Cosmetic and cosmetic raw material sunscreen function evaluation method and application

Abstract

The invention belongs to the field of cosmetic detection, and discloses a cosmetic and cosmetic raw material sunscreen function evaluation method and application. According to the evaluation method, a sunscreen effect test is carried out on a test sample by using a non-contact administration device, wherein the test sample is a cosmetic finished product or a diluted cosmetic raw material; the test comprises evaluation of in-vitro sunscreen effect by skin cells, evaluation of in-vivo sunscreen effect by a zebra fish embryo model and result evaluation. According to the method, the sunburn-resistant effect is evaluated through expression of beta-galactosidase in human dermal fibroblasts and / or secretion of collagen, the sunburn-resistant effect is evaluated through the survival rate of human keratinocytes, the DNA damage condition or inflammatory factor secretion and the lethal / teratogenic condition of zebra fish embryos, and multi-layer and all-around sunburn-resistant effect evaluation is achieved. The method provided by the invention is suitable for evaluating the sunscreen function of various sunscreen products, has accuracy and practicability, and provides an important technical basis for research and development, quality control and performance optimization of sunscreen cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics testing, and specifically discloses a method for evaluating the sunscreen function of cosmetics and cosmetic raw materials and an application thereof. Background Art

[0002] There are three main types of ultraviolet rays: long-wave ultraviolet rays (UVA), medium-wave ultraviolet rays (UVB) and short-wave ultraviolet rays (UVC). UVA and UVB have a greater impact on skin health. UVA can penetrate deep into the dermis of the skin, causing photoaging of the skin, while UVB mainly causes sunburn. Sunscreen products (such as sunscreen) are an important means of protecting against ultraviolet rays. At present, the evaluation methods for the sunscreen effect of sunscreen products mainly rely on ultraviolet spectrometer method, human SPF value method and in vitro reconstructed skin model method.

[0003] The UV spectrometer method uses a UV spectrometer to measure the UV absorbance and transmittance of sunscreen samples to quickly evaluate the sunscreen effect. Although this method is easy to operate, it is difficult for consumers to intuitively understand the results because they are relatively abstract, and they cannot fully simulate the physiological response of the skin, lacking a direct connection with the actual sunscreen effect. The human SPF value method evaluates the effectiveness of sunscreen products by testing the sunscreen effect on human skin. Although this method has high authority and persuasiveness, the testing process is costly and time-consuming, and it involves human experiments, which poses certain safety hazards. Therefore, it is mostly used for effect verification before sunscreen products are put on the market, but not suitable for screening or initial research and development of sunscreen raw materials. The in vitro reconstructed skin model method uses an in vitro reconstructed skin model to evaluate the sunscreen efficacy, which has high safety and accuracy. However, this method has a long model preparation cycle and high cost, which limits its wide application, and is not suitable for rapid screening of a large number of sunscreen raw materials or preliminary pre-experiments. Some patents disclose the use of quartz glass plates combined with mRNA expression detection methods, using quartz glass plates as a medium for receiving drugs, and evaluating the sunscreen efficacy of raw materials or products by evaluating the mRNA expression level of cells after irradiation. However, this method is mainly used to evaluate physical sunscreens or raw materials, especially physical sunscreen products without transdermal activity. For chemical and biological sunscreens, their safety and efficacy are difficult to be accurately evaluated by this method. The existing methods for evaluating the sunscreen function of cosmetics and cosmetic raw materials have certain limitations. A new evaluation method is urgently needed to overcome the shortcomings of the existing methods and meet the comprehensive evaluation needs of sunscreen raw materials and products. Summary of the invention

[0004] In response to these defects, this invention provides a method and application for evaluating the sunscreen function of cosmetics and cosmetic raw materials. This method is based on cell and zebrafish models, combined with a contactless drug delivery method, and achieves a comprehensive evaluation of sunscreen efficacy through multi-level indicators: after irradiating human dermal fibroblasts with long-wave ultraviolet rays (UVA), the expression of intracellular β-galactosidase and / or the secretion of related collagen is used to characterize the sunscreen efficacy of the test sample; after irradiating human keratinocytes / zebrafish with medium-wave ultraviolet rays (UVB), the sunscreen efficacy of the test sample is characterized by detecting cell survival rate, DNA damage or inflammatory factor secretion and lethality / teratogenicity of zebrafish embryos. This method can not only evaluate the sunscreen efficacy in vitro at the cellular level, but also simulate in vivo ultraviolet damage through a zebrafish model, thereby forming a multi-dimensional evaluation system combining in vitro and in vivo. The sunscreen efficacy evaluation system provided by the present invention is efficient, scientific, and reliable, and provides an important reference for the research and development and performance optimization of sunscreen cosmetics.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for evaluating the sunscreen function of cosmetics and cosmetic raw materials, wherein the evaluation method uses a non-contact administration method to test the sunscreen effect of a test sample, wherein the test sample is a finished cosmetic product or a diluted cosmetic raw material; the test includes evaluating the sunscreen efficacy in vitro with skin cells and / or evaluating the sunscreen efficacy in vivo with a zebrafish embryo model; The non-contact drug administration method is achieved by means of a non-contact drug administration device; the non-contact drug administration device comprises a drug administration carrier layer, the drug administration carrier layer is used to carry the test sample; the test sample and the skin cell or the zebrafish embryo are separated by the drug administration carrier layer; the drug administration carrier layer is a light-transmitting film, the film has or does not have micropores, and the micropores have a pore size of 0.4-25 μm; The skin cell evaluation of in vitro sunscreen efficacy includes human keratinocyte sunburn efficacy detection and human dermal fibroblast sunburn efficacy detection; the human keratinocyte sunburn efficacy detection includes measuring the changes in one or more of the cell survival rate of human keratinocytes, IL-8 content in the culture medium or Tail DNA content caused by the test sample after UVB irradiation; the human dermal fibroblast sunburn efficacy detection includes measuring the changes in collagen expression and / or β-galactosidase staining in human dermal fibroblasts caused by the test sample after UVA irradiation; the zebrafish embryo model evaluation of in vivo sunscreen efficacy includes zebrafish embryo model sunburn efficacy detection; the zebrafish embryo model sunburn efficacy detection includes measuring the changes in zebrafish embryo mortality and teratogenicity caused by the test sample after UVB irradiation; Before measuring the change, the test sample is evenly coated on a drug delivery carrier layer; the skin cells and the zebrafish embryos are placed under the drug delivery carrier layer, and ultraviolet light is irradiated above the drug delivery carrier layer.

[0006] The sunscreen evaluation method provided by the present invention combines the change indicators of skin cells and zebrafish embryo models to achieve a comprehensive multi-level evaluation of sunscreen efficacy in vitro and in vivo. The sunscreen efficacy is evaluated by UVB irradiation, and the survival rate of human keratinocytes, DNA damage (Tail DNA content) or changes in the secretion of inflammatory factors (IL-8), as well as the mortality and teratogenicity of zebrafish embryos are detected; the sunscreen efficacy is evaluated by UVA irradiation, and the secretion of collagen and the expression level of β-galactosidase in human dermal fibroblasts are detected, and the skin cells and embryo models are used to fully simulate the photodamage and photoaging effects of ultraviolet rays on human skin.

[0007] The non-contact drug delivery method provides a stable, accurate and innovative drug delivery method for sunscreen efficacy evaluation. The drug delivery carrier layer of the non-contact drug delivery device has stable light transmittance and can evenly transmit ultraviolet light to the skin cells or zebrafish embryos under the test sample, truly simulating the environment of ultraviolet radiation on human skin. The drug delivery carrier layer achieves permeability through microporous design, allowing the active ingredients in chemical and biological sunscreen cosmetics to diffuse around the test model and contact with skin cells or zebrafish embryos without destroying the cosmetic system. Physical sunscreen cosmetics can be isolated from skin cells or zebrafish embryos through a non-contact drug delivery device without micropores, or a non-contact drug delivery device with micropores can be used to maintain a certain spatial distance between the culture liquid surface of skin cells or zebrafish embryos and the drug delivery carrier layer to achieve physical sunscreen cosmetics isolated from skin cells or zebrafish embryos. The device is compatible with different types of sunscreen products, solves the problem of system adaptation during the test process, and provides scientific and efficient technical support for the efficacy evaluation of various sunscreen products.

[0008] The evaluation method provided by the present invention is applicable to physical sunscreen cosmetics or cosmetic raw materials, including but not limited to titanium dioxide, zinc oxide and octolylene and other sunscreen raw materials and sunscreen products containing these ingredients such as sunscreen creams, sunscreen sticks, sunscreen sprays, etc. These sunscreen substances, which are not easily soluble in water and do not penetrate the skin, effectively prevent ultraviolet radiation through a physical barrier mechanism, usually by reflecting or scattering ultraviolet rays.

[0009] The evaluation method provided by the present invention is also applicable to chemical sunscreen cosmetics or cosmetic raw materials, including but not limited to common chemical sunscreens such as avobenzone, simvastatin and benzophenone, which achieve sunscreen effects by absorbing ultraviolet rays and converting them into heat energy.

[0010] The evaluation method provided by the present invention is also applicable to biological sunscreen cosmetics or cosmetic raw materials, including but not limited to natural plant extracts and microbial fermentation products. Biological sunscreen ingredients provide ultraviolet protection by enhancing the skin's natural barrier function or antioxidant effect.

[0011] The malformations in the teratogenicity rate described in the present invention include: developmental arrest, absence of swim bladder, pericardial cyst, spinal curvature, yolk sac edema, and self-decomposition of the tail and caudal fin, or one or more thereof.

[0012] In some embodiments, the method for evaluating the sunscreen function of cosmetics and cosmetic raw materials provided by the present invention further includes the following operations: Human keratinocyte culture; Human dermal fibroblast culture; Obtaining zebrafish embryos: Sexually mature zebrafish are paired at a female-to-male ratio of 1:2, and after natural mating, the female fish lays eggs to obtain the zebrafish embryos.

[0013] Cosmetic / cosmetic raw material sample processing: Cosmetic raw materials are diluted with appropriate solvents according to the planned addition amount of the product.

[0014] Preferably, the contactless drug delivery device comprises an embedded chamber and a culture plate provided with a groove for accommodating the skin cells or the zebrafish embryo model; the embedded chamber is a cup-shaped structure with an open upper end, and the drug delivery carrier layer constitutes the bottom of the embedded chamber cup; the cavity of the embedded chamber is located in the groove, and the upper port of the embedded chamber is circumferentially provided with a horizontally outwardly extending edge, and the embedded chamber is clamped on the surface of the culture plate through the edge and extends into the groove of the culture plate; a distance is left between the drug delivery carrier layer and the bottom of the groove; when the contactless drug delivery device is used for sun protection, the rest of the embedded chamber except the bottom drug delivery carrier layer needs to be treated to isolate ultraviolet rays.

[0015] The space inside the embedded chamber can be called the upper chamber, and the space inside the culture plate and outside the embedded chamber can be called the lower chamber. The drug delivery carrier layer in the upper chamber carries the test sample, and the skin cells and zebrafish embryos are placed in the culture plate in the lower chamber. The embedded chamber, except for the bottom drug delivery carrier layer, needs to be shielded from ultraviolet rays to ensure that the bottom drug delivery carrier layer is the only path for ultraviolet rays to irradiate skin cells or zebrafish embryos. One or a combination of treatment methods such as the selection of ultraviolet-proof materials, surface coating treatment, physical shielding design or dyeing treatment can be used to achieve the need for ultraviolet isolation of the remaining parts of the embedded chamber except for the bottom drug delivery carrier layer.

[0016] The culture plate can be a common cell culture plate, and the groove of the culture plate is the culture chamber of the cell culture plate. The embedded chamber can be made into various specifications that are compatible with the culture plate according to the test requirements and the experimental requirements, such as 96-well plate specifications, 48-well plate specifications, 24-well plate specifications and 6-well plate specifications. At the same time, without affecting the test results, the embedded chamber can be cleaned and sterilized by ultraviolet irradiation and reused.

[0017] The drug delivery carrier layer of the embedded chamber can be made of materials with stable light transmittance, such as inorganic glass and traditional chemical plastics. These materials can be processed by microporous processing technology to form a structure with uniform light transmittance and permeability, meeting the requirements of ultraviolet light transmission and diffusion of effective ingredients. The material of the drug delivery carrier layer of the embedded chamber includes but is not limited to optical quartz glass, ultraviolet transparent black glass, polymethyl methacrylate, polystyrene, polycarbonate, and polyethylene terephthalate.

[0018] These materials have excellent light transmittance and can efficiently transmit ultraviolet light, truly simulating the environment in which ultraviolet light irradiates human skin. At the same time, they have good processing properties, support the precise formation of microporous structures, have good chemical stability, and are non-toxic to cells, ensuring the reliability and accuracy of experimental results.

[0019] More preferably, the embedded chamber drug delivery carrier layer has no micropores and is made of polymethyl methacrylate.

[0020] Polymethyl methacrylate (PMMA) has extremely high transmittance in the ultraviolet band and can effectively transmit ultraviolet light energy; its surface is smooth and its internal molecular structure is uniform, which can reduce light scattering; it is chemically stable and will not react with physical sunscreen cosmetics. At the same time, the design without micropores can well shield the contact between physical sunscreen cosmetics and model cells. Therefore, using PMMA as a carrier layer without micropores can effectively detect the shielding effect of the protective film formed by physical sunscreen cosmetics or raw materials on the surface, thereby evaluating its protective ability.

[0021] More preferably, the embedded chamber drug delivery carrier layer has micropores and is made of polyethylene terephthalate.

[0022] Polyethylene terephthalate (PET) has good light transmittance and chemical stability. Through specific processing techniques, such as foaming, microporous injection molding or compounding with other porous materials, PET materials can have a microporous structure. The drug delivery carrier layer with microporous PET material can control the release of sunscreen ingredients, simulate the penetration and absorption under actual use conditions, and is suitable for the sunscreen efficacy testing of chemical and biological sunscreen cosmetics.

[0023] More preferably, the distance between the drug delivery carrier layer and the bottom of the culture plate is 0.5-1 cm.

[0024] More preferably, the surface of the material of the embedded chamber drug delivery carrier layer has a certain concave-convex texture and a thickness of 1-4 mm.

[0025] Using a PMMA sheet with a certain concave-convex texture, a certain roughness, and a thickness of 1-4 mm can better simulate the roughness and wrinkles of human skin.

[0026] Preferably, the method for detecting the sunburn protection efficacy of human keratinocytes comprises the following steps: (1) Human keratinocytes in the logarithmic growth phase were inoculated into a culture plate, and when the density of the human keratinocytes reached 60%-80%, the test sample was administered to the human keratinocytes in a non-contact administration manner, which was recorded as the experimental group; a blank group and a model group were also set up; (2) The experimental group and the model group were irradiated with UVB, with a dose range of 50-500 mJ / cm 2 , the blank group was protected from light for the same period of time; after UVB irradiation, the culture medium was replaced and cultured for 24-48 hours before index detection; the indexes included one or more of the survival rate of human keratinocytes, the content of IL-8 in the culture medium and the content of Tail DNA; (3) Quality control: When the quality control conditions of the corresponding indicators are met, the results obtained are used to evaluate the performance of the test sample's sunburn protection efficacy; the quality control condition for the survival rate of human keratinocytes is that the survival rate of cells in the model group is significantly lower than that in the blank group; the quality control condition for the IL-8 content in the culture medium is that the survival rate of cells in the model group is ≥70%, and the IL-8 content in the model group is significantly higher than that in the blank group; the quality control condition for the Tail DNA content is that the survival rate of cells in the model group is ≥70%, and the Tail DNA (%) in the model group is significantly higher than that in the blank group; (4) Result evaluation: Calculate the difference in the index test results between the test sample group and the model group to evaluate the sunburn protection effect of the test sample.

[0027] More preferably, the method for evaluating the results of step (4) is: calculating the difference in the test results of any one of the indicators of the test sample group and the model group. If p<0.05, it is proved that the test sample has sunburn protection effect.

[0028] Preferably, the method for detecting the sunscreen efficacy of human dermal fibroblasts comprises the following steps: (1) Human dermal fibroblasts in the logarithmic growth phase were inoculated into a culture plate and cultured until the density of the human keratinocytes reached 60%-80%. The test sample was administered to the human dermal fibroblasts in a non-contact manner and cultured, which was recorded as the experimental group; a blank group and a model group were also set up; (2) The experimental group and the model group were irradiated with UVA, with the irradiation dose ranging from 15-30 J / cm 2 , the blank group was protected from light for the same period of time; after UVA irradiation, the culture medium was replaced and cultured for 24-48 hours before index detection; the indexes included the collagen content in the culture medium and / or the rate of β-galactosidase-stained positive cells; (3) Quality control: When the quality control conditions of the corresponding indicators are met, the results obtained are used to test the performance evaluation of the sample's sunscreen efficacy; the quality control conditions for the collagen content in the culture medium are that the cell survival rate in the model group is ≥70%, and the collagen content in the culture medium in the model group is significantly lower than that in the blank group; the quality control conditions for the β-galactosidase staining positive cell rate are that the cell survival rate in the model group is ≥70%, and the β-galactosidase positive cell rate in the model group is significantly higher than that in the blank group; (4) Result evaluation: Calculate the difference in the test results of the test sample group and the model group to evaluate the sun protection effect of the test sample.

[0029] More preferably, the method for evaluating the results of step (4) is: calculating the difference in the test results of any one of the indicators of the test sample group and the model group. If p<0.05, it is proved that the test sample has the effect of sun protection.

[0030] Preferably, the method for detecting the sunburn prevention efficacy of the horsefish embryo model comprises the following steps: (1) 72 hpf zebrafish embryos were placed in a culture plate; the test sample was administered to the zebrafish embryos in a non-contact manner, which was recorded as the experimental group; a blank group and a model group were also set up; (2) The experimental group and the model group were irradiated with UVB, with the irradiation dose ranging from 400 to 1600 mJ / cm 2 The blank group was protected from light for the same period of time; the culture was continued for 24-72 hours after the UVB irradiation, and the mortality and teratogenicity rates of the zebrafish embryos were recorded at different times; (3) Quality control: In the zebrafish embryo model sunburn prevention efficacy test, the survival rate of zebrafish embryos in the blank group is ≥90%, and the survival rate of zebrafish embryos in the model group is significantly lower than that in the blank group. The obtained results are used as the evaluation results of the sunburn prevention efficacy of the test sample; (4) Result evaluation: Calculate the difference in the index test results between the test sample group and the model group to evaluate the sunburn protection effect of the test sample.

[0031] More preferably, the method for evaluating the results of step (4) is: calculating the difference in the test results of any one of the indicators of the test sample group and the model group. If p<0.05, it is proved that the test sample has sunburn protection effect.

[0032] More preferably, the non-contact drug administration method is that after completing skin cell culture or zebrafish embryo modeling in the culture plate of the non-contact drug administration device, the culture medium in the culture plate of the experimental group is replaced with PBS, the test sample is added to the carrier layer of the embedded chamber drug layer, and the test sample is evenly dispersed on the drug layer carrier layer using a sterile cotton swab or a pipette tip; the culture medium in the culture plate of the blank group and the model group is replaced with PBS, and a dilution solvent with a mass volume equal to that of the test sample is added to the carrier of the embedded chamber drug layer, and the dilution solvent is evenly dispersed on the drug layer carrier layer using a sterile cotton swab or a pipette tip.

[0033] In a second aspect, the present invention also provides the use of any of the above-mentioned sunscreen function evaluation methods in evaluating the sunscreen function of cosmetics or screening sunscreen cosmetics.

[0034] The sunscreen function evaluation method provided by the present invention can be widely used in the screening and function verification of sunscreen cosmetics and cosmetic raw materials. In the process of sunscreen product development, the evaluation method can be used to optimize the formula and improve the sunscreen performance of the product. In the quality control link, it can be used as a standardized testing tool to ensure the consistency and stability of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 It is a schematic diagram of the contactless drug administration method and the contactless drug administration device structure; Figure 2 The effect of different UVB irradiation intensities on keratinocyte survival rate in Example 3; Figure 3 The effect of different UVA irradiation intensities on dermal fibroblast senescence in Example 3; Figure 4 The effects of different UVB irradiation intensities on the teratogenicity and lethality of zebrafish embryos in Example 3; Figure 5 The stability test results of the evaluation method provided by the present invention in Example 4; (*p<0.05, **p<0.01 vs. model group); Figure 6 Detection of the protective effect of sunscreen cosmetics on cell DNA in Example 5 (*p<0.05, **p<0.01 vs model group); Figure 7This is the test of the protective effect of the sunscreen cosmetics on zebrafish in Example 6 (*p<0.05, **p<0.01 vs the model group); Figure 8 The β-galactosidase staining result in Example 7; Fig. 9 This is the detection of the protective effect of the sunscreen cosmetics on human dermal fibroblasts in Example 7 (*p<0.05, **p<0.01 vs the model group). DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] The sunscreen cosmetics used in the present invention are all commercially available.

[0040] The culture medium used in the embodiments of the present invention is: DMEM medium: DMEM medium 85%-90%, fetal bovine serum 10-15%, antibiotic 1%, pH 7.0-7.4.

[0041] E3 medium: NaCl 5 mM, KCl 0.17 mM, CaCl 2 ·2H 2 O 0.33 mM, MgSO 4 7H 2 O 0.33mM. Example 1

[0042] This embodiment provides a contact-free drug delivery device.

[0043] The non-contact drug delivery device includes an embedded chamber and a culture plate; the embedded chamber is a cup-shaped structure with an opening at the upper end, and the drug delivery carrier layer constitutes the bottom of the embedded chamber cup, and the drug delivery carrier layer is made of PMMA with a thickness of 1 mm; the upper end of the embedded chamber is circumferentially provided with a horizontally extending edge, and the embedded chamber is connected to the plate surface of the culture plate through the edge and extends into the groove of the culture plate, and a 1 cm gap is left between the drug delivery carrier layer and the bottom of the culture plate. The space inside the embedded chamber is the upper chamber, and the space inside the culture plate hole and outside the embedded chamber is the lower chamber.

[0044] Non-contact drug administration method: The embedded chamber is covered with aluminum film except for the drug administration carrier layer at the bottom to block ultraviolet rays. The test sample is coated on the drug administration carrier layer, and the skin cells or the zebrafish embryos are placed in the culture plate, below the drug administration carrier layer. Example 2

[0045] This embodiment provides a non-contact drug delivery device. The difference from Embodiment 1 is that the drug delivery carrier layer is made of PET, has evenly distributed micropores with a pore size of 10 μm, and a thickness of 4 mm; and a 0.5 cm gap is left between the drug delivery carrier layer and the bottom of the culture plate. Example 3 Irradiation dose

[0046] 1. Detection of sunburn protection efficacy of human keratinocytes The digested human keratinocytes were cultured in a 24-well culture plate and divided into a blank group, a model group 1 (the non-contact drug delivery device provided in Example 1), and a model group 2 (the non-contact drug delivery device provided in Example 2), with three replicates in each group. When the density of human keratinocytes reached 80%, the culture medium was removed, and 50 μL PBS was added to the upper and lower chambers of the blank group for light protection; 50 μL PBS was added to the upper chamber and 50 μL PBS was added to the lower chamber of the model group 1, and no light protection was performed; 50 μL PBS was added to the upper chamber and 50 μL PBS was added to the lower chamber of the model group 2, and no light protection was performed. The above three groups were respectively treated with 50 mJ / cm 2 、100mJ / cm 2 , 150 mJ / cm 2 , 250 mJ / cm 2 、300 mJ / cm 2 UVB, 500 mJ / cm 2 After UVB irradiation, the embedded chamber of the model group was removed, and 500 μL of 10% FBS DMEM medium was added to the culture plates of each group and the cell survival rate was measured after further culture for 24 hours. Figure 2 shown.

[0047] like Figure 2 As shown in the figure, PMMA material has better light transmittance than PET material, but both can achieve the purpose of irradiating the cells below by adjusting the UVB irradiation dose. 2 When the irradiation intensity is higher than 100 mJ / cm, the cell survival rate under the PMMA material decreases significantly. Therefore, when using the PMMA material drug delivery carrier layer to test the sunburn protection effect of human keratinocytes, 100-150 mJ / cm is selected. 2 As the best modeling condition, 500 mJ / cm 2As the best modeling conditions.

[0048] 2. Human dermal fibroblasts sunscreen efficacy test (UVA) The digested human dermal fibroblasts were cultured in a 6-well culture plate and divided into a blank group, a model group 1 (the non-contact drug delivery device provided in Example 1), and a model group 2 (the non-contact drug delivery device provided in Example 2), with three replicates in each group. When the fibroblast density reached 60%, the culture medium was removed, and 200 μL PBS was added to the upper and lower chambers of the blank group for light protection; 200 μL PBS was added to the upper chamber and 200 μL PBS was added to the lower chamber of the model group 1, and no light protection was performed; 200 μL PBS was added to the upper chamber and 200 μL PBS was added to the lower chamber of the model group 2, and no light protection was performed. The above three groups were respectively exposed to 15 J / cm 2 , 20 J / cm 2 , 30 J / cm 2 After irradiation, the embedded chamber of the model group was removed, and 2 mL of 15% FBS DMEM medium was added to each group of culture plates and cultured for 24 h before β-galactosidase staining. Figure 3 shown.

[0049] like Figure 3 As shown in the figure, the UVA transmission effect on PMMA and PET is similar, and when 20 J / cm 2 When the irradiation intensity is higher than 20 J / cm, the rate of β-galactosidase positive cells in human dermal fibroblasts increases significantly, so 20-30 J / cm 2 As the optimal irradiation dose range for UVA to induce cell senescence.

[0050] 3. Testing the sunburn protection efficacy of zebrafish embryo model 72 hpf zebrafish embryos were placed in a 6-well culture plate for modeling and culture, 30 per well, 3 wells per group, divided into a blank group, model group 1 (non-contact drug delivery device provided in Example 1), model group 2 (non-contact drug delivery device provided in Example 2), and three replicates per group. The blank group was protected from light, while the model group 1 had 250 μL E3 culture medium added to the upper chamber and 250 μL E3 culture medium added to the lower chamber, without protection from light; the model group 2 had 250 μL E3 culture medium added to the upper chamber and 250 μL E3 culture medium added to the lower chamber, without protection from light. The above three groups were treated with 400 mJ / cm 2 、800 mJ / cm 2 UVB, 1600 mJ / cm 2UVB was used for irradiation. After irradiation, the embedded chamber and E3 culture medium of the model group were taken out and replaced with new E3 culture medium. After culturing for 48 h, the teratogenicity and mortality rates of zebrafish were calculated.

[0051] like Figure 4 As shown in the figure, PMMA and PET show different UVB transmittance effects, that is, PMMA has a better UVB transmittance than PET, but both can achieve the purpose of irradiating the zebrafish embryos below by adjusting the UVB irradiation dose. 2 When the irradiation intensity is higher than 400 mJ / cm, the deformity or mortality of zebrafish under the PMMA material increases significantly. Therefore, when using PMMA as a drug delivery carrier layer to test the sunburn protection efficacy of zebrafish embryo models, 400-800 mJ / cm is selected. 2 When using PET as a drug delivery carrier layer to test the sunburn protection efficacy of zebrafish embryo models, 800-1600 mJ / cm 2 as modeling conditions. Example 4 Testing of method stability

[0052] The evaluation method provided by the present invention was tested for stability using zinc oxide raw materials (hereinafter described as "sunscreen raw materials") and the contactless drug delivery devices provided in Examples 1 and 2.

[0053] Detection method: Take the digested human keratinocytes and culture them in a 24-well culture plate, and divide them into a blank group, a model group, and a sunscreen raw material group, with three replicates in each group. When the density of human keratinocytes reaches 80%, remove the culture medium, add 50 μL PBS to the lower chamber of the blank group, and add 50 μL PBS to the upper chamber, and protect from light; add 50 μL PBS to the lower chamber of the model group, and add 50 μL PBS to the upper chamber, without protecting from light; add 50 μL PBS to the lower chamber of the sunscreen raw material group, and evenly apply 50 μL PBS containing 10% sunscreen raw materials to the drug delivery carrier layer, without protecting from light. Perform UVB irradiation, and use 100 mJ / cm 2 UVB was used for irradiation, and 500 mJ / cm 2 UVB was used for irradiation. After irradiation, the embedded chamber was removed and replaced with 500 μL 10% FBS DMEM medium for 24 h, and then the cell survival rate was measured; and the above test method was repeated three times using different batches of human keratinocytes. The results are shown in Figure 5 .

[0054] Figure 5 A is the stability test result of the contactless drug delivery device provided in Example 1; Figure 5B is the stability test result of the contactless drug delivery device provided in Example 2.

[0055] like Figure 5 As shown, the survival rates of the model group and the sunscreen raw material group in the three test results are close, indicating that there is consistency between the parallel results. The survival rates of the cells in the model group using the non-contact drug delivery device provided in Example 1 (the drug delivery carrier layer is made of PMMA) are 69.02%, 73.02% and 68.98%, respectively, and the cell survival rates of the sunscreen raw material group are 96.84%, 99.48% and 103.19%, respectively. The survival rates of the cells in the model group using the non-contact drug delivery device provided in Example 2 (the drug delivery carrier layer is made of PET) are 72.09%, 71.92% and 74.09%, respectively, and the cell survival rates of the sunscreen raw material group are 92.91%, 92.38% and 92.24%, respectively. The above results prove that the cosmetic sunscreen evaluation method provided by the present invention has good stability and can obtain stable and reliable experimental data. Example 5

[0056] In the experiment of this embodiment, the non-contact drug delivery device provided in Example 2 was used, and chemical sunscreen formulation 1 (containing 5% octocrylene, hereinafter described as "chemical sunscreen 1") and chemical sunscreen formulation 2 (containing 15% octocrylene, hereinafter described as "chemical sunscreen 2") were used as test samples.

[0057] Digested human keratinocytes were cultured in 6-well culture plates and divided into blank group, model group, chemical sunscreen group 1 and chemical sunscreen group 2, with three replicates in each group. When the density of human keratinocytes reached 80%, the culture medium was removed, 200 μL PBS was added to both the upper and lower chambers of the blank group, and the cells were protected from light; 200 μL PBS was added to both the upper and lower chambers of the model group without protecting them from light; 200 μL PBS was added to the lower chamber of the chemical sunscreen group, and 200 μL of the corresponding chemical sunscreen formula was added to the upper chamber without protecting them from light. A UV crosslinker was used for 50 mJ / cm 2 UVB irradiation was performed. After irradiation, the embedded chamber was removed and each group was replaced with 2 mL 10% FBS DMEM medium and cultured for 3 h. The DNA damage in the cells was detected by comet electrophoresis kit, and the obtained image data was analyzed by CASP.

[0058] like Figure 6As shown in the figure, UVB irradiation causes DNA damage in human keratinocytes, and the group using chemical sunscreen 1 (containing 5% octocrylene) rarely has DNA damage compared to the model group, proving that the chemical sunscreen has sunscreen efficacy. However, in the group using chemical sunscreen 2 (containing 15% octocrylene), DNA damage was not alleviated but aggravated. This is because excessive octocrylene is used as a protective agent in chemical sunscreen 2, and octocrylene is reported to have potential skin penetration, causing the risk of DNA damage in skin cells. Therefore, when using a permeable drug delivery carrier layer, not only can the sunscreen efficacy of octocrylene used in moderation be detected, but also the toxic side effects of sunscreens when excessive risk sunscreens are used can be monitored. Example 6

[0059] This embodiment provides a method for evaluating the sunburn prevention effect of sunscreen cosmetics. The evaluation method uses the non-contact drug delivery device provided in Example 1. A physical sunscreen cosmetic is used as a test sample.

[0060] 72 hpf zebrafish embryos were placed in a 6-well culture plate for modeling and culture, 30 per well, 3 wells per group, divided into a blank group, a model group, and a sunscreen cosmetics group. After modeling, the lower chamber of the blank group was replaced with 250 μL of normal E3 medium and protected from light; the lower chamber of the model group was replaced with 250 μL of normal E3 medium without protecting from light; the lower chamber of the sunscreen cosmetics group was replaced with 250 μL of normal E3 medium, and 250 mg of sunscreen cosmetics stock solution was evenly applied on the drug delivery carrier layer of the upper chamber without protecting from light. A UV crosslinker was used for 800 mJ / cm 2 After UVB irradiation, the embedded chamber and E3 medium were removed and replaced with new E3 medium. The number of zebrafish teratogenicity and lethality at 48 h was recorded. Figure 7 shown.

[0061] Tests have shown that this physical sunscreen cosmetic can protect against UVB damage to zebrafish embryos. Comparative Example 1

[0062] This embodiment provides a method for evaluating the sunscreen efficacy of sunscreen cosmetics. The difference from Example 6 is that no embedded chamber is used. After the modeling is completed, 250 μL of normal E3 culture medium is replaced in the blank group culture plate and the plate is protected from light; 250 μL of normal E3 culture medium is replaced in the model group culture plate without protecting from light; 250 μL of E3 culture medium containing 10% sunscreen cosmetics is replaced in the sunscreen cosmetics group culture plate without protecting from light. Use a UV crosslinker to perform 800 mJ / cm 2 After UVB irradiation, the E3 medium was removed and replaced with new E3 medium. The number of zebrafish teratogenic and lethal at 48 h was recorded, and the teratogenic rate and lethality were calculated. Figure 7 shown.

[0063] It can be seen from the results that when the traditional method of dosing is used, direct contact between zebrafish embryos and sunscreen cosmetics causes teratogenicity and mortality. Even when diluted sample liquid is used, the teratogenicity and mortality of zebrafish in the group are still much higher than those in the model group, resulting in unusable results and failure to reflect the sunburn prevention effect of cosmetics. In addition, during the experiment, the operation is cumbersome and difficult to observe. In contrast, using the evaluation method provided by the present invention, there is no need to dilute the sunscreen cosmetics, and there is basically no negative impact on the activity of zebrafish. There is no significant difference in teratogenicity and mortality between the model group of the non-contact dosing method and the model group of the traditional dosing method, which further proves the reliability and applicability of the method of the present invention in irradiation experiments. Example 7

[0064] In the experiment of this embodiment, the non-contact drug delivery device provided in Examples 1 and 2 was used. A biological sunscreen formulation (composed of biological sunscreen raw materials such as plant extracts, hereinafter described as "biological sunscreen") was used as a test sample.

[0065] After culturing human dermal fibroblasts for 2-3 generations, the digested human dermal fibroblasts were taken and cultured in 6-well culture plates and divided into blank group, model (PMMA) group, model (PET) group, biological sunscreen (PMMA) group and biological sunscreen (PET) group, with three replicates in each group. When the density of human dermal fibroblasts reached 60%, the culture medium was removed, and 200 μL PBS was added to the upper and lower chambers of the blank group, and the cells were protected from light; 200 μL PBS was added to the upper and lower chambers of the model group without protecting them from light; 200 μL PBS was added to the lower chamber of the biological sunscreen group, and the biological sunscreen was evenly applied on the drug delivery carrier layer in the upper chamber without protecting it from light. A UV cross-linking instrument was used for 20 J / cm 2 UVA irradiation was performed. After irradiation, the embedded chamber was removed and each group was replaced with 2 mL 15% FBS DMEM medium and cultured for 24 h. Senescent cells were stained with a β-galactosidase staining kit, and the β-galactosidase positive cell rate was statistically analyzed.

[0066] The results are as follows Figure 8As shown, there is no significant difference in the β-galactosidase positive cell rate between the model (PMMA) group and the model (PET) group, indicating that there is no significant difference in the permeability of PMMA material and PET material to UVA, and this conclusion is consistent with the test conclusion of Example 3. The β-galactosidase positive cell rate of the biological sunscreen (PET) group is significantly lower than that of the model (PET) group, while the β-galactosidase positive cell rate of the biological sunscreen (PMMA) group is not significantly different from that of the model (PMMA) group. Biological sunscreens usually exert their sunscreen effect by enhancing the cells' own ability to resist UVA, so the sunscreen evaluation of biological sunscreens is suitable for the evaluation method with a permeable drug delivery carrier layer.

[0067] In summary, the present invention relates to a test method suitable for evaluating the sunscreen efficacy of cosmetics or cosmetic raw materials, which has the advantages of simple operation, wide applicability, stable results, etc., and is suitable for sunscreen efficacy testing of cosmetics / cosmetic raw materials.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for evaluating the sunscreen function of cosmetics and cosmetic raw materials, characterized in that: The evaluation method uses a non-contact administration method to test the sunscreen effect of the test sample, and the test sample is a finished cosmetic product or a diluted cosmetic raw material; the test includes skin cells to evaluate the sunscreen efficacy in vitro and / or zebrafish embryo models to evaluate the sunscreen efficacy in vivo; The non-contact drug delivery method is achieved by means of a non-contact drug delivery device; the non-contact drug delivery device comprises a drug delivery carrier layer, the drug delivery carrier layer is used to carry the test sample; the test sample and the skin cell or the zebrafish embryo model are separated by the drug delivery carrier layer; the drug delivery carrier layer is a light-transmitting film, the film has or does not have micropores, and the micropores have a pore size of 0.4-25 μm; The skin cell evaluation of in vitro sunscreen efficacy includes human keratinocyte sunburn efficacy detection and human dermal fibroblast sunburn efficacy detection; the human keratinocyte sunburn efficacy detection includes measuring the changes in one or more of the cell survival rate of human keratinocytes, IL-8 content in the culture medium or Tail DNA content caused by the test sample after UVB irradiation; the human dermal fibroblast sunburn efficacy detection includes measuring the changes in collagen expression and / or β-galactosidase staining in human dermal fibroblasts caused by the test sample after UVA irradiation; the zebrafish embryo model evaluation of in vivo sunscreen efficacy includes zebrafish embryo model sunburn efficacy detection; the zebrafish embryo model sunburn efficacy detection includes measuring the changes in zebrafish embryo mortality and teratogenicity caused by the test sample after UVB irradiation; Before measuring the change, the test sample is evenly coated on the drug delivery carrier layer; The skin cells and the zebrafish embryo model are placed below the drug delivery carrier layer, and ultraviolet light is irradiated above the drug delivery carrier layer.

2. The sunscreen function evaluation method according to claim 1, characterized in that: The contactless drug delivery device comprises an embedded chamber and a culture plate provided with a groove for accommodating the skin cells or the zebrafish embryo model; the embedded chamber is a cup-shaped structure with an opening at the upper end, and the drug delivery carrier layer constitutes the bottom of the embedded chamber cup; the cavity of the embedded chamber is located in the groove, and the upper port of the embedded chamber is circumferentially provided with a horizontally outwardly extending edge, and the embedded chamber is clamped on the plate surface of the culture plate through the edge and extends into the groove of the culture plate; a distance is left between the drug delivery carrier layer and the bottom of the groove; when the contactless drug delivery device is used for sun protection, the rest of the embedded chamber except the bottom drug delivery carrier layer needs to be treated to isolate ultraviolet rays.

3. The sunscreen function evaluation method according to claim 2, characterized in that: The drug delivery carrier layer of the embedded chamber has no micropores and is made of polymethyl methacrylate.

4. The sunscreen function evaluation method according to claim 2, characterized in that: The drug delivery carrier layer of the embedded chamber has micropores and is made of polyethylene terephthalate.

5. The sunscreen function evaluation method according to claim 2, characterized in that: The distance between the drug delivery carrier layer and the bottom of the culture plate is 0.5-1 cm.

6. The sunscreen function evaluation method according to claim 2, characterized in that: The drug delivery carrier layer has a surface with a concave-convex texture and a thickness of 1-4 mm.

7. The sunscreen function evaluation method according to claim 1, characterized in that: The method for detecting the sunburn prevention efficacy of human keratinocytes comprises the following steps: (1) Human keratinocytes in the logarithmic growth phase were inoculated into a culture plate, and when the density of the human keratinocytes reached 60%-80%, the test sample was administered to the human keratinocytes in a non-contact administration manner, which was recorded as the experimental group; a blank group and a model group were also set up; (2) The experimental group and the model group were irradiated with UVB, with a dose range of 50-500 mJ / cm 2 , the blank group was protected from light for the same period of time; after UVB irradiation, the culture medium was replaced and cultured for 24-48 hours before index detection; the indexes included one or more of the survival rate of human keratinocytes, the content of IL-8 in the culture medium and the content of Tail DNA; (3) Quality control: When the quality control conditions of the corresponding indicators are met, the results obtained are used to evaluate the performance of the test sample's sunburn protection efficacy; the quality control condition for the survival rate of human keratinocytes is that the survival rate of cells in the model group is significantly lower than that in the blank group; the quality control condition for the IL-8 content in the culture medium is that the survival rate of cells in the model group is ≥70%, and the IL-8 content in the model group is significantly higher than that in the blank group; the quality control condition for the Tail DNA content is that the survival rate of cells in the model group is ≥70%, and the Tail DNA (%) in the model group is significantly higher than that in the blank group; (4) Result evaluation: Calculate the difference in the index test results between the test sample group and the model group to evaluate the sunburn protection effect of the test sample.

8. The sunscreen function evaluation method according to claim 1, characterized in that: The method for detecting the sunscreen aging efficacy of human dermal fibroblasts comprises the following steps: (1) Human dermal fibroblasts in the logarithmic growth phase were inoculated into a culture plate and cultured until the density of the human keratinocytes reached 60%-80%. The test sample was administered to the human dermal fibroblasts in a non-contact manner and cultured, which was recorded as the experimental group; a blank group and a model group were also set up; (2) The experimental group and the model group were irradiated with UVA, with the irradiation dose ranging from 15-30 J / cm 2 , the blank group was protected from light for the same period of time; after UVA irradiation, the culture medium was replaced and cultured for 24-48 hours before index detection; the indexes included the collagen content in the culture medium and / or the rate of β-galactosidase-stained positive cells; (3) Quality control: When the quality control conditions of the corresponding indicators are met, the results obtained are used to test the performance evaluation of the sample's sunscreen efficacy; the quality control conditions for the collagen content in the culture medium are that the cell survival rate in the model group is ≥70%, and the collagen content in the culture medium in the model group is significantly lower than that in the blank group; the quality control conditions for the β-galactosidase staining positive cell rate are that the cell survival rate in the model group is ≥70%, and the β-galactosidase positive cell rate in the model group is significantly higher than that in the blank group; (4) Result evaluation: Calculate the difference in the test results of the test sample group and the model group to evaluate the sun protection effect of the test sample.

9. The sunscreen function evaluation method according to claim 1, characterized in that: The method for detecting the sunburn prevention efficacy of the zebrafish embryo model comprises the following steps: (1) 72 hpf zebrafish embryos were placed in a culture plate; the test sample was administered to the zebrafish embryos in a non-contact manner, which was recorded as the experimental group; a blank group and a model group were also set up; (2) The experimental group and the model group were irradiated with UVB, with the irradiation dose ranging from 400 to 1600 mJ / cm 2 The blank group was protected from light for the same period of time; the culture was continued for 24-72 hours after the UVB irradiation, and the mortality and teratogenicity rates of the zebrafish embryos were recorded at different times; (3) Quality control: In the zebrafish embryo model sunburn prevention efficacy test, the survival rate of zebrafish embryos in the blank group is ≥90%, and the survival rate of zebrafish embryos in the model group is significantly lower than that in the blank group. The obtained results are used as the evaluation results of the sunburn prevention efficacy of the test sample; (4) Result evaluation: Calculate the difference in the index test results between the test sample group and the model group to evaluate the sunburn protection effect of the test sample.

10. Use of the sunscreen function evaluation method according to any one of claims 1 to 9 in evaluating the sunscreen function of cosmetics or screening sunscreen cosmetics.

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