Marine bioactive peptide, preparation method and application of marine bioactive peptide in skin ultraviolet injury repair
By screening and preparing the active peptides PFRVF and PPPR from the flesh of the red snail, the defects of existing UV protection methods were solved, high-efficiency antioxidant and skin barrier repair were achieved, and safe and stable UV protection effects were provided.
Patent Information
- Application Number
- CN202510714529.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-10-17
AI Technical Summary
Existing UV protection methods such as physical sunscreen, chemical sunscreen and natural antioxidants have their own defects and cannot effectively protect the skin from UV damage, and there is little research on skin barrier repair.
The active peptides PFRVF and PPPR from the flesh of the red snail were screened out, and by optimizing the extraction and purification process, active peptides with high antioxidant activity and stability were prepared. They can be used to prepare drugs or cosmetics against UV damage, inhibit the expression of MMP-1, reduce the release of inflammatory factors, and relieve skin inflammation caused by UV rays.
It achieves highly effective anti-oxidation, reduces free radical levels, improves cellular antioxidant capacity, inhibits collagen degradation, reduces skin inflammation and erythema, and provides safe and stable UV protection.
Smart Images
Figure CN120795070A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biopharmaceuticals, and particularly relates to a marine active peptide and its application in repairing skin ultraviolet damage. BACKGROUND
[0002] Skin is the first defense barrier of the human body, and long-term exposure to ultraviolet radiation can cause photoaging, inflammation and DNA damage. Current ultraviolet protection methods mainly include: 1. Physical sun protection (such as titanium dioxide, zinc oxide, etc.), which reduces the penetration of ultraviolet rays by reflecting or scattering them, but has problems such as affecting skin texture, easily blocking pores, heavy skin feel, affecting air permeability, etc. 2. Chemical sun protection (such as avobenzone, octocrylene, etc.), which protects the skin by absorbing ultraviolet rays and converting them into heat energy, but some ingredients can cause skin allergies, have potential toxicity, and even have potential endocrine disrupting risks. 3. Natural antioxidants (such as vitamin C, astaxanthin, resveratrol, etc.), which have certain antioxidant and anti-inflammatory effects, but have poor stability and are easily degraded, affecting skin care effects.
[0003] In recent years, marine active peptides have become a new direction for research on ultraviolet damage resistance due to their low toxicity, high activity and good stability. The active peptides that have been studied the most include: oyster peptides (with antioxidant and anti-inflammatory effects); cod skin collagen peptides (reduce the expression of MMP-1 and inhibit collagen degradation); sea cucumber peptides (regulate the Nrf2 signaling pathway and increase antioxidant enzyme activity).
[0004] However, the ultraviolet damage protection mechanisms of these active peptides are not clear, and research on skin barrier repair is still limited.
[0005] The present application aims to screen out active peptides from Thais punctata meat and study their anti-ultraviolet damage mechanisms through molecular biology, providing a safe and efficient natural sunscreen ingredient. The main goals include: 1. Screening active peptides with high anti-ultraviolet damage activity and optimizing their extraction and purification processes. 2. Verifying the effects of active peptides on antioxidant, anti-inflammatory, and collagen protection through cell and animal experiments. SUMMARY
[0006] To overcome the problem of skin damage caused by ultraviolet (UV) radiation, the present application aims to design and provide a method and application based on Thais punctata meat active peptides and their anti-ultraviolet damage.
[0007] To achieve the above purpose, the present application adopts the following technical solutions: On the one hand, the present application provides a Thais punctata meat active peptide or its derivative, wherein the Thais punctata meat active peptide is an amino acid sequence as shown in SEQ ID NO. 1-19 or its derivative.
[0008] The one kind of Thais clavigera meat active peptide or its derivative, the Thais clavigera meat active peptide is the amino acid sequence as SEQ ID NO.8 or SEQ ID NO.16 or its derivative.
[0009] Secondly, the application provides a preparation method of the one kind of Thais clavigera meat active peptide or its derivative, comprising the following steps: (1) weigh Thais clavigera meat, dissolve in water, add alkaline protease for enzymolysis reaction, obtain enzymolysis product; (2) ultrafiltration, select the component with the highest activity; (3) the component with the highest activity obtained in the above step (2) is further purified, and the component with high DPPH clearance rate is separated; (4) the component with the highest DPPH clearance rate obtained in the above step (3) is separated by reverse phase high performance liquid chromatography, and the component with high DPPH clearance rate is selected; (5) sequence identification is carried out on the component with high DPPH clearance rate obtained in the above step (4), and the sequence of the active peptide is obtained.
[0010] The preparation method, the conditions of the enzymolysis reaction are as follows: the mass / volume ratio of Thais clavigera meat and water is 8-12:1 mL / g, the enzymolysis reaction time is 2-4 h, and the addition amount of alkaline protease is 2-4%. The purification method in step (3) is dextran gel chromatography; The separation method in step (4) is reverse phase high performance liquid chromatography.
[0011] Thirdly, the application provides a nucleic acid encoding any one of the Thais clavigera meat active peptide or its derivative.
[0012] Fourthly, the application provides a vector comprising the nucleic acid.
[0013] Fifthly, the application provides a host cell transfected with the vector.
[0014] Sixthly, the application provides the application of any one of the Thais clavigera meat active peptide or its derivative in the preparation of a medicine or a cosmetic for resisting aging caused by ultraviolet light, resisting skin inflammation caused by ultraviolet light or resisting oxidation. Preferably, the skin inflammation is selected from erythema or peeling phenomenon.
[0015] Seventhly, the application provides a pharmaceutical composition or a cosmetic, wherein the pharmaceutical composition comprises the Thais clavigera meat active peptide or its derivative.
[0016] Compared with the prior art, the present application has the following beneficial effects: 1. High efficiency against ultraviolet damage: the antioxidant activity of the active peptide PFRVF of the present application is better than that of various natural ultraviolet-resistant substances reported at present; reduces the level of free radicals (ROS) and improves the antioxidant capacity of cells.
[0017] 2. Safe and non-toxic: the toxicity prediction results show that the active peptides PFRVF and PPPR of the present application are non-toxic and suitable for long-term use.
[0018] 3. Good stability: compared with polyphenol antioxidants, the active peptides PFRVF and PPPR of the present application have better stability and can be used for the development of cosmetics or medical skin care products.
[0019] 4. Inhibition of collagen degradation: prevents skin relaxation and wrinkle formation caused by ultraviolet rays.
[0020] 5. Regulation of inflammatory response: reduces the release of inflammatory factors (such as IL-1β, IL-6 and TNF-α) and alleviates chronic inflammation caused by ultraviolet rays. Reduces skin inflammation, erythema and peeling caused by ultraviolet rays. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 is the contour line and response surface graph of the influence of the interaction of different two factors on DPPH clearance rate; Figure 2 is the DPPH clearance rate of each ultrafiltration component; Figure 3 is the G-25 dextran gel chromatography elution curve of the ultrafiltration component; Figure 4 is the DPPH clearance rate of components P1 and P2; Figure 5 is the reverse high performance liquid chromatogram of P1 component; Figure 6 is the DPPH clearance rate of P1-1, P1-2, P1-3 and P1-4; Figure 7 is the total ion flow (TIC) chromatogram of the peptide; Figure 8 is the PFRVF and MMP-1 interaction graph; Figure 9 is the PFRVF and MMP-1 molecular docking graph; Figure 10 is the PPPR and MMP-1 interaction graph; Figure 11 is the PPPR and MMP-1 molecular docking graph; Figure 12Effects of PFRVF and PPPR on cell viability, note: **P<0.01 compared with the blank group; Figure 13 Effects of PFRVF and PPPR on ROS levels in cells after UVB radiation, wherein A: blank group, B: model group, C: PFRVF low-dose group, D: PFRVF medium-dose group; E: PFRVF high-dose group, F: PPPR low-dose group, G: PPPR medium-dose group, H: PPPR high-dose group; Figure 14 Effects of PFRVF and PPPR on antioxidant capacity in cells after UVB radiation, wherein (A): SOD; (B): CAT; (C): GSH-Px; (D): MDA; Figure 15 Effects of PFRVF and PPPR on MMP-1 in cells after UVB radiation; Figure 16 Effects of PFRVF on the back skin of mice; Figure 17 HE staining of the back skin of mice (400x); Figure 18 Masson staining of the back skin of mice (400x); Figure 19 Siro red staining of the back skin of mice (400x); Figure 20 Contents of TNF-α, IL-1β and IL-6 in the back skin of mice, wherein A is the TNF-α content index, B is the IL-1β content index, and C is the IL-6 content index. DETAILED DESCRIPTION
[0022] The application will be further described below in combination with the accompanying drawings and examples.
[0023] Example 1: Preparation of active peptide Raw material: fresh Thais clavigera meat.
[0024] 1. Step 1: The raw material was subjected to enzymolysis to obtain an alkaline protease hydrolysate, wherein the enzymolysis method was as follows: Alkaline protease hydrolysis was adopted, and the enzymolysis conditions were as follows: liquid material ratio 10:1, enzymolysis time 3 h, and enzyme amount 3%.
[0025] The response surface experiment was used, the liquid material ratio of double distilled water and Thais clavigera meat was 10:1 mL / g, the enzyme hydrolysis time was 3 h, the alkaline protease enzyme amount was 3%, at this time the antioxidant effect of the enzyme hydrolysis product was better. In order to improve the enzyme hydrolysis effect, the response surface experiment was used to further optimize the enzyme hydrolysis conditions. The data in table 1 was analyzed, and the effects of (A) liquid material ratio, (B) enzyme hydrolysis time, (C) enzyme amount on the DPPH clearance rate of alkaline protease enzyme hydrolysis product were established.
[0026] Table 1 response surface design and test results
[0027] The quadratic polynomial regression equation is as follows: R=60.824 + 1.1925 * A + 4.0375 * B - 1.1775 *C - 4.4075 * AB - 2.6275 * AC - 1.3275 * BC - 6.86075 * A2 - 3.87575 * B2 -6.04575 * C2 According to the regression equation, the optimal enzyme hydrolysis preparation conditions of Thais clavigera meat are as follows: liquid material ratio is 9.98:1, enzyme hydrolysis time is 3.51 h, enzyme amount is 2.92%, at this time the DPPH clearance rate is 61.99%.
[0028] Response surface regression model result analysis. As shown in table 2, the p-value of the regression model is less than 0.0001 ( P < 0.05), which indicates that the regression model of the response surface experiment of Thais clavigera meat hydrolysis has significance, and the p-value of the lack of fit is 0.2638 ( P > 0.05), which has no significant difference. Therefore, the regression model can be used to predict the relationship between the DPPH clearance rate of Thais clavigera meat alkaline protease enzyme hydrolysis product and the enzyme hydrolysis time, liquid material ratio and enzyme amount.
[0029] As shown in table 2, the influence degree of each factor on the DPPH clearance rate of Thais clavigera meat enzyme hydrolysis product is in descending order: time> liquid material ratio> enzyme amount. Among them, the effect of time on the DPPH clearance rate of Thais clavigera meat enzyme hydrolysis product is the largest.
[0030] Table 2 regression model results and analysis
[0031] The response surface graph and contour graph of this experiment can reflect the interaction between each factor, as well as the influence degree of enzyme hydrolysis time, liquid material ratio and enzyme amount on the DPPH clearance rate of Thais clavigera meat enzyme hydrolysis product. Figure 1The shape of the contour lines is close to an ellipse, indicating that the interaction between the two factors is more significant. It can be seen that the interaction between liquid-solid ratio and time has the greatest effect on the DPPH scavenging rate of the enzymatic hydrolysate of Thais clavigera meat. When optimizing the enzymatic hydrolysis process of Thais clavigera meat, the interaction between liquid-solid ratio and enzymatic hydrolysis time has the greatest effect on the DPPH scavenging rate of the enzymatic hydrolysate of Thais clavigera meat.
[0032] 2. Step 2: The alkaline protease hydrolysate obtained in the previous step was subjected to ultrafiltration (<1 kDa molecular weight) to obtain the H5 component with the highest activity.
[0033] To further separate the enzymatic hydrolysate of Thais clavigera meat, ultrafiltration was used to separate the enzymatic hydrolysate. According to the molecular weight, the enzymatic hydrolysate was divided into five components: H1 (MW > 10 kDa), H2 (10 kDa > MW > 5 kDa), H3 (5 kDa > MW > 3 kDa), H4 (3 kDa > MW > 1 kDa), and H5 (MW < 1 kDa). After collecting the five components, they were freeze-dried and prepared to a final concentration of 10 mg / mL, and then their DPPH scavenging rates were determined. As shown in Figure 2 , the DPPH scavenging rate of the H5 component was the highest, reaching 63.51 ± 0.79 %, which was significantly higher than that of the other components ( P < 0.05). Therefore, the H5 component was selected for further separation and purification experiments.
[0034] 3. Step 3: The H5 component was further purified using G-25 Sephadex gel chromatography to obtain the P1 component.
[0035] The H5 component was further separated by G-25 Sephadex gel chromatography, and the absorbance was determined and plotted. As shown in Figure 3 , two separated components were obtained, named P1 and P2.
[0036] As shown in Figure 4 , the DPPH scavenging rate of P1 was significantly stronger than that of P2 ( P < 0.05), and the DPPH scavenging rate of P1 reached 64.66 ± 0.40 %. Therefore, P1 was selected for subsequent separation and purification.
[0037] 4. Step 4: RP-HPLC was used to separate P1 to obtain P1-2 component (DPPH scavenging rate 66.04%).
[0038] RP-HPLC was used to further separate the P1 component, and the absorbance was determined at a UV wavelength of 220 nm. The results are shown in Figure 5 , a total of 4 characteristic absorption peaks were detected, named P1-1, P1-2, P1-3, and P1-4.
[0039] As shown in Figure 6 Figure 1, the DPPH scavenging rate of P1-2 was significantly higher than that of P1-1, P1-3 and P1-4 (P < 0.05), and the DPPH scavenging rate of P1-2 reached 66.04 ± 0.34%. Therefore, P1-2 was selected for peptide sequence identification. P < 0.05), and the DPPH scavenging rate of P1-2 reached 66.04 ± 0.34%. Therefore, P1-2 was selected for peptide sequence identification.
[0040] 5、Step 5: LC-MS / MS was used to identify the peptide sequence of P1-2, and 19 highly active peptides were screened out, and PFRVF and PPPR were screened out as the two optimal peptides through molecular docking.
[0041] The P1-2 component pair with the best antioxidant activity was subjected to LC-MS / MS structure identification, and the mass spectrum information obtained from the detection was compared with the corresponding protein database analysis. The total ion current (TIC) chromatogram of the peptide is shown in Figure 7 Figure 2, and the P1-2 component contains 297 peptide segments.
[0042] Peptide Ranker software was used to score the pulse red spiral meat peptides obtained by mass spectrometry to screen peptides that may have biological activity. The Peptide Ranker software score is between 0 and 1, and a score higher than 0.5 indicates that the peptide has good biological activity, and a score higher than 0.9 indicates that the peptide has excellent biological activity. As shown in Table 3, there are 19 peptide sequences with a Peptide Ranker score of more than 0.9 in the pulse red spiral meat peptides, which have high biological activity potential.
[0043] Table 3 Activity score of pulse red spiral meat peptides
[0044] Prot Param software was further used to analyze the hydrophobicity, hydrophilicity and isoelectric point of the 19 pulse red spiral meat active peptides, and Toxin Pred software was used to predict the toxicity of the 19 pulse red spiral meat active peptides. As shown in Table 4, the Toxin Pred software predicted that the 19 pulse red spiral meat active peptides were all non-toxic and could be safely used for subsequent activity research.
[0045] Table 4 Toxicity prediction of pulse red spiral meat active peptides
[0046] The binding energy of the 19 active peptides of Thais pulloides meat and MMP-1 was predicted by molecular docking, as shown in Table 5 below. Two active peptides of Thais pulloides meat with a binding energy less than -7 kcal / mol were screened, namely PFRVF and PPPR. The final two screened peptide sequences were PFRVF and PPPR. These two active peptides were selected for further activity verification to evaluate their role in anti-UV damage.
[0047] Table 5 Binding energy of active peptides of Thais pulloides meat and MMP-1
[0048] Example 2: Molecular docking screening The binding energy of the peptide and MMP-1 was analyzed by molecular docking to ensure the stability and targeting of the active peptide.
[0049] Molecular docking can effectively study the interaction between small molecule ligands and target proteins. Among the types of interaction between small molecule ligands and target proteins, the interaction force includes hydrogen bond, intermolecular interaction force and ionic force, etc. As shown in Figure 8 , 9 , in the interaction diagram of PFRVF and MMP-1, PFRVF forms five hydrogen bonds with ILE-232, ALA-234, SER-243 (three hydrogen bonds) and other amino acids on MMP-1, respectively, among which the hydrogen bond with SER-243 is the shortest, about 2.6 Å. The molecular docking results predict that the interaction energy of the stable conformation formed by PFRVF and MMP-1 is -7.39 kcal / mol, which indicates that PFRVF can form a stable structure with MMP-1 and inhibit its activity.
[0050] As shown in Figure 10 , 11 , in the interaction diagram of PPPR and MMP-1, PPPR forms seven hydrogen bonds with GLY-179, ASN-180 (three hydrogen bonds), LEU-181, HIS-218 and TYR-210 and other amino acids on MMP-1, respectively, among which the hydrogen bond with LEU-181 is the shortest, about 2.8 Å. The molecular docking results predict that the interaction energy of the stable conformation formed by PPPR and MMP-1 is -7.14 kcal / mol, which indicates that PPPR can form a stable structure with MMP-1 and inhibit its activity.
[0051] Example 3: Cell experiment 1. Cell model: HaCaT cells (human immortalized keratinocytes) were used to establish a UVB-induced skin damage model (30 mJ / cm²). HaCaT cells (2×10 4HaCaT cells were seeded in 96-well plates (2 x 10 4 cells / well) and incubated for 24 h. After washing with PBS for 3 times, 20 μL PBS was added to cover the cells, and different doses of UVB radiation (10-50 mJ / cm2) were applied. After radiation, 200 μL DMEM was added for 24 h, and 20 μL CCK-8 reagent was added for 2 h. The absorbance was detected at 450 nm to evaluate the cell viability.
[0052] 2. Experiment: (1) Experimental process: HaCaT cells were seeded in 96-well plates (2 x 10 4 cells / well) and incubated for 24 h. After washing with PBS for 3 times, 20 μL PBS was added to cover the cells, and different doses of UVB radiation (10-50 mJ / cm2) were applied. After radiation, 200 μL DMEM was added for 24 h, and 20 μL CCK-8 reagent was added for 2 h. The absorbance was detected at 450 nm to evaluate the cell viability.
[0053] The results of the effect of PFRVF and PPPR on the viability of HaCaT cells are shown in Figure 12 Table 2. The cell viability of PFRVF and PPPR at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL was higher than that of the blank group. Compared with the blank group, PFRVF at concentrations of 100 μg / mL and 200 μg / mL could significantly increase the cell viability of HaCaT cells (P < 0.01). The above results showed that the two active peptides had no toxicity to cells at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, and PFRVF and PPPR could significantly improve the cell survival rate (P < 0.01).
[0054] (2) Experimental process: HaCaT cells in the logarithmic growth phase were seeded in 6-well plates (2 x 10 6 cells / well) and incubated for 24 h. After washing with PBS for 3 times, the blank group was not irradiated, and the other groups (model group, PFRVF and PPPR different dose groups) were irradiated with UVB (30 mJ / cm2). After radiation, serum-free DMEM medium containing 10 μM DCFH-DA was added, and incubated at 37°C for 20 min in the dark. After washing with PBS for 3 times, the fluorescence microscope was used to take pictures and analyze the ROS content of each group.
[0055] UV light can interact with chromogenic groups and photosensitizers in cells to produce ROS. The results are shown in Figure 13 Figure 4. After UVB radiation, the ROS fluorescence area of the model group increased compared with the blank group, indicating that the ROS content in the model group cells increased significantly. Compared with the model group, the fluorescence area of the PFRVF and PPPR dose groups decreased with the increase of the concentration of active peptides. The results of fluorescence microscope detection showed that PFRVF and PPPR could reduce the content of ROS.
[0056] (3) Experimental process: HaCaT cells in the logarithmic growth phase were seeded in 6-well plates (2×10 6 Cells were cultured for 24 hours and then washed three times with PBS. After irradiation with 30 mJ / cm² UVB, the cells in each group were treated with UVB. The blank group was not irradiated. The model group, PFRVF, and PPPR groups (50, 100, and 200 μg / mL) were then treated with DMEM basal medium or solution and incubated for 24 hours. The cell supernatant was aspirated and the levels of SOD, GSH, CAT, and MDA were determined according to the kit instructions.
[0057] like Figure 14 As shown in the figure, after UVB radiation, the content of lipid peroxide malondialdehyde (MDA) in the model group increased significantly compared with the blank group ( P <0.01); after administration of 50 μg / mL, 100 μg / mL, and 200 μg / mL of PFRVF and PPPR, the MDA content was significantly decreased compared with the model group ( P <0.01). This indicates that PFRVF and PPPR can increase the activities of antioxidant enzymes (SOD, GSH-Px, CAT).
[0058] The above results indicate that PFRVF and PPPR can enhance the antioxidant capacity of cells after UVB radiation, reduce the production of MDA, and have a certain effect on resisting UV damage.
[0059] (4) Experimental process: HaCaT cells were seeded in 6-well plates (2×10 6 Cells were cultured for 24 hours and washed with PBS. Each group (blank, model, PFRVF, and PPPR low-, medium-, and high-dose groups) was then irradiated with 30 mJ / cm² UVB. After 24 hours of culture, cells were washed with PBS, lysed, and centrifuged at 10,000 × g for 5 minutes. The supernatant was then assayed for MMP-1 content according to the kit instructions.
[0060] MMP-1 degrades collagen and causes the formation of skin wrinkles. Figure 15 As shown, under UVB radiation, the MMP-1 content of the model group cells increased significantly compared with the blank group ( P <0.01). After administration of 50 μg / mL, 100 μg / mL, and 200 μg / mL of PFRVF and PPPR, the MMP-1 content was significantly decreased compared with the model group ( P<0.01), the degree of decrease of MMP-1 content was proportional to the concentration of PFRVF active peptide. PFRVF and PPPR could reduce the content of MMP-1 after UVB radiation, thereby reducing the degradation of collagen and alleviating the skin damage caused by ultraviolet radiation to a certain extent. Under the same concentration, the effect of PFRVF was better than that of PPPR. It was shown that PFRVF and PPPR could inhibit the expression of MMP-1 and reduce the degradation of collagen.
[0061] Example 4: Animal experiment 1. Model: The UVA / UVB-induced mouse skin ultraviolet damage model was used to observe the erythema, peeling, water content and other indicators of the mouse skin.
[0062] 2. Experiment: (1) Experimental process: 60 mice were randomly divided into blank group (ultrapure water 0.1 mL), model group (UVA+UVB+ultrapure water, 0.1 mL), positive control group (UVA+UVB+WNLNP, 10 mg / mL), PFRVF low-dose group (UVA+UVB+PFRVF, 5 mg / mL), medium-dose group (UVA+UVB+PFRVF, 10 mg / mL) and high-dose group (UVA+UVB+PFRVF, 20 mg / mL), 10 mice in each group. Except for the blank group, the remaining mice were depilated on the back and then received ultraviolet radiation (UVA 0.4 mW / cm², UVB 0.1 mW / cm²; lamp distance 40 cm), 10 min per day during weekdays in the 0-2 weeks (cumulative ultraviolet radiation dose in the 0-2 weeks: UVA 2.4 J / cm², UVB 0.6 J / cm²), 20 min per day during weekdays in the 2-4 weeks (cumulative ultraviolet radiation dose in the 2-4 weeks: UVA 4.8 J / cm², UVB 1.2 J / cm²), 30 min per day during weekdays in the 4-6 weeks (cumulative ultraviolet radiation dose in the 4-6 weeks: UVA 7.2 J / cm², UVB 1.8 J / cm²), 5 times per week. After radiation, the model group was applied with 0.1 mL of ultrapure water, the positive group was applied with 0.1 mL of WNLNP solution (10 mg / mL), and the administration group was applied with 0.1 mL of PFRVF solution of the corresponding concentration. The cumulative ultraviolet radiation dose of the mice was: UVA 14.4 J / cm², UVB 3.6 J / cm².
[0063] The results are as follows Figure 16As shown in the figure, the back skin of the blank group mice is relatively flat and smooth, the skin texture is normal, the skin is elastic and has no wrinkles, and there is no erythema or peeling phenomenon. After 6 weeks of combined radiation of UVA and UVB, the back skin of the model group mice appears severe erythema and wounds, accompanied by obvious peeling and wrinkle phenomenon. This shows that the combined radiation of UVA and UVB can induce ultraviolet radiation damage to the back skin of mice.
[0064] The back skin of the positive control group mice has partial erythema, but compared with the model group, the wrinkles of the back skin of the mice are reduced, and the skin is more flat. After the mice in the low, medium and high dose groups were given different doses of PFRVF, with the increase of the dose, the characteristics of ultraviolet radiation damage gradually weakened, and the erythema, wrinkles and dryness caused by UVA and UVB gradually weakened. No peeling phenomenon was observed on the back skin of the mice in the low dose group, but there was still partial erythema. The erythema of the back skin of the mice in the medium and high dose groups almost disappeared, and no peeling phenomenon was observed, but there were still some wrinkles. This shows that PFRVF can significantly reduce the skin damage caused by ultraviolet radiation.
[0065] (2) Experimental process: Take pictures of the back skin of mice in each group. After the mice were sacrificed, the skin tissue in the ultraviolet radiation area of the back was taken out. A part of the skin tissue was cut and soaked in 4% paraformaldehyde for HE staining, Masson staining and Sirius red staining.
[0066] Ultraviolet radiation can cause the epidermis to thicken and the sebaceous glands to enlarge. The significant increase in epidermal thickness is an adaptive response of the body to ultraviolet radiation, which is used to reduce the stimulation of the skin. The enlargement of the sebaceous glands is because ultraviolet radiation stimulates the sebaceous glands, prompting an increase in sebum secretion on the skin surface.
[0067] The results are shown in Figure 17 The epidermis of the back skin of the model group mice is obviously thickened, and pathological hyperplasia occurs. The cuticle layer of the skin appears damaged and scabbed, and some of the basal layer cells of the epidermis appear vacuole-like lesions, and some hair follicles and sebaceous glands appear swollen. Compared with the model group, the epidermis of the back skin of the mice in the low, medium and high dose groups is thinner, the cuticle layer is not obviously damaged, the morphology of the sebaceous glands is more complete, and the swelling phenomenon is alleviated. The improvement effect of the back skin of the mice in the medium dose group is similar to that of the positive control group. These phenomena show that the ultraviolet radiation damage to the back skin of the mice is improved, and the improvement of the back skin of the mice in the high dose group is the most obvious. It can be seen that the active peptide PFRVF from Thais pullorella can improve the ultraviolet radiation-induced skin morphological changes.
[0068] As shown in the figure, Figure 18As shown, the collagen fibers in the dermis of the back skin of mice in the blank group appeared uniformly dark blue and arranged in a wavy pattern, which was relatively regular and normal. The collagen fibers in the dermis of the back skin of mice in the model group stained light blue, indicating a decrease in collagen density. Some collagen fibers were also broken and missing, indicating that some collagen fibers were degraded, which further triggers skin aging. After PFRVF was administered to the back skin of mice in the low, medium, and high-dose groups, the irregular arrangement and missing collagen fibers improved, with the most significant improvement in the back skin of mice in the high-dose group. This suggests that PFRVF can effectively inhibit the degradation of collagen fibers and has a protective effect on skin exposed to ultraviolet light.
[0069] In the picrosirius red staining of the mouse back skin, type I collagen appears bright red or orange, and type III collagen appears green. Figure 19 As shown, mice in the blank group had more bright red and orange-yellow areas and were rich in type I collagen. In the model group, the bright red and orange areas in the dorsal skin decreased, and the type I collagen content was reduced. In the low, medium, and high-dose groups, treatment with different concentrations of PFRVF reduced the bright red and orange areas and significantly increased the type I collagen content. This suggests that PFRVF can inhibit the degradation of type I collagen in the skin caused by long-term ultraviolet radiation.
[0070] The above results show that PFRVF can increase skin moisture content, reduce wrinkles and collagen degradation (HE staining, Masson staining, and picrosirius red staining).
[0071] (3) Experimental procedure: 300 mg of skin tissue was accurately weighed and minced with surgical scissors. 2.7 mL of normal saline was added. The tissue was homogenized using a tissue homogenizer and centrifuged at 3000 rpm for 20 min. The supernatant was collected and the TNF-α, IL-6, and IL-1β levels were determined according to the ELISA kit instructions.
[0072] The results are as follows Figure 20 As shown in the results, the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the back skin of mice in the model group were 123.7 ± 8.3 pg / mL, 69.2 ± 4.9 pg / mL, and 185.2 ± 8.9 pg / mL, respectively, which were significantly increased compared with the blank group ( P <0.01). These data indicate that long-term ultraviolet radiation stimulates the production of inflammatory cytokines and triggers an inflammatory response. However, with the administration of PFRVF, the levels of inflammatory cytokines IL-1β, IL-6, and TNF-α decreased, and significant improvement was observed in the high-dose group. The expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the back skin of mice in the high-dose group was significantly lower than that in the model group (P <0.01), the contents of which were reduced to 88.5 ± 4.2 pg / mL, 53.7 ± 2.8 pg / mL and 125.8 ± 3.7 pg / mL, respectively. It was shown that PFRVF was able to reduce inflammatory factors (IL-1β, IL-6, TNF-α).
Claims
1. An active peptide from the flesh of a conch or its derivatives, characterized in that: The active peptide of the red snail meat is an amino acid sequence as shown in SEQ ID NO. 1-19 or a derivative thereof.
2. The active peptide from the flesh of the red snail or its derivative according to claim 1, characterized in that: The active peptide from the flesh of red snail is an amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.16 or a derivative thereof.
3. The method for preparing the active peptide from Psoralea corylifolia meat or its derivatives according to claim 1, wherein: The following steps are involved: (1) Weigh the flesh of the red snail, dissolve it in water, add alkaline protease to perform enzymatic hydrolysis, and obtain an enzymatic hydrolysis product; (2) Perform ultrafiltration to select the component with the highest activity; (3) further purifying the most active fraction obtained in step (2) to separate a fraction with a high DPPH clearance rate; (4) using reversed-phase high performance liquid chromatography to separate the fraction with the highest DPPH scavenging rate obtained in step (3) above, and selecting the fraction with the highest DPPH scavenging rate; (5) Sequence identification is performed on the component with high DPPH scavenging rate obtained in the above step (4) to obtain the sequence of the active peptide.
4. The preparation method according to claim 3, wherein The conditions for the enzymatic hydrolysis reaction are: the mass volume ratio of the vein red snail meat to water is 8-12:1 mL / g, the enzymatic hydrolysis reaction time is 2-4 h, and the amount of alkaline protease added is 2-4%.
5. The preparation method according to claim 3, wherein The purification method in step (3) is dextran gel chromatography; The separation method in step (4) is reverse phase high performance liquid chromatography.
6. A nucleic acid, characterized in that It encodes the active peptide of the red snail meat or its derivative as claimed in claim 1 or 2.
7. A carrier, characterized in that Comprising the nucleic acid according to claim 6.
8. A host cell, characterized in that The vector according to claim 7 was transfected.
9. Use of the active peptide from the flesh of the red snail or its derivatives as claimed in claim 1 or 2 in the preparation of drugs or cosmetics for resisting aging caused by ultraviolet light, resisting skin inflammation caused by ultraviolet light or resisting oxidation.
10. A pharmaceutical composition or cosmetic, characterized in that: The pharmaceutical composition comprises the active peptide from Rhizoma Cynoglossum or its derivatives as claimed in claim 1 or 2.