CN110632303A

CN110632303A - Method for evaluating skin anti-aging efficacy based on combination of enzymology system and multiple in-vitro models

Info

Publication number: CN110632303A
Application number: CN201910799522.5A
Authority: CN
Inventors: 杜娟; 程树军; 冯鉴鸿
Original assignee: Guangzhou Huadai Biological Technology Co Ltd
Current assignee: Guangzhou Huadai Biological Technology Co Ltd
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2019-12-31

Abstract

The invention discloses a method for evaluating skin anti-aging efficacy by combining multiple in-vitro models based on an enzymology system, which comprises the following steps: (one) chemical level-based prediction of anti-aging efficacy; (II) predicting anti-aging efficacy based on cell level; (III) anti-aging efficacy prediction based on the 3D skin model; (IV) establishing a prediction model for evaluating the skin anti-aging efficacy by combining a plurality of in-vitro models based on an enzymology system; the method can comprehensively reflect the multi-level expression of the tested substance in an enzymology system, can carry out non-animal test, can comprehensively predict the anti-aging efficacy of the tested substance, has rich data information, and can be widely applied.

Description

Method for evaluating skin anti-aging efficacy based on combination of enzymology system and multiple in-vitro models

Technical Field

The invention belongs to the technical field of skin anti-aging evaluation, and particularly relates to a method for evaluating skin anti-aging efficacy based on combination of an enzymology system and a plurality of in-vitro models.

Background

The skin is used as a barrier for the human body to contact with the outside, and plays an important role in protecting the health of the human body. Skin aging is a complex process with a combination of multiple factors. Skin aging is classified into intrinsic aging and extrinsic aging, wherein natural aging caused by aging is intrinsic aging. It is mainly manifested as dry skin, wrinkles, sagging and drooping. Exogenous aging is mainly caused by environmental factors, including ultraviolet radiation, air pollutants, fine particulate matter (PM2.5, P10), ozone, aerosol, smoking, exposure to chemically harmful substances, and the like. Among them, ultraviolet radiation and PM2.5 cause extrinsic major factors of skin aging, and skin aging caused by long-term exposure to ultraviolet light is also called skin photoaging. Usually manifested as skin pigmentation, leatheriness, dryness, deeper wrinkles.

The endogenous mechanisms of skin aging are very complex, and the representative aging theories mainly include a free radical theory, an injury theory, a cross-linking theory, a neuroendocrine theory and a telomere theory. The exogenous process is mainly caused by ultraviolet irradiation, and the generated ways mainly comprise induction of activation of a protein kinase signal channel, induction of generation of active oxygen, activation of epidermal growth factors and induction of expression of metalloproteinases. The specific action mechanism is as follows: after the action of ultraviolet rays, the level of reactive oxygen species in skin tissue is significantly increased, directly resulting in DNA damage or inhibition of tyrosine phosphatase (PTP), and activation of Matrix Metalloproteinase (MMP) genes. Active protein-1 (AP-1) is a transcription factor complex comprising a c-Fos and c-Jun hybrid dimer, and ultraviolet light can also directly up-regulate the transcription factor c-Jun and down-regulate Retinoic Acid (RA) receptors, so that the inhibition effect of RA on AP-1 is reduced, and excessive activation of AP-1 causes MMP expression increase and collagen degradation. In addition, ultraviolet light can also alter nuclear factor kappa B (NF-kappa B) and transforming growth factor beta (TGF-beta) expression, and the expression of these molecules is related to collagen synthesis, degradation and inflammatory cytokine production, wherein the change of various enzyme activities such as hyaluronidase, collagenase and the like are involved. In addition, the absorption of ultraviolet light by the skin can directly result in oxidative damage to cells, including cell walls, lipid membranes, mitochondria and DNA, ultimately leading to photoaging of the skin.

Skin anti-aging has become a hotspot of research on medicines and skin care cosmetics, and a plurality of products for promoting and delaying skin aging appear on the market, but a standardized evaluation system for judging whether the product can achieve the declared effect or not is not provided so far. At present, for the anti-aging efficacy of raw materials or products in cosmetics, corresponding in-vitro methods are not required to evaluate the efficacy by regulations, and human body evaluation and animal experiments are more adopted. If the safety and effectiveness of the product can be preliminarily evaluated by adopting an in-vitro method before human body evaluation or animal experiments, the method not only can provide support for laboratory data, but also is beneficial to improving the development efficiency of the product, and has better guarantee for subsequent human body evaluation and product marketing. Therefore, the establishment of a standardized anti-aging in-vitro detection screening system and method is beneficial to the evaluation of the efficacy of cosmetic raw materials and products.

Pharmacological and toxicological testing using appropriate biological systems (cells, animal or human tissues) is a need for health safety assessment of cosmetics, pharmaceuticals, food additives and raw materials, pesticides, biologicals, chemicals. In the safety and effectiveness evaluation of personal care products, the traditional evaluation experiment is usually carried out by animals, the test period is long, the cost is high, great pain can be caused to the animals, and the correlation between the animal test result and human is poor in view of the difference between the skin structure function of the animals and the skin of the animals. In addition, the object of human body evaluation is a human body, and ethical, safety, scientificity and the like are inevitably faced with. For the last 20 years, cosmetic skin safety and efficacy testing has gradually shifted to toxicology systems no longer based on animal models, but rather to skin models derived from normal human skin cells or tissues, or reconstructed in vitro using human skin cells.

In vitro anti-aging efficacy tests can be divided into three dimensions, chemical, cellular and 3D skin model levels, depending on the system used. Chemical tests are mainly divided into two main categories, one is to aim at free radicals playing a key role in the stress process, and the capacity of detecting the reduction of the level of the free radicals or eliminating the free radicals is taken as evidence of anti-aging efficacy. For example, the free radical scavenging ability test (DPPH) is a chemical method, and the test system is formed by mixing DPPH with a test substance, comparing the absorbance after 30min incubation, and expressing the scavenging efficiency of free radicals of the test substance as the percentage of DPPH decolorization. The other is to test the inhibition of the test object on elastase, matrix metalloproteinase, collagenase and hyaluronidase and to evaluate the anti-aging efficacy of the product on the cleaning capacity of advanced glycosylation end products. The chemical test is carried out in a laboratory artificial simulation environment, the reaction system is single, and the chemical test result is different from the in-vivo actual application effect because a living biological system is not involved.

The cell test adopts human primary fibroblasts and keratinocytes, and the cell line is close to cells derived from normal human bodies in the aspects of morphology and gene structure, so that the primary separated skin cells can effectively represent the actual situation of use of the human bodies. For example, the elastase inhibition assay is a cellular assay using physical means (UVA or UVB irradiation) or chemical means (H)₂O₂) And (3) establishing a cell aging model, adding a test object for protection before the aging model is carried out, and calculating the inhibition rate of the elastase. The cell test is based on two-dimensional level screening of products, and has the advantages of simple operation, low cost and large-scale application. However, the planar growth of cells cannot construct a true three-dimensional microenvironment and cannot reflect the transdermal property of the sample, so further verification is required.

The tissue engineering skin reconstructed in vitro is another way for evaluating toxicity and efficacy, at present, skin models are divided into an epidermis model, a dermis model and a full-layer model according to complexity, and the epidermis model is relatively common and is mainly used for medicines, cosmetics, skin irritation tests and the like. For example, Bila et al applied a moisturizer to an EpiDerm model for free radical absorption testing. A few of the three-dimensional reconstructed tissue engineering skins are commercially available, a material source is provided for evaluating the anti-aging effect of the skin, but the price is high, large-scale application cannot be realized in a short period, and the anti-aging full-layer skin model has the defects of immature multi-cell co-culture technology, simpler tissue structure, poor differentiation of each cell layer, irregular expression of markers and the like, so that further improvement is needed.

The in vitro method for evaluating the anti-aging efficacy of the three-dimensional cosmetic raw materials and the final product has certain limitations, lacks weight analysis and decision strategies, and needs to deeply research and establish a layered gradual model or develop a feasible integration test strategy. In addition, the aging mechanism is very complex, and the selection and reasonable combination of evaluation endpoints are two major factors in the practicability of the solution. The anti-aging strategy based on the enzymology system is based on the limited zymogram change in the aging process, and key enzymes are selected for combined test. Provides a solution for evaluating in-vitro anti-aging product efficacy products, and has important practical value.

Disclosure of Invention

The invention aims to provide a method for evaluating skin anti-aging efficacy by combining a plurality of in-vitro models based on an enzymology system, which combines three-dimensional in-vitro systems of a chemical test, a cell test and a 3D skin model test based on an enzymology principle in the skin aging process, establishes a weight analysis and decision strategy, screens raw materials such as cosmetics and plant extracts and products for anti-aging safety and efficacy evaluation in the enzymology models at different levels, and has important significance for realizing preliminary screening of product efficacy, improving the accuracy of evaluation and saving the cost of detection.

The above object of the present invention is achieved by the following technical solutions: a method for evaluating skin anti-aging efficacy based on an enzymology system combined with a plurality of in vitro models comprises the following steps:

(one) prediction of anti-aging efficacy at the chemical level based on enzymatic systems:

adopting an elastase inhibition test, a collagenase inhibition test, a matrix metalloproteinase-1 inhibition test or a hyaluronidase inhibition test to determine whether the tested substance has an anti-aging effect, setting the tested substances with different concentrations, adding the different enzymes, wherein the tested substances with different concentrations and the enzyme level change to present a dose-effect relationship, and the change level has statistical significance (P is less than 0.05), which shows that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect;

(II) predicting anti-aging efficacy at the cellular level based on an enzymatic system:

(2.1) isolation, culture and characterization of primary cells from normal human skin: separating, culturing and identifying an isolated foreskin as a raw material to obtain primary cells of normal human skin;

(2.2) preparation of the test substance and determination of the concentration of the test substance: preparing a group of solution with concentration gradient from a test object, when the cell density of normal human primary cells is up to 80%, adding the group of test objects with concentration gradient into a plurality of normal human primary cells respectively to form a test object group, setting blank control and negative control respectively, testing the activity of the cells according to an MTT method, testing the absorbance of each group by using an enzyme-labeling instrument according to a formula: the cell activity is (test substance absorbance value-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) × 100%, the test substance concentration with the cell activity more than 80% is selected as the highest test concentration, and the highest test concentration or 2-3 concentrations which are continuously diluted downwards are used for testing;

(2.3) determination of ultraviolet-inducing radiation dose: setting UVA or UVB irradiation quantity with gradient when the cell density of the primary normal human cells is 80%, respectively irradiating a plurality of primary normal human cells to form irradiation groups according to the UVA or UVB irradiation quantity with gradient, setting blank control and negative control, testing the activity of the cells according to an MTT method, testing the absorbance of the irradiation groups, the blank control and the negative control by using a microplate reader, and according to a formula: the cell activity (the absorbance value of an irradiation group-the absorbance value of a blank control)/(the absorbance value of a negative control-the absorbance value of a blank control) is multiplied by 100 percent, and an irradiation dose with the cell activity of 70 percent is selected for carrying out an anti-aging effect test;

(2.4) confirmation of anti-aging effect of test substance: according to the concentration of the test substance determined in the step (2.2) and the UVA or UVB irradiation dose determined in the step (2.3), when the primary cells of normal people grow to 80%, respectively setting up a blank control, a negative control, a positive control, an irradiation group and an experimental group, testing the levels of elastase, collagenase, matrix metalloproteinase or hyaluronidase in the same step (one) of each group by using an enzyme labeling instrument, and judging the anti-aging effect of each product according to the test result, wherein compared with the irradiation group, the test substance with different concentrations and the level change of cellular enzyme present a dose-effect relationship, and one or more concentration change levels have a statistical significance (P <0.05), which indicates that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect;

(III) anti-aging efficacy prediction in 3D skin models based on enzymatic systems:

(3.1)3D skin model establishment: 3D skin models were purchased or self-contained in the laboratory according to conventional methods;

(3.2) preparation of the test substance and determination of the concentration of the test substance: the steps are the same except that the 3D skin model is adopted (2.2);

(3.3) determination of ultraviolet-inducing radiation dose: the steps are the same except that the 3D skin model is adopted (2.3);

(3.4) confirmation of anti-aging effect of test substance: the same steps are carried out except that the 3D skin model is adopted (2.4);

(IV) establishing a prediction model for evaluating the skin anti-aging efficacy by combining various in-vitro methods based on an enzymology system:

in the method for evaluating the anti-aging efficacy of skin based on the combination of various in vitro models of an enzymology system:

optionally, the test substance in the steps (one) to (three) of the present invention is a raw material, a medicine or a product that may have an anti-aging effect on the skin, for example, the test substance is a cosmetic, a plant extract, a medicine, a food additive, a pesticide, a biological product or a chemical.

Alternatively, the solvent for dissolving the test substance may be MEM medium/DMEM medium, PBS, serum-free medium, dimethyl sulfoxide, or absolute ethanol in order of preference, and the specific solvent and dissolving method are selected according to the characteristics of the test substance when applied.

Alternatively, the present invention may employ any one of the following conventional elastase inhibition assay, collagenase inhibition assay, matrix metalloproteinase-1 inhibition assay, and hyaluronidase inhibition assay to determine whether a subject has anti-aging effects.

Among them, the following are listed as one preferred embodiment:

elastase inhibition assay: mixing 100 μ L of 0.2mol/L tris-HCl buffer (Ph 8.0), 25 μ L of 10mmol/L MAAPVN (N-methoxysuccinyl-alanine-proline-valine-4-nitroaniline, english name N- (methoxysuccinyl) -ala-pro-val-4-nitroanilide, which is a substrate for elastase) and 50 μ L of the test substance, and incubating at 25 ℃ for 15 min; further 25. mu.L of elastase (highest in activity) at 0.3U/mL was added and incubation continued for 15 min. The OD value was measured by a microplate reader at 410nm, and the inhibition ratio was calculated by the following formula. EGCG (Epigallocatechin gallate) was the positive control. The inhibition rate (%) (control absorbance-sample absorbance)/control absorbance) x 100%. The test substance with different concentrations and the change of the enzyme level show a dose-effect relationship, and the change level has statistical significance (P <0.05), which shows that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect.

Collagenase inhibition test: the amount of peptide after fragmentation of the test substance by collagenase was monitored by ninhydrin reaction, and collagen I (commercially available collagen I, derived from bovine achilles tendon or mouse tail, which is a substrate for collagenase) was mixed with TES (pH7.5, containing Tris-HCl, EDTA and SDS) buffer, diluted test substance, and collagen IV, incubated at 37 ℃ for 5 hours, and then centrifuged at 2000rpm for 5 minutes. Collecting supernatant, mixing with ninhydrin, heating at 80 deg.C for 10min, and cooling. The solution was mixed with isopropanol at a ratio of 1:1 and centrifuged at 12000rpm for 10min at 4 ℃. The supernatant was added to a 96-well plate, and the absorbance thereof was measured at 600 nm. And calculating the inhibition rate. EGCG was used as a positive control. The inhibition rate (%) (control absorbance-sample absorbance)/control absorbance) x 100%. The test substance with different concentrations and the change of the enzyme level show a dose-effect relationship, and the change level has statistical significance (P <0.05), which shows that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect.

Matrix metalloproteinase-1 inhibition assay: mu.L of the inhibitor NNGH (N-isobutyl-N-4-methoxyphenylsulfonyl-glycyl hydroxamic acid, substrate for matrix metalloproteinase-1) was diluted with 200. mu.L of buffer. mu.L of substrate was added to each well, followed by 20. mu.L of diluted matrix metalloproteinase. The mixture was warmed to room temperature. The assay buffer was pipetted into the plates as follows: blank control (without MMP1) was 90 μ L, control (without inhibitor) was 70 μ L, and inhibitor (NNGH) was 50 μ L. After equilibrating the plates to 37 ℃, 20 μ L of MMP1 (excluding the blank) was added to each well, followed by 20 μ L of inhibitor in NNGH inhibitor wells. After addition of the inhibitor to be tested to each well, the plates were incubated at 37 ℃ for 30min for reaction of the inhibitor with the enzyme. Finally, 10. mu.L of substrate was added to each well and read every 1min interval at 412nm for 10 min. Calculating an inhibition rate: the inhibition rate (%) (control absorbance-sample absorbance)/control absorbance) x 100%. The test substance with different concentrations and the change of the enzyme level show a dose-effect relationship, and the change level has statistical significance (P <0.05), which shows that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect.

Hyaluronidase inhibition assay: mu.L of hyaluronidase, 100. mu.L of PBS buffer, 50mmol/L of NaCl and 0.01% BSA were mixed with 50. mu.L of the sample solution to prepare a culture medium, and incubated in the culture medium at 37 ℃ for 10 min. Then, 100. mu.L of hyaluronidase solution (0.03%, 300mmol/L phosphate solution, pH 5.35) was added as the start of the reaction, and the mixture was incubated at 37 ℃ for 45 min. Unhydrolyzed hyaluronic acid was precipitated with 1.0mL of acid albumin solution (0.1% BSA solution 24mmol/L sodium acetate and 79mmol/L acetic acid, pH 3.75). The mixture was then left at room temperature for 10min, and the absorbance of the mixture was measured at 600nm with a microplate reader. The test group without enzyme was the control for maximum inhibition and ascorbic acid was the positive control. Calculating an inhibition rate: the inhibition rate (%) (control absorbance-sample absorbance)/control absorbance) x 100%. The test substance with different concentrations and the change of the enzyme level show a dose-effect relationship, and the change level has statistical significance (P <0.05), which shows that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect.

Optionally, the separating and culturing of the normal human primary cells in the step (2.1) refers to separating and culturing to obtain normal human primary cells by taking an isolated foreskin as a raw material; the normal human primary cells are normal human primary keratinocytes and normal human primary fibroblasts, and the cells are isolated and cultured by a conventional method.

The cell identification of the primary cells of the normal human skin refers to the characteristic identification of the primary human keratinocytes and fibroblasts, and can be carried out by a characteristic protein immunohistochemistry or a genetic method. The conventional keratinocyte immunohistochemical identification is positive for broad-spectrum keratin (P-CK) and positive for human keratin 17(CK 17); the human fibroblast immunohistochemical identification should be positive for human Vimentin (Vimentin).

Optionally, the experimental groups in step (2.2) to step (2.3) are respectively: the blank control group contained only cell culture fluid; the test object group contains a test object, normal human primary cells and a cell culture solution; the negative control group contained normal human primary cells and cell culture medium.

Optionally, when the reagent is formulated and the reagent concentration is determined in step (2.2), as a preferred embodiment, the following process may be specifically adopted: when the cell density in the cell bottle is as high as 80%, the cell density is 1 × 10⁴The cells are paved in a 96-well plate, the 96-well plate is put in an incubator to be incubated for 24 hours, the concentration range is set according to the physical and chemical characteristics of a tested object, the diluted skin anti-aging substance is added into the 96-well plate, a blank control, a negative control and a tested object group are arranged, and each group is provided with 6 multiple wells. For example, the final concentration of the cytotoxic substance such as DMSO and ethanol in the diluted solution is not higher than 0.5%. Adding skin anti-aging substances, placing the mixture into an incubator, continuously incubating for 18-24 hours, adding 20 mu L of 5mg/ml MTT solution into each hole after the incubation is finished, adding DMSO to dissolve crystals after the mixture acts for 4 hours, uniformly mixing and oscillating for 10min, and measuring the reading of each hole at the position of 570nm by using a microplate reader.

Calculating the activity of the cells: cell activity ═ (skin antioxidant well OD value-blank control well OD value)/(negative control well OD value-blank control well OD value);

the test is carried out by selecting the concentration of the test object with the cell activity of more than 80 percent.

The research on the anti-aging effect of the skin needs to be carried out under the condition that external factors cause the oxidative damage of a skin model, wherein the external factors comprise ultraviolet irradiation or chemical agent damage, and the ultraviolet irradiation is adopted in the invention.

Optionally, upon determination of the uv-induced radiation dose in step (2.3), a uv-induced skin cell photodamage model may be established. As a preferred embodiment, the following process may be specifically adopted: when the cell density in the cell bottle is as high as 80%, the cell density is 1 × 10⁴Cells were plated in 96-well plates, incubated in an incubator for 24 hours, medium was replaced with PBS, 96-well plates were placed on 4 ℃ flat ice bags, UVA or UVB irradiation intensity was measured using a luminometer, and 6 replicates per irradiation dose were set. And after irradiation, replacing PBS with a new culture medium, placing the PBS into an incubator to continuously incubate for 18-24 hours, adding 20 mu L of 5mg/mL MTT solution into each hole after incubation is finished, acting for 4 hours, adding DMSO to dissolve crystals, uniformly mixing and oscillating for 10min, and measuring the reading of each hole at the position of 570nm by using the wavelength of a microplate reader.

Calculating the activity of the cells: the cell activity (uv-irradiated normal cell OD value-blank control well OD value)/(negative control well OD value-blank control well OD value) × 100%. Selecting irradiation dose with cell activity of 70% to perform anti-aging effect test.

The MTT is called 3- (4,5) -dimethylthiohiazolazo (-z-y1) -3, 5-di-phenylytrazoliumromide in English, the chemical name of Chinese is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide, the trade name is: thiazole blue, a yellow dye, is a commonly used method for detecting cell survival and growth by MTT colorimetry.

Alternatively, in the step (2.4), when the anti-aging effect of the test object is confirmed, according to the concentration of the skin anti-aging substance determined in the step (2.2) and the UVA or UVB irradiation dose determined in the step (2.3), when the normal primary human cells grow to 80%, a non-irradiated group (negative control), a skin anti-aging test object group (test object group) added after irradiation and a skin anti-aging test object group (model group) not added after irradiation are set up, after the UVA or UVB irradiation is turned on, the levels of elastase, matrix metalloproteinase, collagenase and hyaluronidase in each group are tested, and according to the test results, the anti-aging effect of the product is judged.

As a preferred embodiment, the cell-level anti-aging process specifically can adopt the following processes: waiting for the cells to be at 75cm²When the cell density in the cell flask of (1) is 80%, the cell density is 6.4X 10⁵The cells are plated in 12-well plates, incubated in an incubator for 24 hours, set up unirradiated dishes, irradiated and then irradiated with and without anti-aging test dishes. The illuminometer measures UVA or UVB irradiation intensity, a culture dish is cleaned by normal-temperature PBS for 1 time before irradiation, the unirradiated dish and the irradiation dish are processed consistently, the irradiation dish uses the PBS to replace a culture medium, and the unirradiated dish is placed in the incubator after a new culture medium is replaced. Placing the irradiation dish on a smooth ice bag at 4 ℃, adding the anti-aging test object after irradiation, keeping out of the sun, and continuously incubating in a cell culture box for 18-24 hours.

According to the test results of the levels of the cellular elastase, the collagenase, the matrix metalloproteinase or the hyaluronidase in each group in the step (2.4), the anti-aging program of the test object is judged as follows through statistical analysis: compared with model control (irradiation group), the test substance with different concentrations and the change of cellular enzyme level show a dose-effect relationship, one or more concentration change levels have statistical significance (P <0.05), and the substance is predicted to have the anti-aging effect, otherwise, the substance does not have the anti-aging effect.

Optionally, in the steps (2.2) - (2.4), the absorbance of each group is respectively tested at 550-570 nm by using a microplate reader.

Optionally, the irradiation dose determined in step (2.3) is: the UVA radiation intensity is 8-10J/cm²The UVB irradiation intensity is 0.6-0.8J/cm²。

Optionally, the culture solution containing normal human primary cells and normal human primary cells in the negative control in steps (2.2) to (2.4) is not irradiated by ultraviolet rays, and the culture solution containing only normal human primary cells in the blank control is not irradiated by ultraviolet rays; the positive control in the step (2.4) contains known anti-aging substances, normal human primary cells and culture solution of the normal human primary cells and is irradiated by ultraviolet rays, the irradiation group contains culture solution of the normal human primary cells and is irradiated by ultraviolet rays, and the test object group contains test objects, culture solution of the normal human primary cells and is irradiated by ultraviolet rays.

Wherein, the negative control (non-irradiated group), the irradiated group (model group without added test substance), the test substance group (added corresponding test substance after irradiation), and the positive control (added positive substance after irradiation; added EGCG after irradiation). These words may correspond to one another.

Optionally, the 3D skin model is purchased or lab-owned in step (3.1), but the model should meet quality control criteria. The kit is 12-hole or 24-hole, a culture plate containing the skin model is taken, 1-2mL of maintenance medium is added into each hole, the skin model is transferred into culture holes containing fresh medium and is cultured overnight at 37 ℃ under 5% CO 2. The quality control standard is as follows: the production date, batch number, pH value, kit temperature, temperature of detection culture solution, temperature of maintenance culture solution and the like of the commercial kit are checked.

Optionally, the step (3.2) of preparing the test substance and determining the concentration of the test substance specifically includes the following steps: preparing a group of solutions with concentration gradient for a tested object, forming the tested object group by the tested object, simultaneously setting a negative control group and a blank control group, taking out the skin model after the skin model reacts with MTT for 3h, respectively adding acidic isopropanol into each group, respectively measuring the absorbance of each group within the range of 550-570 nm by an enzyme-labeling instrument after keeping out of the sun overnight, and calculating the activity of the skin model according to the following formula, wherein the activity of the skin model is [ (tested object absorbance value-blank absorbance value) ] × 100%.

The research on the anti-aging effect of the skin needs to be carried out under the condition that the skin model is oxidized and damaged by external factors, wherein the external factors comprise ultraviolet irradiation or chemical reagent damage, and the concentration of a test object with the skin model activity of more than 80 percent is selected as the highest test concentration; the test is performed at the highest concentration or with 2-3 concentrations serially diluted down.

Optionally, the determination of the dose of the ultraviolet-induced radiation in the step (3.3) specifically includes the following steps: setting UVA or UVB irradiation amount with gradient, respectively irradiating the skin model according to the UVA or UVB irradiation amount with gradient, setting blank control and negative control, respectively testing the absorbance of the ultraviolet irradiation normal skin model, the blank control and the negative control in the wavelength range of 550 nm-570 nm by using an microplate reader, and according to the formula: skin model activity (absorbance value of ultraviolet irradiation normal skin model group-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) x 100%, radiation dose with skin model activity of 70% was selected for anti-aging effect test.

Optionally, the step (3.4) of confirming the anti-aging effect of the subject specifically includes the following steps: and (3) respectively setting a blank control, a negative control, a model control, a positive control and a test object group according to the concentration of the test object determined in the step (3.2) and the UVA or UVB irradiation dose determined in the step (3.3), testing the levels of elastase, collagenase, matrix metalloproteinase and hyaluronidase of each group by taking EGCG added before ultraviolet irradiation of a normal skin model as the positive control, and judging the anti-aging effect of each product according to the test result.

Optionally, in the steps (3.2) to (3.4), the absorbance of each group is respectively tested at 550-570 nm by a microplate reader.

Optionally, the irradiation dose determined in step (3.3) is: the UVA radiation intensity is 8-10J/cm²The UVB irradiation intensity is 0.6-0.8J/cm²。

Optionally, when the result determination in step (3.4) specifically includes: compared with a model control (irradiation group), the test substance with different concentrations and the change of the cellular enzyme level show a dose-effect relationship, and one or more concentration change levels have statistical significance (P <0.05), which indicates that the substance has the anti-aging effect.

Optionally, the culture solution containing the 3D skin model and the 3D skin model in the negative control described in the step (3.2) to the step (3.4) is not irradiated by ultraviolet rays; the blank control only contains the culture solution of the 3D skin model and is not irradiated by ultraviolet rays; the positive control in step (3.4) contains known anti-aging substances, a 3D skin model and a culture solution of the 3D skin model and is irradiated by ultraviolet rays; the irradiation group contains a 3D skin model and a culture solution of the 3D skin model and is irradiated by ultraviolet rays; the test substance group contains a test substance, a 3D skin model and a culture solution of the 3D skin model and is irradiated with ultraviolet rays.

Optionally, the known anti-aging substance is Epigallocatechin gallate (EGCG), vitamin E or vitamin C or other known anti-aging actives.

Optionally, the levels of elastase, collagenase, matrix metalloproteinase or hyaluronidase are tested in step (2.4) and in step (3.4) using an ELISA kit.

Optionally, in the step (four), based on the weight principle, the anti-aging efficacy prediction based on the 3D skin model > the anti-aging efficacy prediction based on the cellular level > the anti-aging efficacy prediction based on the chemical level, and in the case that the three anti-aging efficacy predictions are inconsistent, the anti-aging efficacy prediction with high weight is taken as a main judgment basis.

The invention has the following beneficial effects:

(1) the invention uses a plurality of normal human skin cells cultured in vitro, the culture technology is mature, the primary division proliferation capacity is strong, the standardization degree is high, the difference between batches is small, and the activity and the function are the same as those in vivo;

(2) the result obtained by using normal healthy human skin cells is more reliable than that obtained by using an animal or human cell line; the toxic effect and the anti-aging effect of the substance to be tested are evaluated from the qualitative aspect and the quantitative aspect, and the method has good repeatability;

(3) the invention combines a plurality of in vitro methods, utilizes an aging model of ultraviolet-induced skin cells to simulate the condition that human skin is exposed to ultraviolet rays, and judges whether the induced cells can be protected from photodamage by adding an anti-aging substance. The simulation method can intuitively reflect the ultraviolet ageing effect and the effect of the anti-aging substances on the skin, has high sensitivity and can meet the conventional screening test of the anti-aging substances.

(4) The method is based on the enzyme spectrum principle of photoaging, takes three dimensions of chemistry, cells and 3D skin into consideration of an evidence weight principle, comprehensively judges whether an anti-aging substance has an effect, can meet the requirement of potential toxicity detection of active raw materials, and can analyze the strength and possible mechanism of the antibody aging function.

(5) The combined test strategy established by the invention can replace the skins of living animals and human beings and can be directly used for toxicity and efficacy tests of anti-aging substances in products such as chemicals, cosmetics, medicines and the like; the combined strategy method is rapid, accurate and scientific, and can carry out scientific and effective screening on the substances.

Detailed Description

The raw materials used in the following examples, such as reagents and culture media, are all commercially available products unless otherwise specified.

Example 1 assessment of anti-aging Effect of certain brands of glucosyl rutin based on methods of chemical level, cellular level, 3D skin model

1. Chemical level based prediction of raw material anti-aging efficacy

Elastase inhibition assay: mixing 100 μ L of 0.2mol/L tris-HCl buffer (Ph 8.0), 25 μ L of 10mmol/L MAAPVN (N-methoxysuccinyl-alanine-proline-valine-4-nitroaniline) and 50 μ L of the stock dilution, and incubating at 25 ℃ for 15 min; further 25. mu.L of elastase (highest in activity) at 0.3U/mL was added and incubation continued for 15 min. The OD value was measured by a microplate reader at 410nm, and the inhibition ratio was calculated by the following formula. EGCG (Epigallocatechin gallate ) is a positive control, the inhibition rate (%) (control group absorbance-sample group absorbance)/control group absorbance) is multiplied by 100%, the change of the levels of the test substance and the enzyme at different concentrations presents a dose-effect relationship, and the change level has statistical significance (P <0.05), which indicates that the substance has the anti-aging effect.

Results of the chemical level elastase assay were analyzed as follows:

SEM: standard error of mean P < 0.05P <0.01

The glucosyl rutin has elastase inhibiting effect and antiaging effect at 50 mg/ml.

2. Cellular level based prediction of glucosyl rutin anti-aging efficacy

(1) Isolation, culture and characterization of normal human skin fibroblasts: separating, culturing and identifying in vitro foreskin as raw material to obtain normal human skin fibroblast;

(2) preparation of test substance and determination of test substance concentration: the final concentration of glucosyl rutin is 100mg/mL, and is diluted into 31.6mg/mL, 10mg/mL, 3.2mg/mL, 1mg/mL and 0.32mg/mL in sequence. Diluted test substances were added to 96-well plates, with a blank control, a negative control, and 6 replicate wells per concentration. A multichannel pipettor was used to reduce the inter-well variation. And placing the mixture in an incubator to incubate for 18-22 hours. After the incubation was completed, the plate was removed from the incubator, and 20. mu.l of 5mg/mL MTT solution (thiazole blue) was added to each well, protected from light. Placing in an incubator to continue hatching for 4 h. After incubation, DMSO (dimethyl sulfoxide) was added in an amount of 100. mu.L per well, and the mixture was placed on a shaker for 10 min. The absorbance of viable cells per well was measured using a microplate reader at 570 nm. Calculating IC by formula₈₀(i.e., the concentration of the test substance at 80% cell activity), according to the formula: cell activity ═ [ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value)]×100％。

Cytotoxicity assay: the test results were obtained as follows: the cell survival rate at 100mg/L was 48.89%, at 31.6 mg/L56.89%, at 10.01 mg/L80.91%, at 3.16 mg/L89.69%, at 1.00 mg/L92.36%, at 0.32 mg/L97.26%. The calculated glucosyl rutin is obtained₈₀The concentration of the test substance with a cell activity of more than 80% was selected as the highest concentration tested for anti-aging test at 10.58 mg/mL.

(3) Determination of dose of ultraviolet-induced radiation

When the density of primary skin fibroblasts of normal people is as high as 80%, the density is 0.8 multiplied by 10⁵Inoculating the mixture into a 96-well plate, setting 6-8J UVA irradiation quantity, irradiating a normal primary human cell group according to gradient, setting blank control and negative control, and respectively testing the absorbance of the normal primary human cell group, the blank control and the negative control in the wavelength range of 550-570 nm by using an enzyme-labeling instrument according to a formula: the cell activity (absorbance value of ultraviolet irradiation normal primary human cell group-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) x 100%, and the irradiation dose with the cell activity of 70% is selected for carrying out the anti-aging effect test;

selecting UVA 8J/cm²And (5) irradiating the powder, and performing molding treatment.

(4) Confirmation of anti-aging Effect of test substance

The concentration of the test substance determined according to the step (2) is 10.58mg/mL, and the UVA irradiation dose determined in the step (3) is 8J/cm²When the primary cells of normal people grow to 80%, respectively setting a non-irradiated group, a test object group (10.58mg/mL, 5.29mg/mL and 2.65mg/mL) added after irradiation and a non-irradiated test object group, taking EGCG (Epigallocatechin gallate) added before ultraviolet irradiation of the primary skin cells of the normal people as a positive control, testing the level of elastase of each group, and judging the anti-aging effect of glucosylrutin according to the test result. The levels of elastase are significantly reduced in the test group at different concentrations compared to the test group without post-irradiation (P)<0.05), has statistical significance, and shows that the substance has an anti-aging effect.

P <0.05 x P <0.01 compared to irradiated groups

The experimental result shows that the expression level of elastase of the test object group with different concentrations is obviously reduced (P is less than 0.05) compared with the test object group without adding the test object group after irradiation by 5.29mg/mL glucosyl rutin and 10.58mg/mlL glucosyl rutin, the statistical significance is achieved, and the glucosyl rutin has the anti-aging effect.

3. 3D skin model-based evaluation of anti-aging effect of glucosyl rutin

(1) Processing skin model, purchasing 3D skin model, taking 12-well plate, adding maintenance medium 2 mL/well into each well, transferring skin model into culture well containing fresh medium, placing at 37 deg.C and 5% CO₂The culture was carried out overnight.

(2) Preparation of test substance and determination of test substance concentration

Preparing a group of solutions with concentration gradient for a tested object, forming the tested object group for the tested object, simultaneously setting a positive control group, a negative control group and a blank control group, taking out the skin model after the skin model reacts with MTT for 3h, respectively adding acidic isopropanol into each group, respectively measuring the absorbance of each group in the range of 550-570 nm by using an enzyme labeling instrument after keeping out of the light overnight, and calculating the activity of the skin model according to the following formula, wherein the activity of the skin model is (the absorbance value of the tested object-the blank absorbance value) ÷ (the absorbance value of the negative control-the blank absorbance value) ] × 100%.

The research on the anti-aging effect of the skin needs to be carried out under the condition that the skin model is oxidized and damaged by external factors, wherein the external factors comprise ultraviolet irradiation or chemical reagent damage, the embodiment adopts the ultraviolet irradiation, and the concentration of a test substance with the activity of more than 80 percent of the skin model is selected as the highest test concentration, so that the concentration of the test substance of glucosylrutin is 1mg/mL and 10 mg/mL;

(3) determination of dose of ultraviolet-induced radiation

Setting UVA irradiation amount with gradient, respectively irradiating the skin model according to the UVA irradiation amount with gradient, setting blank control and negative control, respectively testing the absorbance of the normal skin model, the blank control and the negative control in the wavelength range of 550 nm-570 nm by using an microplate reader, and according to the formula: skin model activity (absorbance value of ultraviolet irradiation normal skin model group-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) x 100%, selecting irradiation dose with skin model activity of 70% to perform anti-aging effect test;

the experimental result shows that the selection is 10J/cm²An anti-aging effect test is performed.

(4) Confirmation of anti-aging Effect of test substance

Respectively setting a non-irradiated group, a group added with the test object after irradiation and a group not added with the test object after irradiation according to the concentration of the test object determined in the step (3) and the UVA irradiation dose determined in the step (4), adding epigallocatechin gallate before ultraviolet irradiation of a normal skin model as a positive control, testing elastase of each group, and judging the anti-aging effect of each product according to the test result. The experimental result shows that compared with the test substance group without adding the test substance after irradiation, the elastase level of the test substance group with different concentrations is obviously reduced (P is less than 0.05), thereby having statistical significance and showing that the substance has the anti-aging effect.

P <0.05 x P <0.01 compared to irradiated groups

The experimental result shows that the glucosyl rutin has the anti-aging effect at the chemical level, the cellular level and the 3D skin model, and the final experimental conclusion shows that the glucosyl rutin has the anti-aging effect.

Example 2 assessment of anti-aging Effect of certain brands of tocopherol derivatives based on chemical-level, cellular-level, 3D skin model

1. Chemical level based prediction of raw material anti-aging efficacy

Hyaluronidase inhibition assay: mu.L of hyaluronidase, 100. mu.L of PBS buffer, 50mmol/L of NaCl and 0.01% BSA were mixed with 50. mu.L of the sample solution to prepare a culture medium, and incubated in the culture medium at 37 ℃ for 10 min. Then, 100. mu.L of hyaluronidase solution (0.03%, 300mmol/L phosphate solution, pH 5.35) was added as the start of the reaction, and the mixture was incubated at 37 ℃ for 45 min. Unhydrolyzed hyaluronic acid was precipitated with 1.0mL of acid albumin solution (0.1% BSA solution 24mmol/L sodium acetate and 79mmol/L acetic acid, pH 3.75). The mixture was then left at room temperature for 10min, and the absorbance of the mixture was measured at 600nm with a microplate reader. The test group without enzyme was the control for maximum inhibition and ascorbic acid was the positive control. Calculating an inhibition rate: the inhibition rate (%) (control absorbance-sample absorbance)/control absorbance) x 100%. The test substance with different concentrations and the change of the enzyme level show a dose-effect relationship, and the change level has statistical significance (P <0.05), which indicates that the substance has the anti-aging effect.

The results of the chemical level hyaluronidase assay were analyzed as follows:

p <0.05 x P <0.01 compared to the test group without added enzyme

The experimental result shows that at the chemical level, the tocopherol derivative has no hyaluronidase inhibition effect and no anti-aging effect.

2. Tocopherol derivative anti-aging efficacy prediction based on cellular levels

(1) Isolation, culture and characterization of normal human keratinocytes: separating, culturing and identifying in vitro foreskin as raw material to obtain normal human keratinocyte;

(2) preparation of test substance and determination of test substance concentration: the final concentration of the tocopherol derivative was 100mg/mL, and the tocopherol derivative was diluted to 31.6mg/mL, 10mg/mL, 3.2mg/mL, 1mg/mL, and 0.32mg/mL in this order. Diluted test substances were added to 96-well plates, with a blank control, a negative control, and 6 replicate wells per concentration. A multichannel pipettor was used to reduce the inter-well variation. Is arranged in an incubatorAnd (4) incubating for 18-22 hours. After the incubation was completed, the plate was removed from the incubator, and 20. mu.l of 5mg/mL MTT solution (thiazole blue) was added to each well, protected from light. Placing in an incubator to continue hatching for 4 h. After incubation, DMSO (dimethyl sulfoxide) was added in an amount of 100. mu.L per well, and the mixture was placed on a shaker for 10 min. The absorbance of viable cells per well was measured using a microplate reader at 570 nm. Calculating IC by formula₈₀(i.e., the concentration of the test substance at 80% cell activity), according to the formula: cell activity ═ [ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value)]×100％

Cytotoxicity assay: the test results were obtained as follows: the cell survival rate at 100mg/L was 38.94%, 46.24% at 31.6mg/L, 65.25% at 10.01mg/L, 84.69% at 3.16mg/L, 86.36% at 1.00mg/L, and 95.32% at 0.32 mg/L. The IC80 of the tocopherol derivative is 3.28mg/mL through statistics, and the concentration of the test substance with the cell activity of more than 80% is selected as the highest test concentration of the anti-aging test.

(3) Determination of dose of ultraviolet-induced radiation

The density of normal human keratinocytes is up to 80%, 1X 10⁵Inoculating the cells into a 96-well plate, setting 0.6-0.8J UVB irradiation dose to irradiate a normal primary skin fibroblast group, setting blank control and negative control, and respectively testing the absorbances of the normal primary skin fibroblast group, the blank control and the negative control by using an microplate reader within the wavelength range of 550-570 nm according to a formula: the cell activity (absorbance value of ultraviolet irradiation normal primary human cell group-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) x 100%, and the irradiation dose with the cell activity of 70% is selected for carrying out the anti-aging effect test;

selecting UVB 0.8J/cm²And (5) irradiating the powder, and performing molding treatment.

(4) Confirmation of anti-aging Effect of test substance

The test object determined according to the step (2)The concentration is 3.28mg/mL, and the UVB irradiation dose determined in the step (3) is 0.8J/cm²When the primary cells of normal people grow to 80%, respectively setting a non-irradiated group, a test object group (3.28mg/mL, 1.64mg/mL and 0.82mg/mL) after irradiation and a non-irradiated test object group, adding EGCG (Epigallocatechin gallate) before ultraviolet irradiation of the primary skin cells of the normal people, as a positive control, testing the hyaluronidase level of each group, and judging the anti-aging effect of the tocopherol derivative according to the test result. The experimental result shows that compared with the test group without adding the test substance after irradiation, the hyaluronidase inhibition rates of the test substance groups with different concentrations have no statistical significance, and the tocopherol derivative has no anti-aging effect.

P <0.05 x P <0.01 compared to irradiated groups

3. Evaluation of anti-aging effects of tocopherol derivatives based on 3D skin model

The research on the anti-aging effect of the skin needs to be carried out under the condition that the external factors cause the oxidative damage of the skin model, wherein the external factors comprise ultraviolet irradiation or chemical agent damage, and the concentration of a test object with the activity of more than 80 percent of the skin model is selected as the highest test concentration, so that the concentration of the test object of the tocopherol derivative is 1mg/mL and 10 mg/mL.

(3) Determination of dose of ultraviolet-induced radiation

Setting UVB irradiation with gradient, respectively irradiating the skin model according to the UVB irradiation with gradient, setting blank control and negative control, respectively testing the absorbance of the normal skin model, the blank control and the negative control in the wavelength range of 550-570 nm by using an microplate reader, and according to a formula: skin model activity (absorbance value of ultraviolet irradiation normal skin model group-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) x 100%, selecting irradiation dose with skin model activity of 70% to perform anti-aging effect test;

the experimental result shows that the selection is 0.8J/cm²An anti-aging effect test is performed.

(4) Confirmation of anti-aging Effect of test substance

Respectively setting a non-irradiated group, a irradiated and non-irradiated group and an irradiated and non-irradiated group according to the concentration of the test object determined in the step (3) and the UVB irradiation dose determined in the step (4), adding epigallocatechin gallate before ultraviolet irradiation of a normal skin model as a positive control, testing hyaluronidase of each group, and judging the anti-aging effect of each product according to the test result.

P <0.05 compared to irradiated group

The experimental result shows that compared with a test group without adding the test substance after irradiation, the inhibition rates of the test substance groups with different concentrations have no statistical significance, and the tocopherol derivative has no anti-aging effect.

The tocopherol derivative has no anti-aging effect at chemical level, cellular level and 3D skin model, and the final experimental conclusion shows that the tocopherol derivative has no anti-aging effect.

Example 3 assessment of anti-aging Effect of certain brands of collagen derivatives based on methods of chemical level, cellular level, 3D skin model

1. Chemical level based prediction of raw material anti-aging efficacy

1.1 collagenase inhibition assay: the amount of peptide after fragmentation of the test substance by collagenase is monitored by ninhydrin reaction, collagen I is mixed with TES (pH7.5, containing Tris-HCl, EDTA and SDS) buffer, diluted test substance and collagen IV, incubated at 37 ℃ for 5h, and then centrifuged at 2000rpm for 5 min. Collecting supernatant, mixing with ninhydrin, heating at 80 deg.C for 10min, and cooling. The solution was mixed with isopropanol at a ratio of 1:1 and centrifuged at 12000rpm for 10min at 4 ℃. The supernatant was added to a 96-well plate, and the absorbance thereof was measured at 600 nm. And calculating the inhibition rate. EGCG (Epigallocatechin gallate) is used as a positive control, the inhibition rate (%) is [ (control group absorbance-sample group absorbance)/control group absorbance ]. times.100%, the inhibition rate level of a test object group with different concentrations is obviously increased (P <0.05), and the statistical significance is realized, so that the substance has the anti-aging effect.

Chemical level collagenase test results analysis

SEM: standard error of mean P < 0.05P <0.01

The experimental result shows that at the chemical level, the collagen derivative has the functions of inhibiting collagenase and resisting aging when the collagen derivative is 50 mg/ml.

2. Cellular level based prediction of collagen derivative anti-aging efficacy

(2) preparation of test substance and determination of test substance concentration: the final concentration of the collagen derivative was 100mg/mL, and the collagen derivative was diluted to 31.6mg/mL, 10mg/mL, 3.2mg/mL, 1mg/mL, and 0.32mg/mL in this order. Diluted test substances were added to 96-well plates, with a blank control, a negative control, and 6 replicate wells per concentration. A multichannel pipettor was used to reduce the inter-well variation. And placing the mixture in an incubator to incubate for 18-22 hours. After the incubation was completed, the plate was removed from the incubator, and 20. mu.L of 5mg/mL MTT solution (thiazole blue) was added to each well, protected from light. Placing in an incubator to continue hatching for 4 h. After incubation, DMSO (dimethyl sulfoxide) was added in an amount of 100. mu.L per well, and the mixture was placed on a shaker for 10 min. The absorbance of viable cells per well was measured using a microplate reader at 570 nm. Calculating IC by formula₈₀(i.e., the concentration of the test substance at 80% cell activity), according to the formula: cell activity ═ [ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value)]×100％

Cytotoxicity assay: the test results were obtained as follows: the cell survival rate at 100mg/L was 65.52%, 68.89% at 31.6mg/L, 95.65% at 10.01mg/L, 96.58% at 3.16mg/L, 94.23% at 1.00mg/L and 98.26% at 0.32 mg/L. The calculated glucosyl rutin is obtained₈₀The concentration of the test substance with a cellular activity of > 80% was chosen as the highest test concentration for the anti-aging test at 50.64 mg/mL.

(3) Determination of dose of ultraviolet-induced radiation

When the density of primary skin fibroblasts of normal people is as high as 80%, the density is 0.8 multiplied by 10⁵Inoculating into 96-well plate, setting 6-8J UVA irradiation amount, and irradiating according to gradientSetting a blank control and a negative control for the normal human primary cell group, respectively testing the absorbances of the normal human primary cell group, the blank control and the negative control in the wavelength range of 550-570 nm by using an enzyme-labeling instrument, and according to a formula: the cell activity (absorbance value of ultraviolet irradiation normal primary human cell group-blank control absorbance value)/(negative control absorbance value-blank control absorbance value) x 100%, and the irradiation dose with the cell activity of 70% is selected for carrying out the anti-aging effect test;

(4) Confirmation of anti-aging Effect of test substance

The concentration of the test substance determined according to the step (2) is 50.64mg/mL, and the UVA irradiation dose determined in the step (3) is 8J/cm²When the primary cells of normal people grow to 80%, respectively setting an unirradiated group, a test object adding group (50.64mg/mL, 25.32mg/mL and 12.66mg/mL) after irradiation and a test object not adding group after irradiation, taking EGCG (Epigallocatechin gallate) added before ultraviolet irradiation of the primary skin cells of normal people as a positive control, testing the collagenase level of each group, and judging the anti-aging effect of glucosyl rutin according to the test result. Compared with the test object group without adding the test object after irradiation, the test object group with different concentrations has obviously increased inhibition rate level (P)<0.05), has statistical significance, and shows that the substance has an anti-aging effect.

P <0.05 x P <0.01 compared to irradiated groups

The experimental result shows that compared with a test object group which is not added after irradiation, the inhibition rate levels of the test object groups with different concentrations of 50.64mg/mL collagen derivatives and 25.32mg/mL collagen derivatives are obviously reduced (P is less than 0.05), the statistical significance is achieved, and the collagen derivatives have the anti-aging effect.

3. Anti-aging effect of collagen derivative evaluated based on 3D skin model

The research on the anti-aging effect of the skin needs to be carried out under the condition that the external factors cause the oxidative damage of a skin model, wherein the external factors comprise ultraviolet irradiation or chemical reagent damage, and the concentration of a test substance with the activity of more than 80 percent of the skin model is selected as the highest test concentration, so that the concentration of the test substance of glucosyl rutin is 1mg/mL, 10mg/mL and 100 mg/mL.

(3) Determination of dose of ultraviolet-induced radiation

the experimental result shows that 8J/cm is selected²An anti-aging effect test is performed.

(4) Confirmation of anti-aging Effect of test substance

Respectively setting a non-irradiated group, a group added with the test object after irradiation and a group not added with the test object after irradiation according to the concentration of the test object determined in the step (3) and the UVA irradiation dose determined in the step (4), adding epigallocatechin gallate before ultraviolet irradiation of a normal skin model as a positive control, testing elastase of each group, and judging the anti-aging effect of each product according to the test result.

P <0.05 x P <0.01 compared to irradiated groups

The experimental result shows that the collagen derivative has anti-aging effect at chemical level and cell level, the 3D skin model has no anti-aging effect, and the final experimental conclusion shows that the tocopherol derivative has the anti-aging effect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention. The present invention is only illustrative of some of the cosmetic materials. However, other non-single component systems mentioned in the present invention, such as skin care raw materials, plant extracts (crude extract, refined extract and mixtures thereof), etc., which may be anti-aging, may also be tested for skin sensitization using the methods of the present invention. This is not further enumerated here.

Claims

1. A method for evaluating skin anti-aging efficacy based on combination of various in-vitro models of an enzymology system is characterized by comprising the following steps:

(2.4) confirmation of anti-aging effect of test substance: according to the concentration of the test substance determined in the step (2.2) and the UVA or UVB irradiation dose determined in the step (2.3), when the primary cells of normal people grow to 80%, respectively setting up a blank control, a negative control, a positive control, an irradiation group and an experimental group, testing the levels of elastase, collagenase, matrix metalloproteinase or hyaluronidase in the same step (one) of each group by using an enzyme labeling instrument, and judging the anti-aging effect of each product according to the test result, compared with the irradiation group, the test substance with different concentrations and the level change of cellular enzyme present a dose-effect relationship, and one or more concentration change levels have statistical significance (P <0.05), which indicates that the substance has the anti-aging effect, otherwise, the substance does not have the anti-aging effect;

2. the method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: the test substance in the steps (one) to (three) is a cosmetic, a plant extract, a medicine, a food additive, a pesticide, a biological product or a chemical product.

3. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: the primary skin cells of the normal human in the step (2.1) are primary keratinocytes of the normal human or primary fibroblasts of the normal human.

4. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: and (3) respectively testing the absorbance of each group at 550-570 nm by using an enzyme-labeling instrument in the steps (2.2) - (2.4).

5. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: the irradiation doses determined in step (2.3) and step (3.3) are: the UVA radiation intensity is 8-10J/cm²The UVB irradiation intensity is 0.6-0.8J/cm²。

6. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: the negative control in the step (2.2) -step (2.4) contains normal human primary cells and cell culture solution and is not irradiated by ultraviolet rays, and the blank control contains only culture solution containing normal human skin primary cells and is not irradiated by ultraviolet rays; the positive control in the step (2.4) contains known anti-aging substances, culture solution of normal human primary cells and normal human skin primary cells and is irradiated by ultraviolet rays, the irradiation group contains culture solution of normal human primary cells and normal human skin primary cells and is irradiated by ultraviolet rays, and the test group contains test objects, culture solution of normal human primary cells and normal human skin primary cells and is irradiated by ultraviolet rays.

7. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: the culture solution containing the 3D skin model and the 3D skin model in the negative control in the steps (3.2) to (3.4) is not irradiated by ultraviolet rays, and the culture solution containing the 3D skin model only in the blank control is not irradiated by ultraviolet rays; and (3.4) the positive control contains known anti-aging substances, a 3D skin model and a culture solution of the 3D skin model and is irradiated by ultraviolet rays, the irradiation group contains the culture solution of the 3D skin model and is irradiated by ultraviolet rays, and the test object group contains a test object, the culture solution of the 3D skin model and is irradiated by ultraviolet rays.

8. The method for evaluating skin anti-aging efficacy based on an enzymatic system combining multiple in vitro models according to claim 6 or 7, characterized in that: the known anti-aging substance is epigallocatechin gallate, vitamin E or vitamin C.

9. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: the levels of elastase, collagenase, matrix metalloproteinase or hyaluronidase in step (2.4) and in step (3.4) were tested using an ELISA kit.

10. The method of claim 1 for evaluating skin anti-aging efficacy based on an enzymatic system in combination with multiple in vitro models, wherein: and (4) in the step (four), based on the weight principle, the anti-aging efficacy prediction based on the 3D skin model is more than the anti-aging efficacy prediction based on the cell level, and more than the anti-aging efficacy prediction based on the chemical level, and under the condition that the three anti-aging efficacy predictions are inconsistent, the anti-aging efficacy prediction with high weight is taken as a main judgment basis.