A method and application for evaluating the skin-whitening effects of safflower filament polysaccharide and seed meal polysaccharide

Abstract

This invention belongs to the field of UVB damage technology and provides a method and application for evaluating the whitening effects of safflower filament polysaccharide and seed meal polysaccharide. A UVB photoaging model of juvenile zebrafish was established to evaluate the whitening effects of the two polysaccharides, including grayscale analysis of melanin in juvenile zebrafish, determination of melanin content, and tyrosinase activity. The results show that both safflower filament polysaccharide and seed meal polysaccharide can reduce melanin content and inhibit tyrosinase activity, thus exhibiting whitening effects.

Description

Technical Field

[0001] This invention belongs to the field of UVB damage technology, and relates to the repair of UVB damage by safflower filament polysaccharide and seed meal polysaccharide. Specifically, it relates to a method and application for evaluating the whitening effects of safflower filament polysaccharide and seed meal polysaccharide. Background Technology

[0002] Photoaging is a type of skin damage induced by ultraviolet radiation, manifested as wrinkles, fine lines, uneven pigmentation, and decreased skin elasticity. Its pathogenesis involves oxidative stress, DNA damage, inflammatory responses, and activation of matrix metalloproteinases (MMPs). UVB radiation induces oxidative stress, DNA damage, and inflammation, leading to wrinkles, impaired barrier function, and increased cancer risk. UV radiation can be absorbed by chromophores within cells, transferring energy to oxygen and generating large amounts of reactive oxygen species (ROS) on mitochondria and cell membranes. Excessive ROS attack and damage cell membranes, proteins, mitochondria, and DNA, disrupting normal cellular function. ROS are key initiators and core drivers of skin aging, especially photoaging. After UV irradiation, keratinocytes secrete more α-MSH. α-MSH binds to the melanocortin 1 receptor (MC1R) on the surface of melanocytes, activating the cAMP / PKA pathway and upregulating microphthalmia-associated transcription factor (MITF). MITF then promotes the expression of tyrosinase (TYR), the rate-limiting enzyme in melanin synthesis. This leads to melanin deposition.

[0003] Zebrafish models are widely used high-throughput, low-cost in vivo screening models. Zebrafish embryos offer advantages such as rapid development, high transparency, clear genetic background, and low rearing costs, allowing for parallel screening of multiple doses and groups within a short period. Melanocytes form during the embryonic stage, and pigment deposition is readily visible, making them suitable for imaging, phenotypic, and quantitative analysis. In photodamage studies, zebrafish are sensitive to UVB, enabling the construction of stable photoaging pigmentation models. Furthermore, image analysis (e.g., ImageJ quantification of pigments) and biochemical detection (e.g., NaOH lysis method for melanin content determination) enable quantifiable and reproducible efficacy evaluation. Compared to in vitro models, zebrafish models better reflect in vivo absorption, metabolism, and synergistic effects, offering significant advantages in the screening of cosmetics and natural active ingredients.

[0004] Safflower (Carthamus tinctorius L.) is abundant, with its filaments and seed meal being the main byproducts. Safflower polysaccharides, as natural high-molecular-weight components, possess advantages such as high safety, stable sources, and sustainable utilization, and have potential value in cosmetics, functional foods, raw material development, and bioactivity screening. Current research mainly focuses on the basic extraction, physicochemical properties, and evaluation of some in vitro activities (such as antioxidant and anti-inflammatory effects) of safflower polysaccharides. However, in vivo evaluation methods related to skin whitening and pigmentation are still unsystematic, lacking unified models and quantitative indicators. Furthermore, as a processing byproduct, the utilization rate of safflower seed meal's polysaccharide resources remains low, and the pathways for productization and efficacy verification are unclear.

[0005] The limitations of existing intervention strategies: Currently, mainstream photoaging protection products on the market are mainly physical / chemical sunscreens, which primarily function by passively shielding or absorbing ultraviolet rays. These strategies suffer from limitations such as a single mode of action, the inability to reverse existing damage, and the potential for some chemical components to cause sensitization. Therefore, developing natural, multi-target, highly effective, and safe active ingredients, and employing novel delivery systems to enhance their efficacy, has significant scientific value and market potential.

[0006] Therefore, this invention establishes an evaluation system for the whitening effects of safflower filament polysaccharides and seed meal polysaccharides, which not only helps to improve the comprehensive utilization rate of safflower resources, but also provides a scientific basis for subsequent product development, efficacy claims and application implementation. Summary of the Invention

[0007] The purpose of this invention is to provide a method and application for evaluating the whitening effects of safflower filament polysaccharide and seed meal polysaccharide. The UVB zebrafish photoaging model is used to evaluate the whitening effects of safflower filament polysaccharide and seed meal polysaccharide, providing a new approach and means for anti-UVB oxidation.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] This invention provides a method for evaluating the skin-whitening effects of safflower filament polysaccharide and seed meal polysaccharide, comprising the following steps:

[0010] S1. Provide AB wild-type zebrafish. After the zebrafish mate and are fertilized, collect, clean and culture the embryos. Establish a UVB zebrafish photoaging model by irradiating the 3dpf period zebrafish juveniles with UVB for 3 min.

[0011] S2. After the UVB zebrafish photoaging model was placed in a non-UVB environment and stabilized for half an hour, safflower filament polysaccharide / seed meal polysaccharide were added to its culture medium for polysaccharide treatment as experimental groups, and zebrafish juveniles were cultured for 24 h. The control group was the UVB zebrafish photoaging model without polysaccharide treatment and was cultured directly for 24 h.

[0012] S3. Efficacy Evaluation: Images were acquired, melanin was extracted, and tyrosinase activity was detected from the tails of zebrafish in the experimental and control groups after cultivation to evaluate the whitening effects of safflower filament polysaccharide and seed meal polysaccharide.

[0013] Preferably, the polysaccharide treatment specifically involves adding safflower filament polysaccharide at a concentration of 80~320 µg / mL to the UVB zebrafish photoaging model culture medium.

[0014] Preferably, the polysaccharide treatment specifically involves adding safflower seed meal polysaccharide at a concentration of 120-160 µg / mL to the UVB zebrafish photoaging model culture medium.

[0015] The present invention also provides a composition for resisting UVB oxidation, the composition comprising safflower filament polysaccharide and / or seed meal polysaccharide.

[0016] The present invention also provides the use of the above-described composition in the preparation of skin repair formulations.

[0017] The beneficial effects of this invention are:

[0018] This invention establishes a UVB zebrafish photoaging model to evaluate the whitening effects of safflower filament polysaccharide and seed meal polysaccharide, including grayscale analysis of melanin in zebrafish juveniles, measurement of melanin content and tyrosinase activity; the results show that both safflower filament polysaccharide and seed meal polysaccharide can reduce melanin content and inhibit tyrosinase activity, and have significant whitening effects. Attached Figure Description

[0019] Figure 1 The changes in ROS fluorescence intensity of zebrafish under different UVB irradiation doses in Examples 1-5 of this invention;

[0020] Figure 2 The mortality rate of zebrafish juveniles under different UVB irradiation doses in Examples 1-5 of this invention;

[0021] Figure 3 The malformation rate of zebrafish juveniles under different UVB irradiation doses in Examples 1-5 of this invention;

[0022] Figure 4 Image J shows the grayscale changes of melanin in zebrafish juveniles exposed to different concentrations of safflower filament polysaccharide after UVB irradiation in Examples 6-11 of this invention. * P < 0.05 ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the Model group; #### P < 0.0001 (comparison between model group and control group).

[0023] Figure 5 The grayscale changes of melanin in zebrafish juveniles exposed to different concentrations of safflower seed meal polysaccharide after UVB irradiation in Examples 12-14 and 16-18 of this invention are shown. *** P < 0.001, **** P < 0.0001 compared with the Model group; #### P < 0.0001 (comparison between model group and control group).

[0024] Figure 6 The results show the effects of different concentrations of safflower filament polysaccharide on melanin content in a UVB photoaged zebrafish model in Examples 8-10 of this invention. ** P < 0.01, *** P < 0.001 compared with the Model group; ### P < 0.001 (comparison between model group and control group).

[0025] Figure 7 The effect of different concentrations of safflower seed meal polysaccharide on melanin content in a UVB photoaged zebrafish model in Examples 14-17 of this invention. ** P < 0.01, *** P < 0.001 compared with the Model group; ### P < 0.001 (comparison between model group and control group).

[0026] Figure 8 These are characterization images of zebrafish juveniles exposed to different concentrations of safflower filament polysaccharide or seed meal polysaccharide after UVB irradiation in Examples 8-10 and 14-17 of this invention.

[0027] Figure 9 The changes in tyrosinase activity in zebrafish juveniles exposed to different concentrations of safflower filament polysaccharide after UVB irradiation in Examples 8-10 of this invention are illustrated below. ** P < 0.01, *** P < 0.001, compared with the Model group; ### P < 0.001 (comparison between model group and control group).

[0028] Figure 10 The changes in tyrosinase activity in zebrafish juveniles exposed to different concentrations of safflower seed meal polysaccharide after UVB irradiation in Examples 14-17 of this invention are shown below. ** P < 0.01, *** P < 0.001, compared with the Model group; ### P < 0.001 (comparison between model group and control group). Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Example 1: Screening of UVB irradiation dose (time)

[0033] S1. Preparation of zebrafish larvae: Provide AB wild-type zebrafish. After the zebrafish mate and are fertilized, collect, clean, and culture the embryos. Establish a UVB zebrafish photoaging model by using 0 J / cm² on zebrafish larvae at the 3 dpf stage.

[0034] S2. Detection: After the zebrafish model was established, the juvenile zebrafish were incubated at 28℃ for 12 h. A DCFH-DA probe was then added to detect ROS fluorescence intensity. After 24 h of incubation, the juveniles were anesthetized, and their lethality and teratogenicity were assessed. The morphology of the zebrafish juveniles was observed under a microscope. Symptoms such as body curvature, pericardial edema, yolk sac cysts, slowed heartbeat, cardiac hemorrhage, and tail swelling and deformation were considered malformations, and the malformation rate was calculated. The absence of heartbeat and blood flow was considered a death, and the mortality rate was calculated.

[0035] Based on Example 1, the effects of different UVB irradiation doses on the morphology and ROS of zebrafish were studied to select a UVB irradiation dose that would not cause death or deformity in zebrafish for subsequent experiments. Example 2: 0.18 J / cm². Example 3: 0.36 J / cm². Example 4: 0.54 J / cm². Example 5: 0.72 J / cm². With a fixed UVB wavelength and light intensity, the irradiation dose is determined by the irradiation time; therefore, this experiment used the irradiation duration to achieve the required irradiation dose.

[0036] This invention statistically analyzed the malformation rate, mortality rate, and ROS fluorescence intensity of zebrafish juveniles under different UVB irradiation doses in Examples 1-5. The results are as follows: Figure 1 , 2As shown in Figure 3, UVB irradiation doses in the range of 0 J / cm²–0.18 J / cm² did not cause mortality in zebrafish juveniles. At a UVB irradiation dose of 0.72 J / cm², the mortality rate of zebrafish juveniles was approximately 24%; at UVB irradiation doses of 0 J / cm²–0.36 J / cm², the deformity rate of zebrafish juveniles was less than 10%; at UVB irradiation doses below 0.36 J / cm², the deformity rate of zebrafish juveniles reached 20%; and at UVB irradiation doses above 0.72 J / cm², the deformity rate of zebrafish juveniles reached 20%. The relative fluorescence intensity of ROS at a UVB irradiation dose of 0.16 J / cm² was 120% higher than that of the 0 J / cm² group. Therefore, a UVB irradiation dose of 0.36 J / cm² was selected for subsequent studies, corresponding to an irradiation time of 3 min.

[0037] Example 6

[0038] S1. Provide AB wild-type zebrafish. After the zebrafish mate and are fertilized, collect, clean and culture the embryos. Establish a UVB zebrafish photoaging model by irradiating the 3dpf period zebrafish juveniles with UVB for 3 min.

[0039] S2. After the UVB zebrafish photoaging model was stabilized in a non-UVB environment for half an hour, the zebrafish were divided into groups. The model group was irradiated with only 5 ml of culture medium, the control group was irradiated with 5 mL of 0.2 mmol / L PTU (PTU group / Positive Control group), and the polysaccharide group was irradiated with 5 mL of 20 µg / mL safflower filament polysaccharide (HS) (experimental group). A blank group was set up without irradiation (Control group). The zebrafish model treated in the above way were incubated in a 28℃ incubator for 24 h. The fish culture medium was used as the solvent for the preparation of the above drugs.

[0040] S3. Efficacy evaluation: Images of the heads of zebrafish in each group after hatching were collected; the melanin content of juvenile fish in the control group and polysaccharide group was analyzed using image analysis software.

[0041] Take a fixed number of zebrafish (10 per group) for each group, wash gently with PBS to remove culture medium residue; centrifuge at 10,000-11,000 rpm for 10-15 minutes at 4℃ with a pulse intensity of 40-45%, a cycle interval of 10-15 seconds, and a total time of 10-20 minutes in PBS containing 1% Triton X-100; collect the precipitate: retain the black precipitate after centrifugation, add 1 M NaOH solution (with 10% DMSO added for dissolution); heat in a water bath at 80-100℃ for 30 minutes to mix thoroughly; vortex intermittently to ensure complete dissolution of melanin; measure the OD value of melanin at 405 nm.

[0042] A fixed number of zebrafish (10 per group) were used in each group. After washing with PBS, lysis buffer was added and the mixture was homogenized. The mixture was centrifuged at 10,000-12,000 rpm for 10 min at 4°C, and the supernatant was collected as the enzyme solution. L-DOPA substrate and reaction buffer were added; the mixture was incubated at 37°C in the dark for 30-60 min, and the absorbance was measured at 475 nm. Background was subtracted from the blank control; relative activity was calculated based on the model group.

[0043] Based on Example 6, this invention investigated the effects of different types and concentrations of polysaccharides used in S3 on melanin content and tyrosinase activity in juvenile zebrafish. The specific polysaccharides used were: Example 7: 40 µg / mL safflower filament polysaccharide; Example 8: 80 µg / mL safflower filament polysaccharide; Example 9: 160 µg / mL safflower filament polysaccharide; Example 10: 320 µg / mL safflower filament polysaccharide; Example 11: 640 µg / mL safflower filament polysaccharide; Example 12: 20 µg / mL safflower seed meal (ZP) polysaccharide; Example 13: 40 µg / mL safflower seed meal polysaccharide; Example 14: 80 µg / mL safflower seed meal polysaccharide; Example 15: 120 µg / mL safflower seed meal polysaccharide; Example 16: 160 µg / mL safflower seed meal polysaccharide; Example 17: 320 µg / mL safflower seed meal polysaccharide; Example 18: 40 µg / mL safflower seed meal polysaccharide; Example 19: 40 µg / mL safflower seed meal polysaccharide; Example 20 ... Safflower seed meal polysaccharide at µg / mL; Example 18: Safflower seed meal polysaccharide at 640 µg / mL.

[0044] Image J shows the experimental results of quantifying melanin in juvenile zebrafish. Figures 4-5 As shown, the safflower filament polysaccharide melanin IntDen is displayed ( Figure 4 Compared with the model group, melanin levels in the safflower seed meal polysaccharide group were significantly reduced, showing an overall dose-dependent trend. After correction with the control group as a blank, the relative melanin IntDen values ​​of the 20 µg / mL, 40 µg / mL, 80 µg / mL, 160 µg / mL, 320 µg / mL, and 640 µg / mL groups were approximately 0.578, 0.530, 0.428, 0.119, 0.054, and 0.131, respectively, corresponding to reduction rates of approximately 42.16%, 47.00%, 57.25%, 88.11%, 94.60%, and 86.91%. Among them, the 320 µg / mL group showed the largest reduction, approaching the blank level, indicating a significant inhibitory effect on melanin deposition. The IntDen results for safflower seed meal polysaccharide showed inconsistent changes. Figure 5After blank correction, the relative melanin levels in the 80 µg / mL and 160 µg / mL groups were approximately 0.676 and 0.584 (reduction rates of approximately 32.45% and 41.59%), respectively, indicating a certain inhibitory effect. However, the relative melanin levels in the 20 µg / mL, 40 µg / mL, 320 µg / mL, and 640 µg / mL groups were all higher than the model (approximately 1.199, 1.179, 1.401, and 1.413), showing an increase in melanin levels. This suggests that the action direction of safflower seed meal polysaccharide is unstable at different dosages or that it has a tendency to promote deposition.

[0045] The above results indicate that the effective repair concentration of safflower filament polysaccharide is 20~640 µg / mL, and the effective repair concentration of safflower seed meal polysaccharide is 80~160 µg / mL.

[0046] Figure 6 The effect of different concentrations of safflower filament polysaccharide on melanin content in a UVB-aged zebrafish model was investigated. The results showed that within the concentration range of 80–320 µg / mL, the effect of safflower filament polysaccharide on melanin content increased with increasing concentration. Figure 7 The effect of different concentrations of safflower seed meal polysaccharide on melanin content in a UVB-aged zebrafish model was investigated. Results showed that within the concentration range of 120–160 µg / mL, the effect of safflower seed meal polysaccharide on melanin content increased with increasing concentration. Figure 8 The corresponding changes in melanin can also be seen in the images.

[0047] Figures 9-10 The relative activity of tyrosinase was measured. In the positive control group (PTU), the inhibition rate was 100.97% (strong inhibition). In the safflower filament polysaccharide group, inhibition increased with increasing dosage, reaching 31.64%, 53.80%, and 85.02% at 80 µg / mL, 160 µg / mL, and 320 µg / mL, respectively. Figure 9 The safflower seed meal polysaccharide group also showed inhibitory effects, with 14.15%, 41.80%, 59.43%, and 22.63% at 80 µg / mL, 120 µg / mL, 160 µg / mL, and 320 µg / mL, respectively. Figure 10 It can be seen that the inhibitory effect of safflower seed meal polysaccharide on tyrosinase activity decreases after the concentration exceeds 160 µg / mL.

[0048] The results above indicate that the effective recovery concentration of safflower filament polysaccharide for UVB damage is 80–320 µg / mL, and the effective recovery concentration of safflower seed meal polysaccharide for UVB damage is 120–160 µg / mL.

[0049] In summary, based on the combined data of InDen, melanin content, and tyrosinase activity inhibition rate, it can be concluded that: safflower filament polysaccharide shows a consistent inhibitory trend across multiple indicators, with the effect increasing with increasing dosage, especially at 320 µg / mL, where it is particularly prominent, and the effective recovery concentration for UVB damage is 80~320 µg / mL; safflower seed meal polysaccharide has a UVB damage recovery effect at a dosage of 120~160 µg / mL. Both can be used as substances for anti-UVB aging.

[0050] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for evaluating the skin-whitening effects of safflower filament polysaccharide and seed meal polysaccharide, characterized in that, Includes the following steps: S1. Provide AB wild-type zebrafish. After the zebrafish mate and are fertilized, collect, clean and culture the embryos. Establish a UVB zebrafish photoaging model by irradiating the 3dpf period zebrafish juveniles with UVB for 3 min. S2. After the UVB zebrafish photoaging model was placed in a non-UVB environment and stabilized for half an hour, safflower filament polysaccharide / seed meal polysaccharide were added to its culture medium for polysaccharide treatment as experimental groups, and zebrafish juveniles were cultured for 24 h. The control group was the UVB zebrafish photoaging model without polysaccharide treatment and was cultured directly for 24 h. S3. Efficacy Evaluation: Images were acquired, melanin was extracted, and tyrosinase activity was detected from the tails of zebrafish in the experimental and control groups after cultivation to evaluate the whitening effects of safflower filament polysaccharide and seed meal polysaccharide.

2. The method according to claim 1, characterized in that, The polysaccharide treatment specifically involves adding safflower filament polysaccharide at a concentration of 80~320 µg / mL to the UVB zebrafish photoaging model culture medium.

3. The method according to claim 1, characterized in that, The polysaccharide treatment specifically involves adding safflower seed meal polysaccharide at a concentration of 120-160 µg / mL to the UVB zebrafish photoaging model culture medium.

4. A composition for resisting UVB oxidation, characterized in that, The composition includes safflower filament polysaccharide and / or seed meal polysaccharide.

5. The use of the composition according to claim 5 in the preparation of skin repair formulations.

Images