Zebra fish model-based non-treatment-purpose method for evaluating anti-photoaging and sunscreen effects of sunscreen cosmetics and application of non-treatment-purpose method for evaluating anti-photoaging and sunscreen effects of sunscreen cosmetics

Abstract

The invention provides a non-treatment-purpose method for evaluating the anti-light aging and sunscreen efficacy of sunscreen cosmetics based on a zebra fish model and application. The method comprises the following steps: setting a control group, a modeling group and a sample group; juvenile zebrafish is placed in the pore plate according to the set groups; preparing an experimental film, wherein only the light-transmitting film of the sample group is uniformly coated with a sunscreen sample to be tested; covering the experimental film on the pore plates of the corresponding groups; performing UVB irradiation on the molding group and the sample group; performing light shielding treatment on the control group; culturing continuously after irradiation, and photographing the tails of the zebrafish; quantifying the tail fin area and / or the tail melanin content through image analysis; and respectively evaluating the anti-light aging and anti-sunburn effects of the sample by comparing the index differences of the sample group and the modeling group. The method has the advantages of high throughput, low cost, short period and high universality, and can be used for scientifically, sensitively and comprehensively evaluating the anti-light aging and sunscreen effects of the sunscreen cosmetics.

Description

Technical Field

[0001] This application belongs to the field of cosmetic testing technology, specifically relating to a method and application for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes. Background Technology

[0002] Ultraviolet (UV) radiation is a major environmental factor contributing to sunburn, photoaging, tanning, and even skin cancer. UV radiation is categorized by wavelength into UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm). UVB, with its higher energy, is a significant factor in causing sunburn, erythema, and skin aging. It can also stimulate melanocyte activity, leading to pigmentation and tanning. Therefore, effective UVB protection is crucial for sun protection. Currently, commercially available sunscreens primarily work by blocking or absorbing UV rays through physical or chemical mechanisms.

[0003] Currently, the main methods for evaluating the efficacy of sunscreen products include: Human efficacy testing: This involves directly testing the sun protection effect of a product on the human body, such as determining the sun protection factor (SPF), water resistance, and UVA protection index. However, this method is expensive, time-consuming, difficult to recruit subjects, has significant individual differences, and raises ethical concerns, making it difficult to conduct large-scale evaluations and screenings.

[0004] In vitro cell models: Skin-derived cell lines such as HaCaT cells are often used in experiments to detect cell activity, inflammatory factors, DNA damage and other indicators after ultraviolet irradiation. However, cells cannot completely simulate the structure of skin tissue, nor can they simulate the film-forming state of sunscreen cosmetics on the skin surface and the complete reaction of the skin.

[0005] In vitro reconstructed skin models: Using 3D skin models to test sunscreen cosmetics can simulate the structure of human skin to a certain extent. Ultraviolet irradiation can cause tissue damage. However, the construction cost of this model is high and the technology is difficult to achieve high-throughput screening. In addition, there are significant differences between 3D skin and living tissue.

[0006] Animal models: Zebrafish are classic model organisms, offering numerous advantages such as low cost, high throughput, short cycle time, high visualization, and high genetic homology with humans. There are already cases of zebrafish models being used in sunscreen product testing.

[0007] Patents CN108956606A, CN108169418A, and CN120586103A disclose a method for evaluating sunscreen products using zebrafish. This method involves adding sunscreen products, or sunscreen products with added biosurfactants, to zebrafish aquaculture water, followed by ultraviolet irradiation, and recording the degree of damage to the pelvic, caudal, and dorsal fins of juvenile fish. However, this method has significant drawbacks: First, it completely fails to simulate the film-forming state of sunscreen products on human skin, severely deviating from real-world usage scenarios; second, sunscreen ingredients (especially chemical sunscreens) typically have high biotoxicity, and direct dissolution in water can cause significant toxic side effects in juvenile zebrafish, interfering with efficacy assessment; furthermore, the introduction of surfactants may itself affect the stability and efficacy of the sunscreen system, and this method has poor adaptability to products that are insoluble in water.

[0008] Patent CN119530335B discloses a method for evaluating sun protection function based on cell and zebrafish models. This method upgrades the drug delivery method to a contactless delivery approach, comprehensively evaluating sun protection efficacy through multi-level indicators (such as cell viability, DNA damage, inflammatory factor secretion, collagen secretion, and lethality / teratogenicity in zebrafish embryos). However, this method primarily focuses on the cell module, as cells cannot fully simulate the state of skin tissue under UV irradiation. The zebrafish module only involves embryo lethality and teratogenicity indicators, using high doses of UVB (400-1600 mJ / cm²). 2 The method involves creating models that cause death or deformity in zebrafish, followed by the application of sunscreen to evaluate its sun protection efficacy. However, this method has significant limitations: death and deformity represent extreme cases of injury, resulting in low sensitivity and difficulty in distinguishing efficacy differences between sunscreens with different SPF values. Furthermore, this indicator only reflects the basic sun protection efficacy of sunscreens and cannot reflect the more comprehensive sun protection effects that are closer to consumer needs, such as anti-photoaging and anti-tanning properties.

[0009] Currently, sunscreen cosmetics are developing in a diversified direction, such as anti-photoaging, prevention of pigmentation, repair, film formation and breathability. Therefore, there is an urgent need for more comprehensive and sensitive evaluation methods for sunscreen efficacy.

[0010] In conclusion, there is an urgent need to develop a high-throughput, low-cost, short-cycle, highly universal, and scientifically sensitive in vivo evaluation method that can comprehensively evaluate the efficacy of anti-photoaging and anti-tanning. Summary of the Invention

[0011] To address the aforementioned technical problems, this application provides a method and application for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes.

[0012] In a first aspect, this application provides a method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes, comprising the following steps: S1: Experimental grouping and zebrafish loading; prepare zebrafish juveniles for the experiment; set up a control group, a model group, and at least one sample group; place the zebrafish juveniles in the multi-well plate according to the set groups; S2: Prepare experimental films; coat the test sunscreen cosmetic samples onto the transparent films of the sample group; do not coat the test sunscreen cosmetic samples onto the transparent films of the control group and the model group; S3: UVB irradiation treatment; the experimental membrane prepared in step S2 is covered on the well plate of the corresponding group set in step S1; the model group and the sample group are irradiated with UVB; the control group is protected from light. S4: Efficacy evaluation; After UVB irradiation, zebrafish fry of each group were cultured; then images of the tails of zebrafish of each group were acquired. The tail fin area of ​​zebra juveniles in each group after UVB irradiation was quantified, and the anti-photoaging efficacy of the sunscreen cosmetic was evaluated by comparing the difference in tail fin area between the sample group and the model group. and / or The melanin content in the tails of zebra fry in each group after UVB irradiation was quantified. The anti-tanning efficacy of the sunscreen cosmetic was evaluated by comparing the difference in tail melanin content between the sample group and the model group.

[0013] Furthermore, in some embodiments, the zebra fry mentioned in step S1 are zebra fry that are 48 to 120 hours after fertilization.

[0014] Furthermore, in some embodiments, the perforated plate in step S1 is a carrier for placing zebrafish fry.

[0015] Furthermore, in some embodiments, the multi-well plate mentioned in step S1 is a commonly used experimental plate, including one of a petri dish, a six-well plate, a twelve-well plate, a twenty-four-well plate, a forty-eight-well plate, and a ninety-six-well plate.

[0016] Furthermore, in some embodiments, the light-transmitting film in step S2 is a film that can transmit UVB; the light-transmitting film is a uniform film with light transmittance.

[0017] Furthermore, in some embodiments, the sunscreen cosmetic sample to be tested in step S2 needs to be applied 30-60 minutes before UVB irradiation, so that the sunscreen cosmetic sample to be tested can form and maintain a film-like state.

[0018] Furthermore, in some embodiments, the UVB irradiation dose in step S3 is 50-200 mJ / cm².

[0019] Furthermore, in some embodiments, in step S3, the experimental membrane of the sample group is covered with the side of the sunscreen cosmetic sample to be tested facing the zebrafish juveniles placed in the well plate or facing the UVB light source.

[0020] Furthermore, in some embodiments, the zebrafish fry are cultured for 12-72 hours in step S4.

[0021] Furthermore, in some embodiments, the caudal fin area of ​​the zebra fry in step S4 is obtained by calculating the area of ​​the caudal fin outline using image analysis software; the melanin content of the zebra fry's tail is obtained by performing grayscale analysis on the tail image using image analysis software.

[0022] Secondly, this application provides the application of the method described in the first aspect for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes in the screening or evaluation of sunscreen cosmetics.

[0023] This application utilizes a zebrafish juvenile model, achieving true in vivo evaluation. The zebrafish's skin structure and gene function are highly similar to those of humans, resulting in evaluation results with greater physiological relevance and predictive value. Furthermore, it boasts significant advantages such as low cost, high throughput, and short cycle time.

[0024] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: This application employs a film-covered drug delivery method, highly simulating real-world conditions and accurately mimicking the actual physical process by which sunscreen products form a film on the human skin surface to exert their effects. This is something that cannot be achieved by directly adding samples to water or conducting cell experiments. It eliminates toxicity interference by physically isolating sunscreen cosmetics from zebrafish larvae, effectively avoiding interference from the toxicity of sunscreen agents (especially chemical sunscreen agents) on experimental results, ensuring the accuracy and reliability of efficacy evaluation. It has strong universality, with no restrictions on the solubility or toxicity of samples. Whether it is a physical, chemical, or biological sunscreen agent, the undiluted solution can be used for testing, making it widely applicable and overcoming many limitations of traditional methods in terms of sample adaptability.

[0025] This application uses tail fin area as an evaluation index for anti-photoaging, which is intuitive and sensitive. Tail fin wrinkling is an intuitive and quantifiable macroscopic phenotype of UV-induced tissue damage and aging, which is more sensitive than indicators such as cell survival rate or lethality / teratogenicity, and can effectively distinguish products with different efficacy intensities. Directly linking changes in tail fin area with the anti-photoaging efficacy of sunscreen products provides a reliable in vivo biological indicator for evaluating this efficacy.

[0026] This application uses the melanin content in the tail area as an evaluation indicator for sun protection against tanning, directly reflecting the core demand and quantifying the most intuitive side effect of ultraviolet radiation—pigmentation—precisely addressing consumers' core need for sun protection against tanning.

[0027] This application employs a sub-damaging irradiation scheme with UVB exposure doses (50-200 mJ / cm²) to induce quantifiable and reversible physiological damage (tail fin wrinkling, melanin production), rather than pathological damage such as lethality or teratogenicity. This ensures the model's sensitivity and discriminative power, accurately reflecting the differences in protective capabilities among different products. The model can clearly and sensitively distinguish subtle efficacy differences between products with different SPF / PA values ​​and different formulation systems, making it suitable for product development and formulation optimization.

[0028] Based on the high-throughput advantage of the zebrafish model and combined with a simplified operating procedure, this application enables large-scale and rapid initial screening of sunscreen raw materials and formulations, significantly reducing the R&D cycle and economic costs.

[0029] This application comprehensively evaluates anti-photoaging and anti-tanning effects, achieving an integrated evaluation of the two core functions of sunscreen products—anti-photoaging and anti-tanning—on a simple and rapid model. It provides more comprehensive efficacy data that is closer to market demands, and its efficiency is far superior to complex systems that require multiple independent tests.

[0030] In summary, this application provides a method and application for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model. Utilizing the zebrafish model to evaluate the sunscreen efficacy, changes in zebrafish tail fin area and melanin content are used as core detection indicators. A film-covered drug delivery method is employed, directly applying the sunscreen concentrate onto the film, which more accurately simulates the actual application of sunscreen cosmetics on human skin. Furthermore, it does not impose limitations on toxicity or solubility, overcoming the limitations of sunscreen cosmetics with significant toxicity, difficulties in administering non-water-soluble sunscreens, and a lack of evaluation indicators. This provides a more sensitive, scientific, efficient, economical, and human-aligned high-throughput screening method. This method is applicable to the efficacy evaluation and ranking of various sunscreen cosmetics and has promising market application prospects. Attached Figure Description

[0031] The present application will be described in further detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will understand that these drawings are drawn only for the purpose of explaining the preferred embodiments and therefore should not be construed as limiting the scope of this disclosure. Furthermore, unless specifically indicated, the drawings are only schematic representations of the composition or structure of the described objects and may contain exaggerated depictions, and the drawings are not necessarily drawn to scale.

[0032] Figure 1 The chart shows the malformation rate of zebrafish juveniles under different UVB irradiation doses in Examples 1-8.

[0033] Figure 2The images show the tail phenotypes of zebrafish juveniles cultured under UVB irradiation in Examples 9-11.

[0034] Figure 3 The chart shows the statistical distribution of the caudal fin area of ​​zebrafish juveniles cultured under UVB irradiation in Examples 9-11.

[0035] Figure 4 The graph shows the melanin content of zebrafish juveniles cultured under UVB irradiation in Examples 9-11.

[0036] Figure 5 This is a schematic diagram of the process for evaluating the anti-photoaging and anti-tanning effects of sunscreen cosmetics based on the zebrafish model in Example 12.

[0037] Figure 6 Example 12: Evaluation of the efficacy of Winona Clear Whitening Sunscreen for treating sunspots. Phenotypic image of the tail of juvenile horsefish.

[0038] Figure 7 Example 12: Evaluation of the efficacy of Winona Clear Whitening Sunscreen for treating sunspots. Statistical chart of the tail fin area of ​​juvenile zebrafish.

[0039] Figure 8 Example 12: Evaluation of the efficacy of Winona Clear Whitening Sunscreen for Spot Removal - Statistical chart of melanin content in zebrafish juveniles.

[0040] Figure 9 Phenotypic image of the tail of juvenile zebrafish used in Example 13, evaluating the efficacy of Winona Clear Sunscreen Lotion.

[0041] Figure 10 Example 13: Evaluation of the efficacy of Winona Clear Sunscreen Lotion. Statistical chart of the tail fin area of ​​juvenile zebrafish.

[0042] Figure 11 Example 13: Evaluation of the efficacy of Winona Clear Sunscreen Lotion. Statistical chart of melanin content in zebrafish juveniles.

[0043] Figure 12 Example 14: Evaluation of the efficacy of Winona Baby Gentle and Refreshing Sunscreen Lotion. Zebrafish tail phenotype diagram.

[0044] Figure 13 Example 14: Evaluation of the efficacy of Winona Baby Gentle and Refreshing Sunscreen Lotion. Statistical chart of the tail fin area of ​​juvenile zebrafish.

[0045] Figure 14 Example 14: Evaluation of the efficacy of Winona Baby Gentle and Refreshing Sunscreen Lotion. Statistical chart of melanin content in zebrafish juveniles.

[0046] Figure 15 This is a statistical chart showing the survival rate of zebrafish juveniles after administration of Winona Clear Sunscreen Lotion in Example 15.

[0047] Figure 16This is a statistical chart showing the survival rate of zebrafish juveniles after administration of Winona Clear Whitening Sunscreen Lotion in Example 15.

[0048] Figure 17 This is a statistical chart showing the survival rate of zebrafish juveniles after administration of Winona Baby Gentle and Refreshing Sunscreen Lotion in Example 15. Detailed Implementation

[0049] The following is in conjunction with the appendix Figures 1 to 17 This disclosure will be explained in detail.

[0050] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0051] Experimental materials Zebrafish juveniles: Wild-type AB strain, Hangzhou Huante Biotechnology Co., Ltd.

[0052] Translucent film: M400A-HN, Hangzhou Miaojie Daily Chemical Technology Co., Ltd.

[0053] Winona Clear Whitening Sunscreen Lotion: SPF50+ PA+++, Yunnan Bettern Biotechnology Group Co., Ltd.

[0054] Winona Clear Sunscreen Lotion: SPF48 PA+++, Yunnan Betterney Biotechnology Group Co., Ltd.

[0055] Winona Baby Gentle and Refreshing Sunscreen Lotion: SPF34 PA+++, Yunnan Betterney Biotechnology Group Co., Ltd.

[0056] Instruments and equipment Incubator: LRH-250, Shanghai Yiheng Scientific Instruments Co., Ltd.

[0057] UVB irradiation instrument: PE Master UV light exposure system.

[0058] Microscope: Zeiss AXIO Zoom.V16.

[0059] Image analysis software: ImageJ.

[0060] Example 1

[0061] This embodiment studies the effect of UVB irradiation dose on the morphology of zebrafish.

[0062] A control group and a model group were set up. Wild-type AB strain zebrafish juveniles with a 2 dpf were used in the experiment. Normally healthy zebrafish juveniles were randomly selected and aliquoted into 12-well plates, with 15 fish per well and 2 mL of juvenile rearing water added. No sunscreen cosmetic samples were applied to the transparent film of the control group and the model group. The transparent film was then placed over the well plates of the control group and the model group and subjected to 100 mJ / cm² pressure. 2 The model group was irradiated with UVB, while the control group was protected from light and not irradiated. After UVB irradiation, the juvenile fish were placed in a 28°C biochemical incubator overnight. After incubation, the juvenile fish were anesthetized, and the morphology of the zebrafish juveniles in the control and model groups was observed under a microscope. When the zebrafish juveniles showed symptoms such as body curvature, pericardial edema, yolk sac cysts, slowed heartbeat, cardiac hemorrhage, and swollen and deformed tail, they were judged as deformed, and the deformity rate was counted.

[0063] Based on Example 1, the effects of different UVB irradiation doses on the morphology of zebrafish were studied to select UVB irradiation doses that would not cause death or deformities in zebrafish for subsequent experimental implementation. Specific UVB irradiation dose parameters are shown in Table 1.

[0064]

[0065] This application statistically analyzed the malformation rate of zebrafish juveniles under different UVB irradiation doses in Examples 1-8, and the results are as follows: Figure 1 As shown. By Figure 1 UVB irradiation dose is 50 mJ / cm 2 -200 mJ / cm 2 Within this range, it will not cause deformities in juvenile zebrafish. UVB radiation dose is 300 mJ / cm². 2 At that time, the deformity rate of zebrafish juveniles was approximately 30%; the UVB radiation dose was 400 mJ / cm. 2 At that time, the deformity rate of zebrafish juveniles exceeded 40%; the UVB radiation dose was 600 mJ / cm. 2 At that time, the deformity rate of zebrafish juveniles exceeded 60%; the UVB radiation dose was 800 mJ / cm. 2 At the above levels, the deformity rate of zebrafish juveniles reached 100%. Therefore, a UVB irradiation dose of 50 mJ / cm² was selected. 2 -200 mJ / cm 2 Further research will be conducted.

[0066] Example 9

[0067] In this embodiment, a light-transmitting film was used to conduct an experiment to test the modeling effect of UVB irradiating juvenile zebrafish through the light-transmitting film.

[0068] A control group and a model group were set up. Wild-type AB strain zebrafish juveniles with a 2 dpf growth factor were used in the experiment. Normally healthy zebrafish juveniles were randomly selected and aliquoted into 12-well plates, with 10 fish per well and 2 mL of juvenile rearing water added. No sunscreen cosmetic samples were applied to the transparent film of the control group and the model group. The transparent film was then placed over the well plates of the control group and the model group, and 100 mJ / cm² was used. 2 The model group was irradiated with UVB, while the control group was protected from light and not irradiated. After UVB irradiation, the juvenile fish were placed in a 28°C biochemical incubator overnight. After incubation, the juvenile fish were anesthetized, and the tails of the zebrafish juveniles in both the control and model groups were photographed using a microscope. Image analysis software was used to analyze the tail fin area and melanin content of the juvenile fish in the control and model groups; the differences in tail fin area and melanin content between the model and control groups were compared.

[0069] Based on Example 9, this application uses different UVB irradiation doses, as shown in Table 2.

[0070]

[0071] The experimental results of Examples 9-11 are as follows: Figure 2 , Figure 3 , Figure 4 As shown. After covering with a light-transmitting film, UVB irradiation is then applied. UVB can pass through the light-transmitting film at 50 mJ / cm². 2 -200 mJ / cm 2 UVB irradiation doses did not cause death or deformities in zebrafish. ImageJ was used to statistically analyze the caudal fin area and melanin content of zebrafish. The control group was normalized, and a bar chart was created using GraphPad Prism. The ordinate represents the normalized caudal fin area and melanin content of the control group, respectively. GraphPad Prism One-way ANOVA was used for significance analysis; * indicates significance. p <0.05, ** represents p <0.01, indicating that *** represents p <0.001, **** represents p <0.0001, ns represents no significance. (From...) Figure 2 and Figure 3 It can be seen that, compared with the control group, the caudal fins of juvenile zebrafish showed significant wrinkling and a reduced caudal fin area. Figure 2 and Figure 4 It was found that in the control group of juvenile fish, melanin was mainly concentrated at the junction of muscle and fin. After UVB irradiation, melanin diffused, and the melanin level increased. (50 mJ / cm) 2-200 mJ / cm 2 The UVB irradiation doses can all induce the above phenotypes, which are suitable modeling conditions, ensuring the purposefulness and accuracy of model construction.

[0072] Compared to existing technologies (such as CN119530335B), which require high doses (400-1600 mJ / cm²) to induce extreme phenotypes such as death and teratogenicity for evaluation, this method has a weaker correlation with the chronic, cumulative photoaging and pigmentation targeted by daily sun protection. The UVB irradiation dose selected in this application can more sensitively and safely reflect the subtle differences in the efficacy of sunscreen products in preventing early and daily photodamage.

[0073] Example 12

[0074] This embodiment uses Winona Clear Whitening Sunscreen Lotion for experimentation to test its sun protection efficacy. Figure 5 As shown.

[0075] S1: Experimental grouping and zebrafish loading; preparation of zebrafish juveniles for the experiment; setting up control group, model group and sample group; placing zebrafish juveniles in multi-well plates according to the set groups; in this embodiment, wild-type AB strain zebrafish juveniles with 2 dpf were used for the experiment. Normal and healthy zebrafish juveniles were randomly selected, divided into 12-well plates, 10 fish per well, and 2 mL of juvenile fish culture water was added.

[0076] S2: Prepare the experimental film; coat the test sunscreen cosmetic sample onto the transparent film of the sample group; do not coat the test sunscreen cosmetic sample onto the transparent film of the control group and the model group; in this embodiment, Winona Clear Whitening Sunscreen Lotion is used as the test sunscreen cosmetic sample; apply Winona Clear Whitening Sunscreen Lotion evenly onto the transparent film of the sample group and let it stand for 30 min to wait for film formation; do not apply Winona Clear Whitening Sunscreen Lotion onto the transparent film of the control group and the model group.

[0077] S3: UVB irradiation treatment; the prepared experimental membrane is covered on the corresponding group of well plates; the model group and sample group are subjected to UVB irradiation; the control group is protected from light; in this embodiment, 100 mJ / cm² is used. 2 The model group and the sample group were irradiated with UVB.

[0078] S4: Efficacy Evaluation; After UVB irradiation, zebrafish juveniles in each group were cultured again; subsequently, images of the tails of the zebrafish in each group were acquired; the tail fin area and / or tail melanin content of the zebrafish juveniles in each group after UVB irradiation were quantified to evaluate the anti-photoaging and / or anti-tanning efficacy of the sunscreen cosmetic. In this embodiment, after UVB irradiation, the zebrafish juveniles in each group were placed in a 28°C biochemical incubator overnight. After incubation, the juveniles were anesthetized, and the tails of the zebrafish juveniles were photographed using a microscope. The tail fin area and melanin content of the juveniles were analyzed using image analysis software; the anti-photoaging efficacy of the sunscreen cosmetic was evaluated by comparing the difference in tail fin area between the sample group and the model group; the anti-tanning efficacy of the sunscreen cosmetic was evaluated by comparing the difference in tail melanin content between the sample group and the model group.

[0079] The calculation method for anti-photoaging efficacy is as follows: ;

[0080] Where A is the normalized average area of ​​the zebrafish tail fin.

[0081] The method for calculating sun protection efficacy is as follows: ;

[0082] Where S is the average value of melanin content in the zebrafish tail after normalization.

[0083] Based on Example 12, this application conducts experiments on different sunscreen cosmetic samples to test their sunscreen efficacy, as shown in Table 3.

[0084]

[0085] The experimental results of Example 12 are as follows Figure 6 , Figure 7 , Figure 8 As shown, the Winona Clear Whitening Sunscreen Lotion group can significantly alleviate the tail fin wrinkling and melanin increase in zebrafish juveniles caused by UVB radiation. After using Winona Clear Whitening Sunscreen Lotion, the tail fin area of ​​zebrafish juveniles was similar to that of the control group, and the melanin level was reduced. This indicates that Winona Clear Whitening Sunscreen Lotion can effectively block UVB, with 91.76% anti-photoaging and 78.54% anti-tanning effects, demonstrating strong comprehensive sun protection capabilities.

[0086] The experimental results of Example 13 are as follows: Figure 9 , Figure 10 , Figure 11As shown, the Winona Clear Sunscreen Lotion group can significantly alleviate the tail fin wrinkling and increased melanin in zebrafish juveniles caused by UVB radiation. After using Winona Clear Whitening Sunscreen Lotion, the tail fin area of ​​zebrafish juveniles was similar to that of the control group, and the melanin level was reduced. This indicates that Winona Clear Sunscreen Lotion can effectively block UVB, with 92.14% anti-photoaging and 72.55% anti-tanning effects, demonstrating strong comprehensive sun protection capabilities.

[0087] The experimental results of Example 14 are as follows: Figure 12 , Figure 13 , Figure 14 As shown, the Winona Baby Gentle and Refreshing Sunscreen Lotion can alleviate the shrinkage of the tail fin and increased melanin in juvenile zebrafish caused by UVB radiation to a certain extent. After using the Winona Baby Gentle and Refreshing Sunscreen Lotion, the tail fin area of ​​the juvenile zebrafish was significantly larger than that of the model group, and the melanin level was reduced. This indicates that the Winona Baby Gentle and Refreshing Sunscreen Lotion can block UVB to a certain extent, with 36.67% anti-photoaging and 46.90% anti-tanning effects, and has a certain comprehensive sun protection ability.

[0088] In summary, the non-therapeutic method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model provided in this application has been successfully applied to the efficacy evaluation of three Winona sunscreen products with different formulations (Clear Whitening Sunscreen Lotion, Clear Sunscreen Lotion, and Baby Gentle Refreshing Sunscreen Lotion). Experimental results show that: The evaluation method described above can sensitively and reliably quantify the differences among various sunscreen products in two core functions: anti-photoaging (indicating tail fin area) and anti-tanning (indicating melanin content). All tested products showed varying degrees of protection, confirming the effectiveness and universality of the evaluation system.

[0089] This method demonstrates excellent efficacy differentiation ability, as detailed below: High-efficacy products: Winona Clear Whitening Sunscreen and Clear Sunscreen demonstrate excellent comprehensive sun protection capabilities, with anti-photoaging efficacy exceeding 90% and anti-tanning efficacy exceeding 70%, proving their strong blocking effect against UVB.

[0090] Medium-efficacy product: Winona Baby Gentle and Refreshing Sunscreen Lotion shows medium protection effect, with anti-photoaging and anti-tanning effects of 36.67% and 46.90% respectively, which is consistent with the product's positioning as an infant and toddler product with a gentler formula.

[0091] The evaluation method established in this application can not only intuitively and quantitatively verify the effectiveness of sunscreen products, but also accurately distinguish the efficacy levels of different products, providing strong data support and scientific basis for the research and development, formulation optimization, market positioning and efficacy claims of sunscreen products.

[0092] Example 15

[0093] In this embodiment, the sunscreen product was directly administered into a well plate to evaluate its toxicity.

[0094] Winona Clear Whitening Sunscreen Lotion, Winona Clear Sunscreen Lotion, and Winona Baby Gentle Refreshing Sunscreen Lotion were prepared into suspensions with concentrations of 100 mg / mL (approximately 10 times the dose of the sunscreen concentrate), 10 mg / mL, and 1 mg / mL, respectively (sunscreen products are special and cannot be completely dissolved in water). Wild-type AB strain zebrafish juveniles with a 72 hpf were used in the experiment. Normal and healthy zebrafish juveniles were randomly selected and divided into 12-well plates, with 15 fish in each well. 2 mL of the sunscreen solution to be tested was added, and the zebrafish were cultured and observed for mortality and deformities.

[0095] Experimental results are as follows Figure 15 , Figure 16 , Figure 17 As shown, zebrafish mortality was high when only sunscreen solution was administered without UVB irradiation. Two hours after administration of a 100 mg / mL sunscreen solution, juvenile zebrafish stopped swimming and became sluggish; they died completely after four hours. All zebrafish died after 12 hours of administration of a 10 mg / mL sunscreen solution. The mortality rate was as high as 86.67% after 24 hours of administration of a 1 mg / mL Winona Baby Mild and Refreshing Sunscreen Lotion. These results indicate that sunscreen products are highly toxic; the undiluted solution cannot be administered directly, and even after dilution, the toxicity remains high, easily leading to death or deformities in zebrafish. This makes it difficult to accurately reflect the sunscreen's efficacy. Furthermore, direct administration requires the sunscreen product to be water-soluble, making it impossible to measure the sunscreen efficacy of water-insoluble sunscreens.

Claims

1. A method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes, characterized in that, Includes the following steps: S1: Experimental grouping and zebrafish loading; prepare zebrafish juveniles for the experiment; set up a control group, a model group, and at least one sample group; place the zebrafish juveniles in the multi-well plate according to the set groups; S2: Prepare the experimental film; coat the test sunscreen cosmetic sample onto the transparent film of the sample group; do not coat the test sunscreen cosmetic sample onto the transparent film of the control group and the model group; S3: UVB irradiation treatment; the experimental membrane prepared in step S2 is covered on the well plate of the corresponding group set in step S1; the model group and the sample group are irradiated with UVB; the control group is protected from light. S4: Efficacy evaluation; After UVB irradiation, zebrafish fry of each group were cultured; then images of the tails of zebrafish of each group were acquired. The tail fin area of ​​zebra juveniles in each group after UVB irradiation was quantified, and the anti-photoaging efficacy of the sunscreen cosmetic was evaluated by comparing the difference in tail fin area between the sample group and the model group. and / or The melanin content in the tails of zebra fry in each group after UVB irradiation was quantified. The anti-tanning efficacy of the sunscreen cosmetic was evaluated by comparing the difference in tail melanin content between the sample group and the model group.

2. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... The zebra fry mentioned in step S1 are zebra fry that are 48-120 hours after fertilization.

3. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... The multi-well plate mentioned in step S1 is a carrier for placing zebrafish fry, including one of the following: petri dish, six-well plate, twelve-well plate, twenty-four-well plate, forty-eight-well plate, and ninety-six-well plate.

4. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... In step S2, the sunscreen cosmetic sample to be tested needs to be applied 30-60 minutes before UVB irradiation to allow the sunscreen cosmetic sample to form and maintain a film-forming state.

5. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... In step S2, the light-transmitting film is a film that can transmit UVB.

6. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... In step S3, the UVB irradiation dose is 50-200 mJ / cm².

7. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... In step S3, the experimental membrane of the sample group is covered with the side of the sunscreen cosmetic sample to be tested facing the zebrafish juveniles placed in the well plate or facing the UVB light source.

8. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... In step S4, the zebrafish fry are cultured for 12-72 hours.

9. The method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on a zebrafish model for non-therapeutic purposes as described in claim 1, characterized in that... In step S4, the caudal fin area of ​​the zebra fry is obtained by calculating the area of ​​the caudal fin outline using image analysis software; the melanin content of the zebra fry's tail is obtained by performing grayscale analysis on the tail image using image analysis software.

10. The application of the method for evaluating the anti-photoaging and anti-tanning efficacy of sunscreen cosmetics based on the zebrafish model for non-therapeutic purposes as described in claim 1 in the screening or evaluation of sunscreen cosmetics.

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