Method for evaluating sunscreen effect and application thereof

Abstract

The invention belongs to the technical field of cosmetic efficacy evaluation, and discloses a sunscreen efficacy evaluation method and application thereof, and the method comprises the following steps: building a blank group; establishing a model group; establishing a sample group; performing constant-temperature incubation on the blank group, the model group and the sample group for a first period of time, recording a swimming track, and calculating swimming distance increment and after-sun burning pain relieving efficiency of each group of zebrafish; performing constant-temperature incubation on the blank group, the model group and the sample group for a second duration, recording the tail fin and dorsal fin area of the zebra fish, and calculating the sunburn prevention efficiency; performing constant-temperature incubation on the blank group, the model group and the sample group for a third period of time, recording the head melanin area of the zebra fish, and calculating the anti-sunburn efficiency; and grading the sunscreen effect of the sample. The method disclosed by the invention has the advantages of low cost, short test period, high reproducibility, high visualization degree, intuitive and understandable test result and the like, and can be used for accurately grading the sunscreen effects of chemical sunscreen agents, water-soluble sunscreen cosmetics and raw materials thereof from the whole animal level.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic efficacy evaluation, and in particular to a method for evaluating sunscreen efficacy and application thereof. Background Art

[0002] Ultraviolet radiation can be divided into three wavelengths: UVA (320–400 nm), UVB (280–320 nm), and UVC (100–280 nm). Most UVC is absorbed by the ozone layer and prevents it from reaching the Earth's surface. Therefore, it is primarily UVA and UVB that cause sunburn and tanning. Compared to UVA, UVB radiation has a more intense effect on the skin and is a common cause of sunburn. UVB is absorbed by the epidermis, causing a series of changes in epidermal cells, including DNA damage, altered gene expression, and increased levels of reactive oxygen species, altering epidermal structure. UVB sensitizes peripheral nerves in damaged skin, increasing the skin's sensitivity to mechanical and thermal stimuli and causing tenderness and heat pain. UVB also stimulates melanin production, leading to tanning. UVB damages the DNA of epidermal melanocytes, activating tyrosinase activity and promoting the production of new melanosomes. These melanosomes are then transported to neighboring keratinocytes via dendritic cells. On the other hand, UVB stimulates epidermal cells to produce a large amount of ROS, which accelerates the reaction between dihydroxyindole and indolequinone in the final stage of melanin production.

[0003] Excessive exposure to UVB rays can cause erythema and scaling on the skin, followed by reactions such as heat, tenderness, and swelling. Localized melanin deposition can persist for weeks to months. These symptoms appear within 1 to 24 hours of UVB exposure, typically peaking between 12 and 24 hours. In severe cases, blisters or bullae may develop, and even systemic symptoms such as fever, chills, weakness, and shock may occur. Long-term exposure to UVB rays not only accelerates skin aging but can also increase the risk of skin cancer.

[0004] In order to avoid excessive exposure of the skin to UVB and to prevent the skin from being sunburned or tanned, sunscreen clothing, sunscreen cosmetics and other means are used to provide photoprotection. In addition to traditional sunscreens such as physical blockers and chemical absorbers, natural and pure cosmetics currently on the market add natural product extracts as photoprotective agents to filter or absorb UVB. Existing sunscreen models mainly include cell models, 3D full-thickness skin models, mouse skin models, and human skin models. Among them, cell models and 3D full-thickness skin models are in vitro models, which are difficult to simulate the interactions between cells in organisms and cannot reflect the actual reaction of products or active ingredients on the skin. The ban on animal testing of cosmetics restricts the use of animal models. Although human testing is the main way to claim the safety and efficacy of cosmetics, the testing cycle is long and the cost is expensive, making it unsuitable for the initial screening of active ingredients.

[0005] As a small vertebrate model organism, zebrafish has a genetic homology of up to 87% with humans, and its skin physiological structure is highly similar to that of humans. At the same time, it has the advantages of transparent juveniles, small size, strong reproductive capacity, and short development cycle. It makes up for the shortcomings of existing methods and provides a useful tool for sunscreen efficacy evaluation. Chinese patents CN201711342756 and CN201810747616 disclose a method for evaluating the sunscreen efficacy of cosmetics using zebrafish, but its evaluation indicators only include the tail fin retraction rate. Chinese patent CN202410463775 discloses a method for constructing a zebrafish ultraviolet damage model. Although its evaluation indicators include the tail fin retraction rate and melanin content, these two indicators are not combined for evaluation. The evaluation indicators of the above-mentioned zebrafish evaluation model are relatively single, and it is impossible to comprehensively evaluate the sunscreen effect of samples or active substances. Summary of the Invention

[0006] The purpose of the present invention is to provide a method and application for evaluating sunscreen efficacy, wherein the evaluation method comprehensively evaluates the sunscreen effect of a sample or active substance from three dimensions: post-sunburn pain relief, sunburn prevention, and suntan prevention.

[0007] The present invention provides a technical solution: a method for evaluating sunscreen efficacy, comprising the following steps:

[0008] Step S1, selecting several zebrafish with the same tail development status and not treating them to establish a blank group;

[0009] Step S2, selecting the same number of zebrafish with the same developmental status as in step S1, stimulating the zebrafish with UVB to establish a model group;

[0010] Step S3, selecting the same number of zebrafish as in step S1 and with the same developmental status, adding the test sample, and stimulating the zebrafish with UVB to establish a sample group;

[0011] Step S4: After the blank group, model group, and sample group are incubated in a constant temperature chamber for a first period of time, the zebrafish are placed in a behavioral recorder preheated to different temperatures for observation. Infrared tracking software is used to record the swimming trajectory and swimming distance of the zebrafish in a dark environment for a specified period of time, and the post-sunburn pain relief efficiency is calculated;

[0012] Step S5, after incubating the blank group, model group, and sample group in the incubator for a second period of time, observing the zebrafish under a microscope, recording the tail fin and dorsal fin areas of the zebrafish, and calculating the sunburn prevention efficiency;

[0013] Step S6, after the blank group, the model group, and the sample group are incubated in the incubator for a third period of time, the zebrafish are observed under a microscope, and the melanin area of ​​the zebrafish's head is recorded to calculate the suntanning efficiency;

[0014] Step S7: Rating the sunscreen efficacy of the sample according to the sunscreen efficacy rating system.

[0015] In the above method for evaluating the efficacy of sunscreen, the zebrafish are wild AB zebrafish of 68-74 hpf. This strain of zebrafish has a clear genome, a high degree of commercialization, and stable embryo quality, which can improve the stability of experimental results. At 27 hpf, zebrafish begin to exhibit swimming behavior. In subsequent developmental stages, zebrafish swim faster and acquire different swimming behaviors. Zebrafish of 68-74 hpf have weaker swimming ability, and the UVB stimulation received by zebrafish is more uniform. At the same time, UVB stimulation does not cause serious motor dysfunction in zebrafish of 68-74 hpf, and can accurately reflect the degree of sting caused by UVB. Therefore, wild AB zebrafish of 68-74 hpf were selected as the test organisms.

[0016] 4. In the above method for evaluating sunscreen efficacy, in steps S2 and S3, the UVB dose is 72 mJ / cm 2 ~81mJ / cm 2 72mJ / cm 2 ~81mJ / cm 2 UVB stimulation lowers the zebrafish's thermal pain threshold, causing a pain hypersensitivity reaction, resulting in a strong pain response under lower temperature stimulation (i.e., an enhanced pain response compared to the control group), manifested as a significantly faster swimming speed. In steps S4 to S6, the temperature of the incubator is set to 28°C ± 1°C; in step S4, the first duration is 4 hours, and the specified duration is 5 minutes. The zebrafish are placed in a behavioral recorder preheated to 28°C ± 1°C and 33°C ± 1°C for observation; in step S5, the second duration is 20 hours, and the microscope is a stereo microscope; in step S6, the third duration is 72 hours, and the microscope is a stereo microscope.

[0017] In the above-mentioned method for evaluating sunscreen efficacy, in step S2, compared with the blank group, the zebrafish in the model group swam an increased distance, decreased tail fin and dorsal fin areas, and increased head melanin area under stimulation at 33°C±1°C. At the same time, the swimming distance data of the sample group and the model group, the tail fin and dorsal fin area data of the sample group and the model group, and the head melanin area data of the sample group and the model group were all less than 0.05 after variance analysis.

[0018] In the above method for evaluating the efficacy of sunscreen, in step S4, the behavioral recorder has been preheated to 28°C ± 1°C and 33°C ± 1°C. High temperatures will activate the capsaicin receptors (TRPV1 receptors) of zebrafish. As the temperature rises, the activity of TRPV1 gradually increases and reaches a peak at around 37°C, which is manifested as a significant increase in swimming speed and an increase in swimming distance within a fixed time. Zebrafish stimulated by UVB show a heat pain hypersensitivity reaction, that is, low temperature stimulation of around 33°C will cause the pain response of zebrafish in the model group to increase. It is manifested as: compared with the blank group, the swimming speed of zebrafish in the model group increased under low temperature stimulation of around 33°C, and the increment of swimming distance within a fixed time increased significantly.

[0019] In the above method for evaluating sunscreen efficacy, in step S4, the post-sunburn pain relief efficiency R 晒后灼痛舒缓 The calculation formula is as follows:

[0020]

[0021] Among them, D 样品组1 D is the average swimming distance of zebrafish in the sample group at 33℃±1℃; 样品组0 D is the average swimming distance of zebrafish in the sample group at 28℃±1℃; 模型组1 D is the average swimming distance of zebrafish in the model group at 33℃±1℃; 模型组1 The average swimming distance of zebrafish in the model group at 28℃±1℃.

[0022] In the above method for evaluating sunscreen efficacy, in step S5, the sunburn prevention efficiency R 防晒伤 The calculation formula is as follows:

[0023]

[0024] Among them, S 样品组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the sample group; S 模型组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the model group; S 空白组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the blank group.

[0025] In the above method for evaluating sunscreen efficacy, in step S6, the sunscreen efficiency R 防晒黑 The calculation formula is as follows:

[0026]

[0027] Among them, S 样品组 is the average area of ​​melanin on the head of zebrafish in the sample group; S 模型组 is the average area of ​​melanin on the head of zebrafish in the model group; S 空白组 is the average area of ​​melanin on the head of zebrafish in the blank group.

[0028] In the above method for evaluating sunscreen efficacy, in step S7, the sunscreen efficacy scoring system is shown in Table 1.

[0029] Table 1. Sunscreen efficacy scoring system

[0030]

[0031]

[0032]

[0033] The samples were rated for their effectiveness in improving skin inflammation and aging based on the scoring results, with 9-12 points considered excellent, 5-8 points considered good, 1-4 points considered average, and 0 points and below considered none.

[0034] In the above method for evaluating the sunscreen efficacy, the swimming distance data of the sample group and the model group were subjected to variance analysis. 晒后灼痛舒缓 When R > 0%, if p < 0.05, it means that there is a significant difference between the two groups of data, the sample group has the effect of protecting the skin from sunburn and thus reducing the stinging effect of sunburn, and the scoring result is valid data; if p ≥ 0.05, it means that there is no significant difference between the two groups of data, the stinging degree caused by sunburn in the sample group is consistent with that in the model group, the scoring result is invalid data, and the score is returned to 0. 晒后灼痛舒缓 When ≤0%, if p < 0.05, it means that there is a significant difference between the two groups of data, the sample group has the effect of increasing the degree of stinging, and the scoring result is valid data; if p ≥ 0.05, it means that there is no significant difference between the two groups of data, the degree of stinging caused by sunburn in the sample group is consistent with that in the model group, the scoring result is invalid data, and the score is returned to 0.

[0035] In the above method for evaluating the sunscreen efficacy, the caudal fin and dorsal fin area data of the sample group and the model group were subjected to variance analysis. When R 防晒伤 >0%, if p<0.05, it means that there is a significant difference between the two groups of data, the sample group has the effect of protecting the skin from sun damage, and the scoring result is valid data; if p≥0.05, it means that there is no significant difference between the two groups of data, the degree of sun damage in the sample group is consistent with that in the model group, the scoring result is invalid data, and the score is returned to 0. 防晒伤When ≤0%, if p < 0.05, it means that there is a significant difference between the two groups of data, the sample group has the effect of aggravating the degree of sun damage, and the scoring result is valid data; if p ≥ 0.05, it means that there is no significant difference between the two groups of data, the degree of sun damage in the sample group is consistent with that in the model group, the scoring result is invalid data, and the score is returned to 0.

[0036] The above method for evaluating the sunscreen efficacy, wherein the head melanin area data of the sample group and the model group were subjected to variance analysis, when R 防晒黑 When R > 0%, if p < 0.05, it means that there is a significant difference between the two groups of data, the sample group has the effect of protecting the skin from pigmentation, and the scoring result is valid data; if p ≥ 0.05, it means that there is no significant difference between the two groups of data, the pigmentation degree of the sample group is consistent with that of the model group, the scoring result is invalid data, and the score is returned to 0. 防晒黑 When ≤0%, if p < 0.05, it means that there is a significant difference between the two groups of data, the sample group has the effect of aggravating the degree of pigmentation, and the scoring result is valid data; if p ≥ 0.05, it means that there is no significant difference between the two groups of data, the degree of pigmentation of the sample group is consistent with that of the model group, the scoring result is invalid data, and the score is returned to 0.

[0037] The present invention also provides the use of the above-mentioned method for evaluating sunscreen efficacy in evaluating the sunscreen efficacy of chemical sunscreens, water-soluble sunscreen cosmetics or cosmetic raw materials.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] Existing sunscreen models have disadvantages such as long testing cycles, high costs, and single evaluation indicators. The present invention simulates a series of changes in the skin such as burning pain, sunburn, and melanin deposition during excessive UVB exposure at the whole animal level. It comprehensively evaluates the sunscreen effect of samples or active substances from three dimensions: post-sunburn relief, sunburn prevention, and sunburn prevention. Combined with the sunscreen efficacy scoring system, it can achieve accurate grading of products or active substances. Among them, the calculation formula for post-sunburn relief efficiency combines the UVB damage model and temperature stimulation for the first time, and uses the incremental transformation of the swimming distance of zebrafish stimulated by UVB under low temperature stimulation to simulate the post-sunburn relief effect of the test substance. It can truly reflect that after the skin is exposed to the sun, UVB induces sensitization of the peripheral nerves of the damaged skin, increases the sensitivity of the skin to thermal stimulation, and causes thermal pain reaction in the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the process of evaluating the sunscreen efficacy of a product of the present invention.

[0041] Figure 2 The swimming trajectories of zebrafish in the blank group, model group and sample group in Example 1.

[0042] Figure 3 The phenotype diagram of zebrafish in the blank group, model group and sample group in Example 1.

[0043] Figure 4 These are the statistical results of the swimming distance increments of the blank group, model group, and sample group in Example 1.

[0044] Figure 5 The statistical results of the caudal fin and dorsal fin areas of zebrafish in the blank group, model group and sample group in Example 1.

[0045] Figure 6 These are the statistical results of melanin area on the zebrafish head in the blank group, model group, and sample group in Example 1.

[0046] Figure 7 The swimming trajectories and phenotypes of zebrafish in the blank group, model group and sample group in comparative example 1.

[0047] Figure 8 The statistical results of the swimming distance increment of zebrafish in the blank group, model group and sample group in comparative example 1 ( Figure 8 Left), caudal fin and dorsal fin area statistics ( Figure 8 Middle) and the statistical results of head melanin area ( Figure 8 right).

[0048] Figure 9 The swimming trajectories of zebrafish in the blank group and model group in Example 1 and Comparative Example 3.

[0049] Figure 10 The statistical results of the swimming distance increment of zebrafish in the blank group and the model group in Example 1 ( Figure 10 Left), statistical results of the swimming distance increment of zebrafish in the blank group and model group in comparative example 3 ( Figure 10 right).

[0050] Figure 11 The swimming trajectories of zebrafish in the blank group and the model group in comparative example 4 ( Figure 11 Left) and the average swimming distance quantification results ( Figure 11 right).

[0051] Figure 12 The swimming trajectories of zebrafish in the blank group and sample group in Comparative Example 5.

[0052] Figure 13 This is the quantitative result of the average swimming distance of zebrafish in the blank group and sample group in Comparative Example 5. DETAILED DESCRIPTION

[0053] In order to enable those skilled in the art to better understand the technical solution of the present invention, its specific implementation methods are described in detail below with reference to the accompanying drawings. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0054] In the following examples, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0055] The names of the samples to be tested in the following examples are shown in Table 2. Except for sample 4, the remaining samples to be tested were purchased directly from the market. Sample 4 was obtained according to the preparation method of the emblica fruit extract described in the specific embodiment of Chinese invention patent CN119454540A. The extract has a polyphenol content of ≥40.0% and a gallic acid content of ≥2.0%.

[0056] Table 2. Names of samples to be tested

[0057] Sample number Name of the sample to be tested Sample 1 to be tested p-Aminobenzoic acid Sample 2 to be tested Polysilicone-15 Sample 3 2-Hydroxy-4-methoxy-5-sulfonic acid benzophenone Sample 4 Emblica officinalis fruit extract Sample 5 glycerin

[0058] <Example 1>

[0059] See also Figure 1 The method for evaluating the sunscreen efficacy of a sample comprises the following steps:

[0060] Step S1: 90 wild-type AB zebrafish (72 hpf) with normal development and consistent condition were randomly selected and placed in a 6-well cell culture plate, with 30 zebrafish per well (3 mL per well). The standard dilution water in the 6-well cell culture plate was replaced with fresh standard dilution water without harming the juvenile fish, and no treatment was performed to establish a blank group.

[0061] Step S2: Select the same number of 72 hpf wild AB zebrafish with the same developmental status as in step S1 and place them in a 6-well cell culture plate, with 30 zebrafish per well and a volume of 3 mL per well. Replace the standard dilution water in the 6-well cell culture plate with fresh standard dilution water without harming the juvenile fish and use 72 mJ / cm 2 UVB was used to stimulate zebrafish to establish a model group;

[0062] Step S3: Select the same number of 72 hpf wild AB zebrafish with the same developmental status as in step S1 and place them in a 6-well cell culture plate, with 30 zebrafish per well and a volume of 3 mL per well. Replace the standard dilution water in the 6-well cell culture plate with the sample solution to be tested without harming the juvenile fish, and use 72 mJ / cm 2 UVB was used to stimulate zebrafish and establish sample groups;

[0063] Step S4: After the blank group, model group, and sample group were incubated in a constant temperature box at 28°C ± 1°C for 4 hours, 24 zebrafish were randomly selected from each group and transferred to a 48-well plate, with one zebrafish per well. The 48-well plate was placed under a behavioral recorder preheated to 28°C ± 1°C and 33°C ± 1°C for observation, and the swimming trajectory and swimming distance of the zebrafish in the dark environment within 5 minutes were recorded using infrared tracking software. The swimming trajectory results are shown in Table 1. Figure 2 First, set the temperature of the behavior recorder temperature control unit to 28℃±1℃. When the water temperature in the observation box is heated to 28℃±1℃, place the 48-well plate in the observation box to track and observe the zebrafish; then set the temperature of the temperature control unit to 33℃±1℃. When the water temperature in the observation box is heated to 33℃±1℃, place the 48-well plate in the observation box to track and observe the zebrafish. Calculate the sunburn relief efficiency (R) of the sample to be tested according to Formula 1. 晒后灼痛舒缓 ):

[0064]

[0065] Among them, D 样品组1 D is the average swimming distance of zebrafish in the sample group at 33℃±1℃; 样品组0 D is the average swimming distance of zebrafish in the sample group at 28℃±1℃; 模型组1 D is the average swimming distance of zebrafish in the model group at 33℃±1℃; 模型组1 The average swimming distance of zebrafish in the model group at 28℃±1℃. 晒后灼痛舒缓 ) is expressed as the relative swimming distance increment, and the results are shown in Figure 4 .

[0066] Step S5: After the blank group, model group and sample group were incubated in a constant temperature box at 28℃±1℃ for 20h, 24 zebrafish were randomly selected from each group, and the pictures of zebrafish in each group were observed and photographed under a stereo microscope. The photographic results are shown in Figure 3 The caudal fin and dorsal fin areas were statistically analyzed using Image J image processing software, and the sunburn protection efficiency (R 防晒伤 ):

[0067]

[0068] Among them, S 样品组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the sample group; S 模型组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the model group; S 空白组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the blank group. 防晒 The results are shown in Table 1. Figure 5 .

[0069] Step S6: After the blank group, model group and sample group were incubated in a constant temperature box at 28℃±1℃ for 72h, 24 zebrafish were randomly selected from each group, and the pictures of zebrafish in each group were observed and photographed under a stereo microscope. The photographic results are shown in Figure 3 The melanin area of ​​the head was statistically analyzed using Image J image processing software, and the suntanning efficiency (R 防晒黑 ):

[0070]

[0071] Among them, S 样品组 is the average area of ​​melanin on the head of zebrafish in the sample group; S 模型组 is the average area of ​​melanin on the head of zebrafish in the model group; S 空白组 is the average area of ​​melanin on the head of zebrafish in the blank group. 防晒 Black) is expressed by the relative area of ​​head melanin, the results are shown in Figure 6 .

[0072] Combined with the sunscreen efficacy scoring system (Table 1), the sample group data were compared with the model group and scored. The results are shown in Table 3. Figure 2 As shown in Figure 2, the black lines represent the swimming trajectories of zebrafish in each group under different temperature stimulations. Figure 3 The area within the dotted line is the statistical area of ​​the zebrafish caudal fin and dorsal fin ( Figure 3 Left), head melanin statistical area ( Figure 3 Right) statistical area.

[0073] Depend on Figure 2 It can be seen that the swimming trajectories of both the blank group and the model group increased under 33°C stimulation, indicating that under temperature stimulation of around 33°C, the capsaicin receptor (TRPV1 receptor) of zebrafish will be activated, causing the swimming distance within a fixed time to increase. Figure 2 Combine Figure 4 It can be seen that under the stimulation of 33℃, the average swimming distance increment of the zebrafish in the model group within a fixed time was 1407mm, which was significantly higher than the average swimming distance increment of the blank group (550mm). This shows that the thermal pain threshold of the zebrafish stimulated by UVB is reduced, causing a thermal pain allergic reaction, resulting in an enhanced pain response to thermal stimulation. Figure 3 Left Associative Figure 5 It can be seen that compared with the blank group, the tail fin and dorsal fin of the zebrafish in the model group showed obvious wrinkling, indicating that UVB stimulation caused sun damage to the zebrafish skin. Figure 3 Right Associative Figure 6It can be seen that compared with the blank group, the model group zebrafish head showed obvious melanin deposition, indicating that UVB stimulation caused the zebrafish skin to tan. In summary, compared with the blank group, the model group had an increased swimming distance, a decreased tail fin and dorsal fin area, and an increased melanin area on the head. The relevant data were analyzed by variance analysis with p values ​​less than 0.05, indicating that 72mJ / cm 2 UVB stimulation can successfully establish the model.

[0074] Figures 2 to 6 Combined with Table 1, it can be seen that the average value of the swimming distance increment of the tested sample 1 is 2011mm, which is higher than that of the model group (1407mm). 晒后灼痛舒缓 <0% and p < 0.05, indicating that the test sample 1 makes the zebrafish's pain response to heat stimulation stronger than that of the model group. The test sample 1 has the effect of aggravating the burning pain after sun exposure. 晒后灼痛舒缓 -2 is the valid data. The degree of wrinkling of the tail fin and dorsal fin of the tested sample 1 is similar to that of the model group. 防晒伤 <0% and p>0.05, indicating that the damage status of the tested sample 1 after sun exposure is consistent with that of the model group, R 防晒伤 The score is invalid data and is set to 0. Compared with the model group, the head melanin of the test sample 1 is significantly reduced. 防晒黑 >0% and p<0.05, indicating that the sample 1 has a sun-proof effect, so R 防晒黑 The score of the sample 1 is 2 points, which is a general rating.

[0075] The average value of the swimming distance increment of the tested sample 2 is 1198 mm, slightly smaller than that of the model group (1407 mm). 晒后灼 Pain relief>0% and p<0.05, indicating that the sample 2 slightly relieved the pain response of zebrafish to heat stimulation. The sample 2 has the effect of relieving the pain after sunburn. Therefore, R 晒后灼痛舒缓 The score of 1 is valid data. Compared with the model group, the wrinkling degree of the tail fin and dorsal fin of the test sample 2 is slightly reduced. 防晒伤 <0% and p<0.05, indicating that the sample 2 has the effect of preventing sun damage, R 防晒伤 Score 2 points as valid data. Compared with the model group, the melanin of the head of the test sample 2 was slightly lighter, R 防晒黑 >0% and p<0.05, indicating that the sample 2 has a sun-proof effect, so R 防晒黑 The score of sample 2 is 5 points, and the rating result is good.

[0076] The average value of the swimming distance increment of the tested sample 3 is 286 mm, which is much smaller than that of the model group (1407 mm). 晒后灼痛Soothing>0% and p<0.05, indicating that the sample 3 can significantly relieve the pain response of zebrafish to heat stimulation. The sample 3 has the effect of soothing the burning pain after sunburn. 晒后灼痛舒缓 The score of 4 points is valid data. Compared with the model group, the wrinkling degree of the tail fin and dorsal fin of the test sample 3 was significantly reduced. 防晒伤 <0% and p<0.05, indicating that the sample 3 has the effect of preventing sun damage, R 防晒伤 4 points are considered valid data. Compared with the model group, the melanin of the head of the sample 3 was slightly reduced. 防晒黑 >0% and p<0.05, indicating that the sample 3 has a sun-proof effect, so R 防晒黑 The score of sample 3 is 10 points, and the rating result is excellent.

[0077] The average value of the swimming distance increment of the tested sample 4 is 852 mm, which is smaller than that of the model group (1407 mm). 晒后灼痛舒缓 >0% and p<0.05, indicating that the sample 4 can alleviate the pain response of zebrafish to heat stimulation. The sample 4 has the effect of relieving the burning pain after sun exposure. 晒后灼痛舒缓 2 points are valid data. Compared with the model group, the wrinkling degree of the tail fin and dorsal fin of the test sample 4 is reduced. 防晒伤 <0% and p<0.05, indicating that the sample 4 has the effect of preventing sun damage, R 防晒伤 A score of 3 points is considered valid data. Compared with the model group, the melanin of the head of the test sample 4 was significantly reduced. 防晒黑 >0% and p<0.05, indicating that the sample 4 has a sun-proof effect, so R 防晒黑 The score of 4 points is valid data. The score of sample 4 is 9 points, and the rating result is excellent.

[0078] The average value of the swimming distance increment of the tested sample 5 is 1668 mm, slightly higher than that of the model group (1407 mm). 晒后灼 Pain relief < 0% and p > 0.05, indicating that the pain response of zebrafish in sample 5 to heat stimulation is the same as that in the model group, and the degree of sunburn in sample 5 is consistent with that in the model group. 晒后灼痛舒缓 The scores are invalid data and are returned to 0. The wrinkling degree of the tail fin and dorsal fin of the tested sample 5 is similar to that of the model group. 防晒伤 <0% and p>0.05, indicating that the damage status of the tested sample 5 after sun exposure is consistent with that of the model group, R 防晒伤 The score is invalid data and is set to 0. The head melanin of the tested sample 4 is consistent with that of the model group, R 防晒黑 >0% and p>0.05, indicating that the tanning degree of the tested sample 5 is consistent with that of the model group, so R 防晒黑The score of the sample 5 is invalid data. The score of the sample 5 is 0 points, and the rating result is none.

[0079] Table 3. Test results of each sample in Example 1

[0080]

[0081]

[0082] Comparative Example 1

[0083] The difference between Comparative Example 1 and Example 1 is that the UVB dose is 54mJ / cm 2 , the rest of the steps are exactly the same.

[0084] pass Figure 7 、 Figure 8 It can be seen that in comparative example 1, compared with the blank group, the model group had a slight increase in swimming distance under low temperature stimulation, but the data was analyzed by variance analysis p = 0.7061> 0.05; the tail fin and dorsal fin area decreased slightly, but the data was analyzed by variance analysis p = 0.0542> 0.05; the head melanin area did not change significantly, and the data was analyzed by variance analysis p = 0.7085> 0.05. The above results show that when the UVB dose is 54mJ / cm 2 When the dose was 50mg / L, the zebrafish in the model group did not show any heat pain allergic reaction, nor did it cause obvious sunburn or tanning on their skin. The modeling failed and the dose was not suitable as a modeling dose.

[0085] Comparative Example 2

[0086] The difference between Comparative Example 2 and Example 1 is that the UVB dose is 108 mJ / cm 2 , the rest of the steps are exactly the same.

[0087] When the UVB dose is 108mJ / cm 2 The mortality rate of the model group was 57%, and subsequent efficacy evaluation was not possible. 2 When the dose is too high, it causes irreparable damage to the zebrafish and is not suitable as a modeling dose.

[0088] Comparative Example 3

[0089] The difference between Comparative Example 3 and Example 1 is that in S4, the 48-well plate is placed under a behavioral recorder preheated to 28°C±1°C and 37°C±1°C for observation, and the remaining steps are exactly the same.

[0090] pass Figure 9 、 Figure 10It can be seen that in Comparative Example 3, compared with the blank group, the incremental swimming distance of the model group under 37℃±1℃ stimulation did not change significantly, and the data were analyzed by variance analysis p=0.9490>0.05. Compared with the blank group of Example 1, the incremental swimming distance of the blank group of Comparative Example 3 under 37℃±1℃ stimulation increased significantly. The above results show that under the temperature stimulation of 37℃±1℃, the TRPV1 receptor of zebrafish will be activated, resulting in an increase in the swimming distance within a fixed time; however, excessive temperature stimulation will cause the swimming speed of the blank group zebrafish to surge, resulting in the swimming distance reaching a peak within a fixed time, and the incremental swimming distance is similar to that of the model group, which cannot accurately reflect the thermal pain hypersensitivity reaction of the zebrafish in the model group, and ultimately leads to modeling failure.

[0091] Comparative Example 4

[0092] The difference between Comparative Example 4 and Example 1 is that in S4, the 48-well plate was placed under a behavioral recorder for observation, and the swimming trajectory and swimming distance of zebrafish in a dark environment within 5 minutes were recorded using infrared tracking software. The post-sunburn pain relief efficiency (R 晒后灼痛舒缓 ):

[0093]

[0094] Among them, D 样品组 is the average swimming distance of zebrafish in the sample group; D 模型组 is the average swimming distance of zebrafish in the model group. The rest of the steps are exactly the same.

[0095] pass Figure 11 As can be seen in Comparative Example 4, the average swimming distance within 5 minutes in the blank group was 327 mm, while that in the model group was 321 mm. Compared to the blank group, the model group's swimming distance trajectory was slightly reduced, but not significantly changed. Analysis of variance revealed a p value of 0.6785 > 0.05. These results suggest that if temperature is ignored and only the effects of UVB stimulation on zebrafish locomotor behavior are considered, the model group's swimming distance within a fixed timeframe is similar to that of the blank group, ultimately leading to modeling failure and making it impossible to further evaluate the sample group's efficacy in alleviating post-sunburn pain.

[0096] Comparative Example 5

[0097] The difference between Comparative Example 5 and Example 1 is that in S4, after the blank group and the sample group were incubated in a constant temperature box at 28°C±1°C for 4 hours, 24 zebrafish were randomly selected from each group and transferred to a 48-well plate, with 1 zebrafish in each well. The blank group was first placed under a behavioral recorder preheated to 28°C±1°C for observation, and the swimming trajectory and swimming distance of the zebrafish in a dark environment within 5 minutes were recorded using infrared tracking software. The blank group and the sample group were then placed under a behavioral recorder preheated to observe, and the swimming trajectory and swimming distance of the zebrafish in a dark environment within 5 minutes were recorded using infrared tracking software. The post-sunburn pain relief efficiency (R 晒后灼痛舒缓 ):

[0098]

[0099] Among them, D 样品组 D is the average swimming distance of zebrafish in the sample group at 33℃±1℃; 空白组1 is the average swimming distance of zebrafish in the blank group at 33℃±1℃, and the rest of the steps are exactly the same.

[0100] Table 4. Test results of each sample in Comparative Example 5

[0101] Group <![CDATA[R 晒后灼痛舒缓 ]]> <![CDATA[R 防晒伤 ]]> <![CDATA[R 防晒黑 ]]> Scoring results Rating results Sample 1 to be tested -2.57％ -14.54％ 82.30％ 4 generally Sample 2 to be tested 2.74％ 28.06％ 45.78％ 4 generally Sample 3 -18.98％ 92.69％ 40.01％ 5 good Sample 4 10.99％ 50.18％ 104.21％ 8 good Sample 5 9.94％ -16.28％ 1.48％ 0 none

[0102] Figure 12 、 13 As shown in Table 4, in Comparative Example 5, the average swimming distance of the blank group at 28°C ± 1°C was 327 mm, while the average swimming distance at 33°C ± 1°C was 897 mm. The data were analyzed by ANOVA with a p value of 0.0001 < 0.05. This indicates that low temperatures around 33°C activate the TRPV1 receptor in zebrafish, increasing their swimming distance within a fixed timeframe.

[0103] The average value of the swimming distance increment of the tested sample 5 is 1668mm, slightly higher than that of the model group (1407mm). 晒后灼痛舒缓 <0% and p>0.05, indicating that the pain response of zebrafish in sample 5 to heat stimulation is the same as that in the model group, and the degree of sunburn in sample 5 is consistent with that in the model group. 晒后灼痛舒缓 The score is invalid data and the score is reset to 0.

[0104] Under the stimulation of 33℃±1℃, the average swimming distance of the test sample 1 was 921mm, slightly higher than that of the blank group (897mm). The data were analyzed by variance analysis with p=0.8936>0.05, indicating that the test sample 1 could not inhibit the stinging reaction caused by the TRPV1 receptor. Under the stimulation of 33℃±1℃, the average swimming distance of the test sample 2 was 873mm, slightly lower than that of the blank group (897mm). The data were analyzed by variance analysis with p=0.8652>0.05, indicating that the test sample 2 could not inhibit the stinging reaction caused by the TRPV1 receptor. Under the stimulation of 33℃±1℃, the average swimming distance of the test sample 3 was 1068mm, higher than that of the blank group (897mm). The data were analyzed by variance analysis with p=0.0005<0.05, indicating that the test sample 3 aggravated the stinging reaction caused by the TRPV1 receptor. Under stimulation at 33°C ± 1°C, the average swimming distance of sample 4 was 799 mm, which was lower than that of the blank group (897 mm). The data were analyzed by variance analysis with p = 0.0255 < 0.05, indicating that sample 4 can inhibit the stinging reaction induced by the TRPV1 receptor. Under stimulation at 33°C ± 1°C, the average swimming distance of sample 5 was 808 mm, which was slightly lower than that of the blank group (897 mm). The data were analyzed by variance analysis with p = 0.0529 > 0.05, indicating that sample 5 cannot inhibit the stinging reaction induced by the TRPV1 receptor.

[0105] As can be seen from the above results, under the stimulation of 33°C ± 1°C, the test results of each sample group are different from the test results of the sample group in the embodiment. This is because if the UVB variable is ignored and only the effect of temperature stimulation on the movement behavior of zebrafish is considered, it can only simulate the effect of temperature activation of the zebrafish TRPV1 receptor on its movement behavior, thereby reflecting the inhibitory effect of the sample on the TRPV1 receptor. However, it cannot simulate the process of post-sunburn pain, that is, excessive UVB induces damage to the epidermis, leading to sensitization of peripheral nerves in the damaged skin, thereby increasing the sensitivity of the damaged skin to thermal stimulation and producing a heat pain allergic reaction. Therefore, it is impossible to further accurately reflect the post-sunburn pain relief effect of the sample group.

[0106] The results of Comparative Examples 4 and 5 clearly demonstrate that the evaluation method for post-sunburn pain relief requires combining UVB damage with temperature stimulation. The change in the incremental swimming distance of UVB-stimulated zebrafish under low-temperature stimulation is used to simulate the post-sunburn pain relief effect of the test substance. Neither UVB stimulation nor temperature stimulation as a single variable can truly reflect the thermal pain response of the skin after sun exposure.

[0107] In summary, this invention simulates the series of skin changes such as burning pain, sunburn, and melanin deposition during excessive UVB exposure in whole animals. It comprehensively evaluates the sunscreen efficacy of samples or active ingredients based on three dimensions: post-sunburn pain relief, sunburn prevention, and sunburn prevention. Combined with a sunscreen efficacy scoring system, it enables precise grading of products or active ingredients. This overcomes the shortcomings of existing sunscreen models, such as long testing cycles, high costs, and single evaluation indicators. It can simply and intuitively reflect the sunscreen efficacy of products or active ingredients, providing scientific and intuitive data support for related product development.

[0108] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for evaluating sunscreen efficacy, characterized in that: The following steps are involved: Step S1, selecting several zebrafish with the same tail development status and not treating them to establish a blank group; Step S2, selecting the same number of zebrafish with the same developmental status as in step S1, stimulating the zebrafish with UVB to establish a model group; Step S3, selecting the same number of zebrafish as in step S1 and with the same developmental status, adding the test sample, and stimulating the zebrafish with UVB to establish a sample group; Step S4, after the blank group, model group, and sample group are incubated in a constant temperature chamber for a first period of time, the zebrafish are placed in a behavioral recorder preheated to different temperatures for observation. Infrared tracking software is used to record the swimming trajectory and swimming distance of the zebrafish in a dark environment for a specified period of time, and the post-sunburn pain relief efficiency is calculated; Step S5, after incubating the blank group, model group, and sample group in the incubator for a second period of time, observing the zebrafish under a microscope, recording the tail fin and dorsal fin areas of the zebrafish, and calculating the sunburn prevention efficiency; Step S6, after the blank group, the model group, and the sample group are incubated in the incubator for a third period of time, the zebrafish are observed under a microscope, and the melanin area of ​​the zebrafish's head is recorded to calculate the suntanning efficiency; Step S7: Rating the sunscreen efficacy of the sample according to the sunscreen efficacy rating system.

2. A method for evaluating sunscreen efficacy according to claim 1, characterized in that: The zebrafish is a wild AB zebrafish of 68 to 74 hpf.

3. The method for evaluating sunscreen efficacy according to claim 1, wherein: In steps S2 and S3, the UVB dose is 72 mJ / cm 2 ~81mJ / cm 2 ; In steps S4 to S6, the temperature of the constant temperature box is set to 28℃±1℃; in step S4, the first time length is 4 hours, the specified time length is 5 minutes, and the zebrafish is placed under a behavioral recorder preheated to 28℃±1℃ and 33℃±1℃ for observation; in step S5, the second time length is 20 hours, and the microscope is a stereo microscope; in step S6, the third time length is 72 hours, and the microscope is a stereo microscope.

4. A method for evaluating sunscreen efficacy according to claim 3, characterized in that: In step S2, the criteria for successfully establishing the model group are: compared with the blank group, the swimming distance increment of the zebrafish in the model group under stimulation of 33°C±1°C increases, the tail fin and dorsal fin area decreases, and the head melanin area increases; at the same time, the swimming distance data of the sample group and the model group, the tail fin and dorsal fin area data of the sample group and the model group, and the head melanin area data of the sample group and the model group are all less than 0.05 after variance analysis.

5. A method for evaluating sunscreen efficacy according to claim 4, characterized in that: In step S4, the post-sunburn pain relief efficiency R 晒后灼痛舒缓 The calculation formula is as follows: Among them, D 样品组1 D is the average swimming distance of zebrafish in the sample group at 33℃±1℃; 样品组0 D is the average swimming distance of zebrafish in the sample group at 28℃±1℃; 模型组1 D is the average swimming distance of zebrafish in the model group at 33℃±1℃; 模型组1 The average swimming distance of zebrafish in the model group at 28℃±1℃.

6. The method for evaluating sunscreen efficacy according to claim 1, wherein: In step S5, the sunburn prevention efficiency R 防晒伤 The calculation formula is as follows: Among them, S 样品组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the sample group; S 模型组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the model group; S 空白组 is the average area of ​​the caudal fin and dorsal fin of zebrafish in the blank group.

7. The method for evaluating sunscreen efficacy according to claim 1, wherein: In step S6, the sun protection efficiency R 防晒黑 The calculation formula is as follows: Among them, S 样品组 is the average area of ​​melanin on the head of zebrafish in the sample group; S 模型组 is the average area of ​​melanin on the head of zebrafish in the model group; S 空白组 is the average area of ​​melanin on the head of zebrafish in the blank group.

8. The method for evaluating sunscreen efficacy according to claim 1, wherein: In step S7, the sunscreen efficacy scoring system is shown in Table 1: Table 1. Sunscreen efficacy scoring system The samples were rated for their effectiveness in improving skin inflammation and aging based on the scoring results, with 9-12 points considered excellent, 5-8 points considered good, 1-4 points considered average, and 0 points and below considered none.

9. The method for evaluating sunscreen efficacy according to claim 8, wherein: The swimming distance data of the sample group and the model group were analyzed by variance analysis. If p < 0.05, the scoring result was valid data; if p ≥ 0.05, the scoring result was invalid data and the score was returned to 0. The data of caudal fin and dorsal fin area of ​​sample group and model group were analyzed by variance analysis, if p < 0.05, the scoring results were valid data; If p ≥ 0.05, the scoring result is invalid data and the score is reset to 0; The head melanin area data of the sample group and the model group were analyzed by variance analysis. If p < 0.05, the scoring results were valid data; If p≥0.05, the scoring result is invalid data and the score is reset to 0.

10. Use of the method for evaluating sunscreen efficacy according to any one of claims 1 to 9 in evaluating the sunscreen efficacy of chemical sunscreens, water-soluble sunscreen cosmetics or cosmetic raw materials.

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