A method for evaluating the sun protection function of a cosmetic and cosmetic raw materials and its application

Abstract

The present invention belongs to the field of cosmetics detection, and discloses a method for evaluating the sun protection function of cosmetics and cosmetic raw materials and its application. This evaluation method uses a non-contact drug delivery device to test the sun protection effect of the test samples, and the test samples are finished cosmetics or diluted cosmetic raw materials; the tests include evaluating the in vitro sun protection efficacy by skin cells, evaluating the in vivo sun protection efficacy by the zebrafish embryo model, and result evaluation. This method evaluates the anti-aging effect of sun protection through the expression of β-galactosidase and / or the secretion of collagen in human dermal fibroblasts, and evaluates the sunburn prevention effect through the survival rate of human keratinocytes, DNA damage, or the secretion of inflammatory factors and the lethal / teratogenic conditions of zebrafish embryos, realizing multi-level and all-round evaluation of the sun protection efficacy. The method provided by the present invention is applicable to the evaluation of the sun protection function of various sun protection products, has accuracy and practicability, and provides an important technical basis for the research and development, quality control, and performance optimization of sun protection cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetic detection, and specifically discloses a method for evaluating the sun protection function of cosmetics and cosmetic raw materials and its application. Background Art

[0002] Ultraviolet rays are mainly divided into three types: 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. Among them, UVA can penetrate deep into the dermis layer of the skin, leading to skin photoaging, while UVB mainly causes skin sunburn. Sunscreen products (such as sunscreen lotions) are important means of protecting against ultraviolet rays. At present, the evaluation methods for the sun protection effect of sunscreen products mainly rely on ultraviolet spectroscopy instrument method, human SPF value method, and in vitro reconstructed skin model method.

[0003] The ultraviolet spectroscopy instrument method measures the ultraviolet absorbance and transmittance of sunscreen samples through an ultraviolet spectrometer to quickly evaluate the sun protection effect. Although this method is easy to operate, due to the relatively abstract results, it is difficult for consumers to intuitively understand, and it cannot fully simulate the physiological reactions of the skin, lacking a direct connection with the actual sun protection efficacy. The human SPF value method evaluates the effectiveness of sunscreen products by testing the sun protection effect on human skin. Although this method has high authority and persuasiveness, the testing process is costly, time-consuming, and involves human experiments, posing certain safety hazards. Therefore, it is mostly used for the effect verification before the launch of sunscreen products and is not suitable for the screening of sunscreen raw materials or initial research and development. The in vitro reconstructed skin model method uses an in vitro reconstructed skin model to evaluate the sun protection efficacy, with high safety and accuracy. However, the model preparation cycle of this method is long and the cost is high, which limits its wide application and is not suitable for quickly screening a large number of sunscreen raw materials or conducting preliminary pre-experiments. Some patents disclose the use of a quartz glass plate combined with mRNA expression detection method, using a quartz glass plate as a medium for receiving drugs, and evaluating the sun protection efficacy of raw materials or products by evaluating the mRNA expression level of cells after irradiation. However, this method is mainly used for the evaluation of physical sunscreens or raw materials, especially physical sunscreen products without transdermal activity. For chemical and biological sunscreens, it is difficult to accurately evaluate their safety and efficacy through this method. The existing methods for evaluating the sun protection function of cosmetics and cosmetic raw materials all have certain limitations, and there is an urgent need for a new evaluation method to overcome the deficiencies of the existing methods and meet the comprehensive evaluation needs of sunscreen raw materials and products. Summary of the Invention

[0004] In view of these deficiencies, the present invention provides a method for evaluating the sun protection function of cosmetics and cosmetic raw materials and its application. Based on cell and zebrafish models, combined with a non-contact drug delivery method, a comprehensive evaluation of sun protection efficacy is achieved through multi-level indicators: after irradiating human dermal fibroblasts with long-wave ultraviolet (UVA), the anti-aging efficacy of the test sample is characterized by the expression of intracellular β-galactosidase and / or the secretion of related collagen; after irradiating human keratinocytes / zebrafish with medium-wave ultraviolet (UVB), the sunburn protection efficacy of the test sample is characterized by detecting the cell viability, DNA damage or secretion of inflammatory factors of the cells and the lethality / teratogenicity of zebrafish embryos. This method can not only evaluate the sun protection efficacy in vitro at the cell level, but also simulate in vivo ultraviolet damage through the zebrafish model, thus forming a multi-dimensional evaluation system combining in vitro and in vivo. The sun protection efficacy evaluation system provided by the present invention is efficient, scientific and reliable, providing an important reference basis for the research and development and performance optimization of sun protection cosmetics.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for evaluating the sun protection function of cosmetics and cosmetic raw materials. The evaluation method uses a non-contact drug delivery method to test the sun protection effect of the test sample, and the test sample is a finished cosmetic product or a diluted cosmetic raw material; the test includes evaluating the in vitro sun protection efficacy of skin cells and / or evaluating the in vivo sun protection efficacy of a zebrafish embryo model.

[0007] The non-contact drug delivery method is realized by means of a non-contact drug delivery device; the non-contact drug delivery device includes a drug delivery carrier layer, and the drug delivery carrier layer is used to carry the test sample; the test sample is separated from the skin cells or the zebrafish embryos by the drug delivery carrier layer; the drug delivery carrier layer is a film with light transmissibility, and the film may or may not have micropores, and the pore size of the micropores is 0.4-25 μm.

[0008] The evaluation of the in vitro sun protection efficacy of skin cells includes detecting the sunburn protection efficacy of human keratinocytes and detecting the anti-aging efficacy of human dermal fibroblasts; the detection of the sunburn protection efficacy of human keratinocytes includes measuring one or more changes in the cell viability of human keratinocytes, the content of IL-8 in the culture medium or the content of Tail DNA caused by the test sample after UVB irradiation; the detection of the anti-aging efficacy of human dermal fibroblasts 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 evaluation of the in vivo sun protection efficacy of the zebrafish embryo model includes detecting the sunburn protection efficacy of the zebrafish embryo model; the detection of the sunburn efficacy of the zebrafish embryo model includes measuring the changes in the lethality rate and teratogenicity rate of zebrafish embryos caused by the test sample after UVB irradiation.

[0009] Before measuring the change, uniformly coat the test sample on the drug delivery carrier layer; place the skin cells and the zebrafish embryos under the drug delivery carrier layer, and irradiate ultraviolet light above the drug delivery carrier layer.

[0010] The sunscreen evaluation method provided by the present invention combines the change indexes of skin cells and zebrafish embryo models to achieve a multi-level comprehensive evaluation of in vitro and in vivo sunscreen efficacy. The sunscreen burn efficacy is evaluated by UVB irradiation, and the survival rate, DNA damage (Tail DNA content) or inflammatory factor (IL-8) secretion change of human keratinocytes, as well as the lethality and teratogenic rate of zebrafish embryos are detected; the sunscreen anti-aging 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 light damage and photoaging effects of ultraviolet rays on human skin are comprehensively simulated by using skin cell and embryo models.

[0011] 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 uniformly transmit ultraviolet light to the skin cells or zebrafish embryos under the test sample, truly simulating the environment of ultraviolet rays irradiating human skin. The drug delivery carrier layer realizes permeability through microporous design, allowing the active ingredients in chemical and biological sunscreen cosmetics to diffuse to the periphery of the test model and contact the skin cells or zebrafish embryos without destroying the cosmetic system. Physical sunscreen cosmetics can achieve isolated contact between physical sunscreen cosmetics and skin cells or zebrafish embryos through a non-contact drug delivery device without micropores, or use a non-contact drug delivery device with micropores to keep a certain spatial distance between the culture liquid surface of skin cells or zebrafish embryos and the drug delivery carrier layer to achieve isolated contact between physical sunscreen cosmetics and skin cells or zebrafish embryos. This 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.

[0012] The evaluation method provided by the present invention is applicable to physical sunscreen cosmetics or cosmetic raw materials, including but not limited to sunscreen raw materials such as titanium dioxide, zinc oxide and octocrylene, as well as sunscreen products such as sunscreen creams, sunscreen sticks and sunscreen sprays containing these ingredients. These sunscreen substances are not easily soluble in water and do not penetrate the skin, and effectively prevent ultraviolet irradiation through a physical barrier mechanism, usually by reflecting or scattering ultraviolet rays.

[0013] 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 sunscreen agents such as avobenzone, simvastatin and benzophenone. These ingredients achieve sunscreen effects by absorbing ultraviolet rays and converting them into heat energy.

[0014] 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, microbial fermentation products, etc. The biological sunscreen ingredients provide ultraviolet protection by enhancing the natural barrier function or antioxidant effect of the skin.

[0015] In the present invention, the deformities in the teratogenic rate include one or more of the following: developmental arrest, absence of swim bladder, pericardial cyst, spinal curvature, yolk sac edema, self-decomposition of tail and caudal fin.

[0016] In some embodiments, the method for evaluating the sunscreen function of a cosmetic and cosmetic raw materials provided by the present invention further includes the following operations:

[0017] Culture of human keratinocytes;

[0018] Culture of human dermal fibroblasts;

[0019] Obtain zebrafish embryos: Pair sexually mature zebrafish at a male-female ratio of 1:2. After natural mating, the female fish lays eggs, and the obtained zebrafish embryos are obtained.

[0020] Treatment of cosmetic / cosmetic raw material samples: Cosmetic raw materials are diluted with an appropriate solvent according to the proposed addition amount of the product.

[0021] Preferably, the non-contact dosing device includes 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 dosing carrier layer forms the bottom of the embedded chamber; the chamber of the embedded chamber is located in the groove, and a horizontally outward extending edge is circumferentially provided at the upper port of the embedded chamber. 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; there is a gap between the dosing carrier layer and the bottom of the groove; when using the non-contact dosing device for sunscreen efficacy, except for the bottom dosing carrier layer, the rest of the embedded chamber needs to be treated to isolate ultraviolet rays.

[0022] 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 dosing 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. Except for the bottom dosing carrier layer, the rest of the embedded chamber needs to be treated to isolate ultraviolet rays to ensure that the bottom dosing carrier layer is the only path for ultraviolet irradiation of skin cells or zebrafish embryos. One or a combination of treatment methods such as selecting ultraviolet-proof materials, surface coating treatment, physical shielding design, or dyeing treatment can be used to meet the requirement of isolating ultraviolet rays for the rest of the embedded chamber except the bottom dosing carrier layer.

[0023] The culture plate can be a common cell culture plate, and the culture chamber of the culture plate is the groove of the culture plate. The embedded chamber can be made into various specifications adapted to the culture plate according to the test requirements, such as 96-well plate specification, 48-well plate specification, 24-well plate specification, and 6-well plate specification. At the same time, on the premise of not affecting the test results, the embedded chamber can be reused after being cleaned and sterilized by ultraviolet irradiation.

[0024] The drug delivery carrier layer of the embedded chamber can be made of materials with stable light transmission performance, such as inorganic glass and traditional chemical plastics. These materials are processed by microporous processing technology to form a structure with uniform light transmission and permeability, meeting the requirements of ultraviolet light transmission and effective ingredient diffusion. The materials of the drug delivery carrier layer of the embedded chamber include but are not limited to optical quartz glass, ultraviolet-transmitting black glass, polymethyl methacrylate, polystyrene, polycarbonate, and polyethylene terephthalate.

[0025] These materials have excellent light transmission performance, can efficiently transmit ultraviolet light, and truly simulate the environment of ultraviolet light irradiating human skin; at the same time, they have good processing performance, support the precise formation of microporous structures, and have good chemical stability, being non-toxic to cells, ensuring the reliability and accuracy of experimental results.

[0026] More preferably, the drug delivery carrier layer of the embedded chamber does not have micropores and is made of polymethyl methacrylate.

[0027] Polymethyl methacrylate (PMMA) has extremely high transmittance in the ultraviolet band, can effectively transmit ultraviolet light energy; its surface is smooth, and the 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 the carrier layer without micropores can effectively detect the shielding effect of physical sunscreen cosmetics or raw materials in forming a protective film on the surface, thereby evaluating their protective ability.

[0028] More preferably, the drug delivery carrier layer of the embedded chamber has micropores and is made of polyethylene terephthalate.

[0029] Polyethylene terephthalate (PET) has good light transmittance and chemical stability. Through specific processing technologies, such as foaming, microporous injection molding, or compounding with other porous materials, the PET material can be made to have a microporous structure. The drug delivery carrier layer made of PET material with micropores can control the release of sunscreen ingredients, simulate the penetration and absorption under actual use conditions, and is suitable for the detection of the sunscreen efficacy of chemical and biological sunscreen cosmetics.

[0030] More preferably, the distance between the drug delivery carrier layer and the bottom of the culture plate is 0.5 - 1 cm.

[0031] More preferably, the surface of the material of the embedded chamber drug delivery carrier layer has certain concave and convex textures, and the thickness is 1-4 mm.

[0032] PMMA plates with certain concave and convex textures, certain roughness on the surface, and a thickness of 1-4 mm can better simulate the roughness and folds of human skin.

[0033] Preferably, the method for detecting the sunburn prevention effect of human keratinocytes includes the following steps:

[0034] (1) Take human keratinocytes in the logarithmic growth phase and inoculate them on a culture plate. When the density of the human keratinocytes reaches 60%-80%, administer the test sample to the human keratinocytes by non-contact drug delivery for culture, which is recorded as the experimental group; at the same time, set a blank group and a model group;

[0035] (2) Irradiate the experimental group and the model group with UVB, and the irradiation dose range is 50-500 mJ / cm 2 , and the blank group is protected from light for the same time; after the UVB irradiation ends, change the culture medium and continue to culture for 24-48 h, and then perform index detection; the indexes include 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;

[0036] (3) Quality control: When the quality control conditions corresponding to the indexes are met, the obtained results are used for the performance evaluation of the sunburn prevention effect of the test sample; the quality control condition for the survival rate of human keratinocytes is that the survival rate of the cells in the model group is significantly lower than that in the blank group; the quality control condition for the content of IL-8 in the culture medium is that the survival rate of the cells in the model group ≥ 70%, and the content of IL-8 in the model group is significantly higher than that in the blank group; the quality control condition for the content of Tail DNA is that the survival rate of the cells in the model group ≥ 70%, and the Tail DNA (%) in the model group is significantly higher than that in the blank group;

[0037] (4) Result evaluation: Calculate the difference in the index detection results between the test sample group and the model group to evaluate the sunburn prevention effect of the test sample.

[0038] More preferably, the method for result evaluation in step (4) is: calculate the difference in any one of the index detection results between the test sample group and the model group. If p < 0.05, it proves that the test sample has a sunburn prevention effect.

[0039] Preferably, the method for detecting the anti-aging effect of human dermal fibroblasts includes the following steps:

[0040] (1) Human dermal fibroblasts in the logarithmic growth phase were inoculated into a culture plate. When the density of the human keratinocytes reached 60%-80%, the test sample was administered to the human dermal fibroblasts by non-contact administration for culture, which was recorded as the experimental group. At the same time, a blank group and a model group were set up.

[0041] (2) The experimental group and the model group were irradiated with UVA, and the irradiation dose range was 15-30 J / cm 2 , and the blank group was treated in the dark for the same time. After the UVA irradiation was completed, the culture medium was changed and the cells were cultured for another 24-48 h, and then the indexes were detected. The indexes included the content of collagen in the culture medium and / or the positive cell rate of β-galactosidase staining.

[0042] (3) Quality control: When the quality control conditions for the corresponding indexes were met, the obtained results were used for the performance evaluation of the anti-aging effect of the test sample. The quality control conditions for the collagen content in the culture medium were that the cell survival rate of the model group was ≥70%, and the collagen content in the culture medium of the model group was significantly lower than that of the blank group. The quality control conditions for the positive cell rate of β-galactosidase staining were that the cell survival rate of the model group was ≥70%, and the positive cell rate of β-galactosidase in the model group was significantly higher than that of the blank group.

[0043] (4) Result evaluation: The anti-aging effect of the test sample was evaluated by calculating the difference in the index detection results between the test sample group and the model group.

[0044] More preferably, the method for result evaluation in step (4) is: calculate the difference in any one of the index detection results between the test sample group and the model group. If p<0.05, it is proved that the test sample has anti-aging effect.

[0045] Preferably, the method for detecting the sunburn prevention effect of the zebrafish embryo model includes the following steps:

[0046] (1) Zebrafish embryos at 72 hpf were placed in a culture plate. The test sample was administered to the zebrafish embryos by non-contact administration for culture, which was recorded as the experimental group. At the same time, a blank group and a model group were set up.

[0047] (2) The experimental group and the model group were irradiated with UVB, and the irradiation dose range was 400-1600 mJ / cm 2 , and the blank group was treated in the dark for the same time. After the UVB irradiation was completed, the embryos were cultured for another 24-72 h, and the lethality rate and teratogenic rate of the zebrafish embryos at different times were recorded.

[0048] (3) Quality control: In the detection of the sunburn prevention efficacy of the zebrafish embryo model, 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 performance of the test sample.

[0049] (4) Result evaluation: The sunburn prevention efficacy of the test sample is evaluated by calculating the difference in the detection results of the indicators between the test sample group and the model group.

[0050] More preferably, the method for result evaluation in step (4) is: calculate the difference in any one of the detection results of the indicators between the test sample group and the model group. If p < 0.05, it proves that the test sample has sunburn prevention efficacy.

[0051] More preferably, the non-contact drug delivery method is as follows: after completing skin cell culture or zebrafish embryo modeling in a culture plate without contacting the drug delivery device, the culture medium in the experimental group culture plate is changed to PBS, and the test sample is added to the drug layer carrier layer of the embedded chamber. Then, a sterile cotton swab or pipette tip is used to evenly disperse the test sample on the drug layer carrier layer; the culture medium in the culture plates of the blank group and the model group is changed to PBS, and a dilution solvent with the same mass and volume as the test sample is added to the drug layer carrier of the embedded chamber. Then, a sterile cotton swab or pipette tip is used to evenly disperse the dilution solvent on the drug layer carrier layer.

[0052] In the second aspect, the present invention also provides the application of the sun protection function evaluation method described in any one of the above in evaluating the sun protection function of cosmetics or screening sun protection cosmetics.

[0053] The sun protection function evaluation method provided by the present invention can be widely applied to the screening and function verification of sun protection cosmetics and cosmetic raw materials. During the research and development process of sun protection products, this evaluation method can be used to optimize the formula and improve the sun protection performance of the products. In the quality control link, it can be used as a standardized test tool to ensure the consistency and stability of product quality. Description of the drawings

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0055] Figure 1 It is a schematic diagram of the non-contact drug delivery method and the structure of the non-contact drug delivery device;

[0056] Figure 2 It is the influence of different UVB irradiation intensities on the survival rate of keratinocytes in Example 3;

[0057] Figure 3 For the effect of different UVA irradiation intensities on the senescence of dermal fibroblasts in Example 3;

[0058] Figure 4 For the effect of different UVB irradiation intensities on the teratogenic and lethal rates of zebrafish embryos in Example 3;

[0059] Figure 5 For 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);

[0060] Figure 6 For the detection of the DNA protection effect of sunscreen cosmetics in Example 5 (*p < 0.05, **p < 0.01 vs model group);

[0061] Figure 7 For the detection of the protection effect of sunscreen cosmetics on zebrafish in Example 6 (*p < 0.05, **p < 0.01 vs model group);

[0062] Figure 8 For the results of β-galactosidase staining in Example 7;

[0063] Figure 9 For the detection of the protection effect of sunscreen cosmetics on human dermal fibroblasts in Example 7 (*p < 0.05, **p < 0.01 vs model group). Detailed implementation manners

[0064] Next, in combination with the implementation manners of the present invention, the technical solutions in the implementation manners of the present invention will be clearly and completely described. Obviously, the described implementation manners are only a part of the implementation manners of the present invention, rather than all of the implementation manners. Based on the implementation manners in the present invention, all other implementation manners obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0065] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0066] The sunscreen cosmetics used in the present invention are all sourced from the market.

[0067] The culture media used in the examples of the present invention:

[0068] DMEM culture medium: 85% - 90% DMEM culture medium, 10% - 15% fetal bovine serum, 1% antibiotic, pH 7.0 - 7.4.

[0069] E3 culture medium: 5 mM NaCl, 0.17 mM KCl, CaCl 2 ·2H 2O 0.33 mM, MgSO 4 ·7H 2 O 0.33 mM. Example 1

[0070] This example provides a non-contact drug delivery device.

[0071] The non-contact drug delivery device includes an embedded chamber and a culture plate; the embedded chamber is a cup-shaped structure with an open upper end, the drug delivery carrier layer forms the bottom of the embedded chamber, the material of the drug delivery carrier layer is PMMA, and the thickness is 1 mm; a horizontally outward extending edge is circumferentially provided at the upper port of the embedded chamber, 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, and there is a 1 cm spacing 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 well and outside the embedded chamber is the lower chamber.

[0072] Non-contact drug delivery method: Except for the bottom drug delivery carrier layer, the rest of the embedded chamber is coated with aluminum film for ultraviolet isolation treatment. The test sample is coated on the drug delivery carrier layer, and the skin cells or the zebrafish embryos are placed below the drug delivery carrier layer inside the culture plate. Example 2

[0073] This example provides a non-contact drug delivery device. The difference from Example 1 is that the material of the drug delivery carrier layer is PET, with uniformly distributed micropores with a pore diameter of 10 μm and a thickness of 4 mm; there is a 0.5 cm spacing between the drug delivery carrier layer and the bottom of the culture plate.

[0074] Example 3 Irradiation dose

[0075] 1. Detection of sunburn prevention efficacy of human keratinocytes

[0076] Take the digested human keratinocytes and culture them in a 24-well culture plate, divided into a blank group, Model Group 1 (the non-contact drug delivery device provided in Example 1), and 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 reaches 80%, remove the culture medium. In the blank group, add 50 μL of PBS to both the upper chamber and the lower chamber for light shielding treatment; in Model Group 1, add 50 μL of PBS to the upper chamber and 50 μL of PBS to the lower chamber without light shielding treatment; in Model Group 2, add 50 μL of PBS to the upper chamber and 50 μL of PBS to the lower chamber without light shielding treatment. The above three groups are respectively irradiated in a UV crosslinker with 50 mJ / cm 2 、100 mJ / cm 2 、150 mJ / cm 2 、250 mJ / cm 2 、300 mJ / cm 2 UVB, 500 mJ / cm2 Irradiate with UVB. After the irradiation is completed, take out the embedded chambers in the model group, and re-add 500 μL of 10% FBS DMEM medium to each culture plate and continue to culture for 24 h, then measure the cell survival rate. The results are as Figure 2 shown.

[0077] As Figure 2 shown, the PMMA material has better light transmittance than the PET material, but both can achieve the purpose of irradiating the cells below by adjusting the UVB irradiation dose. And when the irradiation intensity is 150 mJ / cm 2 and above, the cell survival rate below the PMMA material decreases significantly. Therefore, when using the PMMA material as the drug delivery carrier layer to detect the sunburn prevention effect of human keratinocytes, select 100 - 150 mJ / cm 2 as the optimal modeling condition. When using the PET material as the drug delivery carrier layer to detect the sunburn prevention effect of human keratinocytes, select 500 mJ / cm 2 as the optimal modeling condition.

[0078] 2. Detection of the anti - aging effect of human dermal fibroblasts against UVA

[0079] Take the digested human dermal fibroblasts and culture them in a 6 - well culture plate. Divide them into a blank group, model group 1 (the non - contact drug delivery device provided in Example 1), and model group 2 (the non - contact drug delivery device provided in Example 2), with three replicates in each group. When the density of fibroblasts reaches 60%, remove the medium. Add 200 μL of PBS to both the upper and lower chambers of the blank group for light - shielding treatment; add 200 μL of PBS to the upper chamber and 200 μL of PBS to the lower chamber of model group 1, without light - shielding treatment; add 200 μL of PBS to the upper chamber and 200 μL of PBS to the lower chamber of model group 2, without light - shielding treatment. The above three groups are irradiated in a UVA ultraviolet instrument with 15 J / cm 2 , 20 J / cm 2 , 30 J / cm 2 respectively. After the irradiation is completed, take out the embedded chambers in the model group, and re - add 2 mL of 15% FBS DMEM medium to each culture plate and continue to culture for 24 h, then perform β - galactosidase staining. The results are as Figure 3 shown.

[0080] As Figure 3 shown, the UVA irradiation effect through the PMMA material and the PET material is equivalent, and when the irradiation intensity is 20 J / cm 2 and above, the positive cell rate of β - galactosidase in human dermal fibroblasts increases significantly. Therefore, 20 - 30 J / cm 2 can be selected as the optimal irradiation dose range for UVA - induced cell senescence.

[0081] 3. Detection of the sunburn prevention effect of zebrafish embryo model

[0082] Place 72 hpf zebrafish embryos in a 6-well culture plate for modeling culture, 30 embryos per well, 3 wells per group, divided into a blank group, model group 1 (the non-contact drug delivery device provided in Example 1), and model group 2 (the non-contact drug delivery device provided in Example 2), with three replicates in each group. The blank group is treated in the dark. In model group 1, add 250 μL of E3 medium to the upper chamber and 250 μL of E3 medium to the lower chamber, without dark treatment; in model group 2, add 250 μL of E3 medium to the upper chamber and 250 μL of E3 medium to the lower chamber, without dark treatment. The above three groups are respectively irradiated in a UV crosslinker with 400 mJ / cm 2 、800 mJ / cm 2 UVB, 1600 mJ / cm 2 UVB. After irradiation, take out the inner chamber and E3 medium of the model group, replace with new E3 medium, and continue to culture for 48 h, then count the teratogenic rate and lethality of zebrafish.

[0083] As Figure 4 shown, PMMA and PET showed different UVB transmission effects, that is, the light transmission effect of PMMA on UVB was better than that of PET, but both could achieve the purpose of irradiating the zebrafish embryos below by adjusting the UVB irradiation dose. And when the irradiation intensity was 400 mJ / cm 2 and above, the malformation or mortality rate of zebrafish under PMMA material increased significantly. Therefore, when using PMMA material as the drug delivery carrier layer to detect the sunburn prevention effect of zebrafish embryo model, 400 - 800 mJ / cm 2 is selected as the modeling condition. When using PET material as the drug delivery carrier layer to detect the sunburn prevention effect of zebrafish embryo model, 800 - 1600 mJ / cm 2 is selected as the modeling condition.

[0084] Example 4: Test of method stability

[0085] Use zinc oxide raw material (described as "sun protection raw material" below) and the non-contact drug delivery devices provided in Examples 1 and 2 to test the stability of the evaluation method provided by the present invention.

[0086] Detection method: Take the digested human keratinocytes and culture them in a 24-well culture plate, divided 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. In the blank group, add 50 μL of PBS to the lower chamber and 50 μL of PBS to the upper chamber, and perform light-shielding treatment; in the model group, add 50 μL of PBS to the lower chamber and 50 μL of PBS to the upper chamber, without light-shielding treatment; in the sunscreen raw material group, add 50 μL of PBS to the lower chamber, and evenly apply 50 μL of PBS containing 10% sunscreen raw material on the drug delivery carrier layer, without light-shielding treatment. Perform UVB irradiation, irradiate the non-contact drug delivery device provided in Example 1 with 100 mJ / cm 2 UVB, and irradiate the non-contact drug delivery device provided in Example 2 with 500 mJ / cm 2 UVB. After irradiation, remove the inner chamber and replace it with 500 μL of 10% FBS DMEM culture medium and continue to culture for 24 h, then measure the cell viability; and repeat the above test method three times with different batches of human keratinocytes. The results are shown in Figure 5 .

[0087] Figure 5 A is the stability test result of using the non-contact drug delivery device provided in Example 1; Figure 5 B is the stability test result of using the non-contact drug delivery device provided in Example 2.

[0088] As Figure 5 shown, the survival rates of the model group and the sunscreen raw material group are close in the three test results, indicating the consistency between parallel results. The survival rates of the model group cells using the non-contact drug delivery device (the drug delivery carrier layer material is PMMA) provided in Example 1 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 model group cells using the non-contact drug delivery device (the drug delivery carrier layer material is PET) provided in Example 2 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

[0089] In the experiment of this example, 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 product 1"); chemical sunscreen formulation 2 (containing 15% octocrylene, hereinafter described as "chemical sunscreen product 2") was used as the test sample.

[0090] Take the digested human keratinocytes and culture them in a 6-well culture plate. They are divided into a blank group, a model group, a chemical sunscreen 1 group, and a chemical sunscreen 2 group, with three replicates in each group. When the density of human keratinocytes reaches 80%, remove the culture medium. Add 200 μL of PBS to both the upper and lower chambers of the blank group and perform light avoidance treatment; add 200 μL of PBS to both the upper and lower chambers of the model group without performing light avoidance treatment; add 200 μL of PBS to the lower chamber of the chemical sunscreen group and add 200 μL of the corresponding chemical sunscreen formulation to the upper chamber without performing light avoidance treatment. Use a UV crosslinker for 50 mJ / cm 2 UVB irradiation. After the irradiation is completed, remove the inner chamber. Each group is changed to 2 mL of 10% FBS DMEM culture medium and continue to culture for 3 h. Then, detect the DNA damage in the cells using a comet electrophoresis kit, and analyze the obtained image data through CASP.

[0091] As Figure 6 shown, UVB irradiation causes DNA damage in human keratinocytes. In the chemical sunscreen 1 group (containing 5% octocrylene) compared with the model group, there is little DNA damage, proving that this chemical sunscreen has a sunscreen effect. However, in the chemical sunscreen 2 group (containing 15% octocrylene), the DNA damage situation not only does not alleviate but instead worsens. This is because an excessive amount of octocrylene is used as a protective agent in chemical sunscreen 2, and octocrylene is reported to have potential skin penetration, causing a risk of DNA damage to skin cells. Therefore, when using a permeable drug delivery carrier layer, not only can the sunscreen effect of appropriately using octocrylene be detected, but also the toxic and side effects of the sunscreen agent when using an excessive amount of the risk sunscreen agent can be monitored. Example 6

[0092] This example provides a method for evaluating the sunburn prevention effect of a sunscreen cosmetic. This evaluation method uses the non-contact drug delivery device provided in Example 1. Use a physical sunscreen cosmetic as the test sample.

[0093] Place 72 hpf zebrafish embryos in a 6-well culture plate for modeling culture, 30 embryos per well, 3 wells per group, divided into a blank group, a model group, and a sunscreen cosmetic group. After the modeling is completed, change the lower chamber of the blank group to 250 μL of normal E3 culture medium and perform light avoidance treatment; change the lower chamber of the model group to 250 μL of normal E3 culture medium without performing light avoidance treatment; change the lower chamber of the sunscreen cosmetic group to 250 μL of normal E3 culture medium, and evenly apply 250 mg of the sunscreen cosmetic stock solution on the drug delivery carrier layer of the upper chamber without performing light avoidance treatment. Use a UV crosslinker for 800 mJ / cm 2 UVB irradiation. After the irradiation is completed, remove the inner chamber and the E3 culture medium, and change to a new E3 culture medium. Record the number of teratogenic and lethal zebrafish at 48 h. The results are as Figure 7 shown.

[0094] It was detected that this physical sunscreen cosmetic could resist the damage of UVB to zebrafish embryos. Comparative Example 1

[0095] This example provides a method for evaluating the sunburn prevention efficacy of a sunscreen cosmetic. The difference from Example 6 is that without using the embedded chamber, after the model is established, the blank group culture plate is replaced with 250 μL of normal E3 medium and subjected to light avoidance treatment; the model group culture plate is replaced with 250 μL of normal E3 medium without light avoidance treatment; the sunscreen cosmetic group culture plate is replaced with 250 μL of E3 medium containing 10% sunscreen cosmetic without light avoidance treatment. Use a UV cross-linker for 800 mJ / cm 2 UVB irradiation. After the irradiation is completed, remove the E3 medium and replace it with fresh E3 medium. Record the number of teratogenic and lethal zebrafish at 48 h, and calculate the teratogenic rate and lethal rate. The results are as Figure 7 shown.

[0096] It can be seen from the results that using the traditional method of adding drugs, the direct contact between zebrafish embryos and the sunscreen cosmetic causes teratogenesis and lethality. Even when using the diluted sample solution, the teratogenic rate and lethal rate of zebrafish in its group are still much higher than those of the model group, resulting in unavailable results and being unable to reflect the sunburn prevention efficacy of the cosmetic. Moreover, during the experiment, the operation is cumbersome and not easy to observe. In contrast, using the evaluation method provided by the present invention, there is no need to dilute the sunscreen cosmetic, and it has basically no negative impact on the activity of zebrafish. There is no significant difference in the teratogenic rate and lethal rate between the model group with the non-contact drug delivery method and the model group with the traditional drug addition method, further proving the reliability and applicability of the method of the present invention in the irradiation experiment. Example 7

[0097] In the experiment of this example, 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 product") was used as the test sample.

[0098] After human dermal fibroblasts were cultured for 2-3 generations, take the digested human dermal fibroblasts and culture them in a 6-well culture plate, divided into a blank group, a model (PMMA) group, a model (PET) group, a biological sunscreen product (PMMA) group, and a biological sunscreen product (PET) group, with three replicates in each group. When the density of human dermal fibroblasts reaches 60%, remove the culture medium. 200 μL of PBS is added to both the upper chamber and the lower chamber of the blank group and subjected to light avoidance treatment; 200 μL of PBS is added to both the upper chamber and the lower chamber of the model group without light avoidance treatment; 200 μL of PBS is added to the lower chamber of the biological sunscreen product group, and the biological sunscreen product is evenly applied on the drug delivery carrier layer of the upper chamber without light avoidance treatment. Use a UV cross-linker for 20 J / cm 2After UVA irradiation, the embedded chamber was removed after the irradiation was completed. Each group was changed to 2 mL of 15% FBS DMEM medium and continued to be cultured for 24 h. Then, senescent cells were stained with a β-galactosidase staining kit, and the positive rate of β-galactosidase cells was statistically analyzed.

[0099] The results are as Figure 8 shown. There was no significant difference in the positive rate of β-galactosidase cells between the model (PMMA) group and the model (PET) group, indicating that there was no obvious difference in the UVA transmission effect between PMMA material and PET material. This conclusion was consistent with the test conclusion of Example 3. The positive rate of β-galactosidase cells in the biological sunscreen (PET) group was significantly lower than that in the model (PET) group, while there was no significant difference in the positive rate of β-galactosidase cells between the biological sunscreen (PMMA) group and the model (PMMA) group. Biological sunscreens usually exert their sun protection effects by enhancing the cells' own ability to resist UVA. Therefore, the evaluation method with a permeable drug delivery carrier layer is suitable for the sun protection evaluation of biological sunscreens.

[0100] In summary, a test method for evaluating the sun protection efficacy of cosmetics or cosmetic raw materials involved in the present invention has the advantages of simple operation, wide applicability, and stable results, and is suitable for detecting the sun protection efficacy of cosmetics / cosmetic raw materials.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for evaluating the sunscreen function of cosmetics and a method for evaluating the sunscreen function of 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 is to evaluate the in vitro sunscreen efficacy of skin cells and / or to evaluate the in vivo sunscreen efficacy of zebrafish embryo models; 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 micropores, and the micropores have an aperture size of 0.4-25 μm; The non-contact 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; the drug delivery carrier layer and the bottom of the groove are spaced apart; when the non-contact drug delivery device is used for sunscreen efficacy testing, the embedded chamber, except for the bottom drug delivery carrier layer, needs to be treated to isolate ultraviolet rays; 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 cell survival rate of human keratinocytes, IL-8 content in 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 is a 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 or 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 drug delivery carrier layer of the embedded chamber has micropores and is made of polyethylene terephthalate.

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

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

5. 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 the survival rate of human keratinocytes, the IL-8 content or the Tail DNA content in the culture medium; (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 sunscreen 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 content 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.

6. 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 when the density of the human dermal fibroblasts reached 60%-80%, the test sample was administered to the human dermal fibroblasts 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 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.

7. 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 24-48 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 sample to be tested; (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. Use of the sunscreen function evaluation method according to any one of claims 1 to 7 in screening sunscreen cosmetics.

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