after being replaced by normally cultured fibroblast, the degree of alleviation of cell sugarization by carnosine, An Tang You and Danshen extract is similar, and the anti-sugar effect is equivalent.

[0148] Comparative Example 2

[0149] According to the method of Examples 1-3, the P7 stage photoaging fibroblast model is replaced by P4 stage and P16 stage photoaging fibroblast model, and the same procedure is followed, the sugarization is induced, the fibroblast sugarization model is constructed, and the carnosine, An Tang You and Danshen extract are added for treatment, and the CML, RAGE (cell flow) are detected, and according to the results of Figure 28 , it is found that the P16 stage photoaging fibroblast model treated by carnosine, An Tang You and Danshen extract has no obvious improvement in cell sugarization, and the P4 stage photoaging fibroblast model treated by carnosine, An Tang You and Danshen extract has similar degree of alleviation of cell sugarization by carnosine, An Tang You and Danshen extract, and the anti-sugar effect is equivalent.

[0150] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by ordinary skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for evaluating the in vitro anti-glycation effect, characterized in that, A photo-aged fibroblast model was used. After induction with an inducer, the anti-glycation was evaluated by detecting the content of extracellular and intracellular proteins after the application of the test substance. The inducer included high concentrations of glucose, active carbonyl substances, and AGE-BSA. The extracellular protein included collagen, and the intracellular protein included vimentin.

2. The evaluation method according to claim 1, characterized in that, The inducing agent is an active carbonyl substance, which is methylglyoxal; the extracellular protein is type I collagen, and the intracellular protein is vimentin.

3. The evaluation method according to claim 1, characterized in that, The method for preparing the photo-aged fibroblast model includes the following steps: S1. Human primary fibroblasts were irradiated with UVA and the medium was changed to obtain P1 generation cells. S2. Repeat step S1 with P1 generation cells to obtain P7 generation cells; S3. P7 generation cells were induced with active carbonyl substances, and cell biochemical indicators were detected at different treatment times to verify whether the model was successfully constructed. The active carbonyl substance includes methylglyoxal, and the biochemical indicators include the detection of cell activity level, glycation marker level, cytokine level, cell carbonylation level, or NF-κB protein expression level.

4. The evaluation method according to any one of claims 1-3, characterized in that, The evaluation method also includes using collagen contraction models, cell carbonylation models, or human efficacy models for detection.

5. The evaluation method according to claim 4, characterized in that, The detection includes detecting cell viability levels, glycation marker levels, inflammatory factor levels, cell carbonylation levels, or NF-κB protein expression levels.

6. The evaluation method according to claim 5, characterized in that, The cell viability level was obtained by detecting CCK8.

7. The evaluation method according to claim 5, characterized in that, The glycation markers include CML and RAGE.

8. The evaluation method according to claim 5, characterized in that, The cytokines include IL6 and IL8.

9. The evaluation method according to claim 5, characterized in that, The cell carbonylation level was obtained by detecting carbonylated proteins.

10. The application of the evaluation method according to any one of claims 1-9 in the screening and / or evaluation of anti-glycation substances.

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