A tenebrio molitor-derived polypeptide for resisting ultraviolet-induced skin photoaging and application thereof

Abstract

The application provides a tenebrio molitor-derived anti-ultraviolet-induced skin photoaging polypeptide and application thereof, and relates to the technical fields of bioactive peptides and biomedical engineering. The amino acid sequence of the anti-ultraviolet-induced skin photoaging polypeptide is Trp-Ile-Gly-Gly-Glu. The polypeptide can effectively improve the survival rate of HaCaT cells after UVB damage in a prevention and treatment mode, and has good cell safety at a concentration level of 25-100 mu g / mL, thereby providing a new natural anti-skin photoaging active substance for the fields of skin care products, medical preparations and other related fields.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides and biomedical engineering technology, specifically to a yellow mealworm-derived anti-ultraviolet-induced skin photoaging peptide and its application. Background Technology

[0002] Photoaging of the skin is mainly caused by chronic skin damage resulting from long-term ultraviolet (UV) radiation. UVB irradiation can induce oxidative stress and inflammatory responses in skin cells and promote the degradation of extracellular matrix components such as collagen, leading to dryness, roughness, decreased elasticity, and increased wrinkles. Therefore, developing active ingredients that can reduce UV damage, maintain skin cell vitality, and delay the degradation of the extracellular matrix has significant application value.

[0003] Currently, active ingredients for combating photoaging mainly include vitamins, plant extracts, and antioxidants. However, some of these ingredients suffer from insufficient stability, low bioavailability, or limited functionality. Naturally derived bioactive peptides, on the other hand, are characterized by their small molecular weight, relatively well-defined structure, good biocompatibility, and ease of absorption and utilization. Some bioactive peptides also exhibit multiple functions, including antioxidant, anti-inflammatory, and cell-protective effects, making them promising candidates for development in anti-photoaging cosmetics and skincare products. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a mealworm-derived anti-UV-induced skin photoaging peptide and its application. The screened anti-UV-induced skin photoaging peptide exhibits good cellular safety and anti-skin photoaging effects, providing new natural anti-skin photoaging raw materials for cosmetics, pharmaceuticals and other related fields.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A mealworm-derived anti-UV-induced skin photoaging polypeptide, wherein the amino acid sequence of the anti-UV-induced skin photoaging polypeptide is: Trp-Ile-Gly-Gly-Glu.

[0006] The method for obtaining mealworm-derived anti-UV-induced skin photoaging peptides includes the following steps: S1. Yellow mealworm protein was obtained by alkaline extraction and acid precipitation using defatted yellow mealworm powder as raw material. S2. The mealworm protein was enzymatically hydrolyzed with alkaline protease for 3 hours and then the enzyme was inactivated to obtain the mealworm protein hydrolysate for later use. S3. The mealworm protein hydrolysate was fractionated using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to obtain components with a molecular weight of <1 kDa. S4. Peptide identification and analysis of <1 kDa molecular weight components of yellow mealworm protein hydrolysate were performed using LC-MS / MS peptidomics method. S5. The identified peptides were input into Toxinpred 3.0, plm4Alg, Innovagen, Peptide Ranker, and BIOPEP_UWM for toxicity prediction, sensitization prediction, water solubility prediction, bioactivity scoring, and comparison with activity databases. Finally, the peptides were molecularly docked with Keap1 protein to screen candidate peptides. The candidate peptides were then synthesized in a solid phase chemical process and subjected to a UVB-induced damage prevention experiment on HaCaT cells. The anti-UV-induced skin photoaging peptide was identified as Trp-Ile-Gly-Gly-Glu.

[0007] Preferably, the specific screening method for the candidate peptides in step S5 is as follows: peptides with a Peptide Ranker score greater than 0.5 are classified as potential bioactive peptides, and combined with the non-toxic, non-allergenic, and soluble peptides predicted by the online website, the final score ranking is performed using the molecular docking binding energy with the Keap1 protein, and the top-ranked peptides are selected as candidate peptides for solid-phase chemical synthesis.

[0008] Application of the above-mentioned mealworm-derived anti-UV-induced skin photoaging peptides in the preparation of drugs for the prevention and / or treatment of UVB irradiation-induced skin damage An anti-photoaging peptide composition, said composition containing at least one of a polypeptide with the amino acid sequence Trp-Ile-Gly-Gly-Glu, or a pharmaceutically acceptable salt, derivative, modification, isotope label, and conjugate thereof with a carrier.

[0009] Preferably, the composition is used in the preparation of peptide powders, skin care agents, or pharmaceutical preparations that protect against UV-induced skin damage and aging.

[0010] This invention provides a mealworm-derived anti-UV-induced skin photoaging polypeptide and its application, which has the following advantages compared with the prior art: This invention screens an anti-photoaging peptide, Trp-Ile-Gly-Gly-Glu, using a screening method combining enzymatic hydrolysis-peptidomics technology and experiments. This anti-photoaging peptide can effectively improve the survival rate of HaCaT cells after UVB damage in both preventive and therapeutic modes, and exhibits good cellular safety at concentration levels of 25-100 μg / mL. It provides a new natural anti-photoaging active substance for skin care products, pharmaceutical preparations, and other related fields. Attached Figure Description

[0011] Figure 1This illustrates the effect of polypeptides generated after enzymatic hydrolysis by different proteases in Example 1 of the present invention on the free radical scavenging activities of ABTS and DPPH. Figure 2 This illustrates the effect of different molecular weight peptides of alkaline protease hydrolysate on the free radical scavenging activities of ABTS and DPPH in Example 1 of this invention. Figure 3 The images show the ESI-MS (left) and RP-HPLC (right) spectra of polypeptide TIP1 in Example 1 of this invention. Figure 4 The images show the ESI-MS (left) and RP-HPLC (right) spectra of polypeptide TIP2 in Example 1 of this invention. Figure 5 The images show the ESI-MS (left) and RP-HPLC (right) spectra of polypeptide TIP3 in Example 1 of this invention. Figure 6 The images show the ESI-MS (left) and RP-HPLC (right) spectra of polypeptide TIP4 in Example 1 of this invention. Figure 7 The images show the ESI-MS (left) and RP-HPLC (right) spectra of polypeptide TIP5 in Example 1 of this invention. Figure 8 This is a schematic diagram illustrating the effect of alkaline protease hydrolysate <1 kDa active component and TIP1-5 on HaCaT cell toxicity in Example 1 of the present invention; Figure 9 This is a schematic diagram illustrating the use of alkaline protease hydrolysate <1 kDa active component and TIP1-5 in Example 1 of the present invention for the prevention and treatment of HaCaT cell damage caused by UVB irradiation. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Example 1: I. Obtaining anti-photoaging peptides: 1. Preparation of mealworm protein Defatted mealworm powder was selected as the raw material. The defatted mealworm powder was added to deionized water at a material-to-liquid ratio of 1:20 (g / mL), stirred until homogeneous, and the pH of the solution was adjusted to 12.0. Extraction was carried out at room temperature for 4 h. After extraction, the mixture was centrifuged, the supernatant was collected, and the pH of the supernatant was adjusted to 4.5 to precipitate the mealworm protein. The protein precipitate was collected by centrifugation, washed twice with deionized water, and freeze-dried to obtain mealworm protein.

[0014] 2. Mealworm proteolysis Alkaline protease, trypsin, papain, and bromelain were selected for enzymatic hydrolysis of mealworm protein. The mealworm protein was prepared into a 5% (w / v) protein suspension, and the temperature and pH were adjusted according to the optimal conditions for each protease. The corresponding protease was added at a dosage of 5000 U / g protein, and the hydrolysis time was 3 h. After hydrolysis, the sample was immediately placed in a 100℃ water bath for 10 min to inactivate the protease, obtaining the mealworm protein hydrolysate. Optimal hydrolysis conditions were screened using ABTS and DPPH free radical scavenging rates.

[0015] The ABTS free radical scavenging rate was calculated as follows: An ABTS stock solution was prepared by mixing equal volumes of 7 mM ABTS and 2.45 mM K₂S₂O₈ and allowing it to stand in the dark for 16 h. The solution was then diluted with deionized water to a working absorbance of 0.70 ± 0.02 at 734 nm. 1 μL of enzyme digest and 199 μL of the ABTS working solution were added to each well of a 96-well plate and incubated at room temperature in the dark for 10 min. The absorbance at 734 nm was measured, with deionized water used as a control instead of the sample.

[0016] (1) In equation (1), OD sample The absorbance and OD values ​​are for 1 μL sample + 199 μL ABTS working solution. control The absorbance of 1 μL deionized water + 199 μL ABTS working solution is given by OD. blank The absorbance is the value of 1 μL sample + 199 μL deionized water.

[0017] The DPPH radical scavenging rate was calculated as follows: 20 μL of sample solution and 180 μL of DPPH solution (0.8 mM) were placed in a 96-well plate. The sample was shaken well and allowed to stand in the dark at room temperature for 30 min. The absorbance at 517 nm was then measured. The formula for calculating the DPPH radical scavenging rate is as follows: (2) In equation (2), OD sample The absorbance and OD values ​​are calculated for a 20 μL sample + 180 μL DPPH solution. controlThe absorbance (OD) of 20 μL deionized water + 180 μL DPPH working solution is given. blank The absorbance is for a 20 μL sample + 180 μL deionized water mixture.

[0018] Specific results are as follows Figure 1 As shown, the ABTS radical scavenging activity of the alkaline protease hydrolysate was significantly higher than that of the other three hydrolysates (p < 0.05), reaching 96.49%. The DPPH radical scavenging activity of the alkaline protease was significantly higher than that of bromelain (p < 0.05) and trypsin, while papain showed no significant difference. Considering both ABTS and DPPH radical scavenging activities, alkaline protease hydrolysis for 3 h was determined for further studies.

[0019] 3. Ultrafiltration Fractionation Ultrafiltration membranes with molecular weight cutoffs of 1 kDa, 3 kDa, and 5 kDa were used to fractionate mealworm protein hydrolysates. The hydrolysates were passed through the corresponding molecular weight cutoff membranes and then freeze-dried to obtain ultrafiltration fractions of different molecular weights.

[0020] The inhibition rates of ABTS and DPPH of components with different molecular weights were detected, and the specific results are as follows: Figure 2 As shown: IC50 values ​​for ABTS and DPPH inhibition rates of components below 1 kDa. 50 The values ​​of all components were significantly lower than those of the other three components (p < 0.05), indicating that the component sample with a value below 1 kDa has good in vitro free radical scavenging activity.

[0021] 4. Peptidomics identification of fractions with a molecular weight <1 kDa Peptide identification and analysis of <1 kDa molecular weight components in yellow mealworm protein hydrolysate was performed using LC-MS / MS peptidomics. Peptide identification and analysis of <1 kDa molecular weight components in yellow mealworm protein hydrolysate were performed using LC-MS / MS peptidomics.

[0022] To reduce disulfide bonds in the sample, dithiothreitol (DTT) was added to the sample to a final concentration of 10 mmol / L, and the reaction was carried out at 56 °C in a water bath for 1 h. Iodoacetamide (IAM) was then added to a final concentration of 20 mmol / L, and the reaction was carried out at room temperature in the dark for 40 min to alkylate free thiol groups. After the reaction, DTT was added to a final concentration of 10 mmol / L to quench any remaining IAM in the system. Trifluoroacetic acid (TFA) was then added to acidify the sample to a final concentration of 0.1% (v / v). The acidified sample was desalted using a C18 StageTip, and the resulting eluent was concentrated to dryness under vacuum at a temperature not exceeding 45 °C for subsequent analysis.

[0023] Peptides were separated using an Easy-nLC 1200 nano-scale liquid chromatography system. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and 0.1% formic acid-80% acetonitrile (B). After gradient elution, the samples were injected into a Q Exactive Orbitrap mass spectrometer. Data were acquired in DDA mode (MS1 resolution 70,000, MS2 resolution 17,500, TopN=20, NCE=28). The raw mass spectrometry data were identified using PEAKSStudio software based on the UniProt database (mealworm). The fixed modification was cysteine ​​alkylation, and the variable modifications included methionine oxidation and N-terminal acetylation. The mass errors were set at 20 ppm (MS1) and 0.02 Da (MS2) to achieve high-confidence peptide identification analysis.

[0024] 5. Screening of anti-photoaging peptides 2884 peptides with molecular weights <1 kDa were identified and input into Toxinpred 3.0, plm4Alg, Innovagen, Peptide Ranker, and BIOPEP_UWM for toxicity prediction, sensitization prediction, water solubility prediction, bioactivity scoring, and comparison with bioactivity databases. Finally, the peptides were molecularly docked with the Keap1 protein. Peptides with a Peptide Ranker score greater than 0.5 were classified as potentially bioactive peptides. Combined with online predictions of non-toxicity, non-sensitization, and soluble peptides, the final score and ranking were based on the molecular docking binding energy with the Keap1 protein. Five candidate peptides (see Table 1 below) were selected for solid-phase chemical synthesis.

[0025] Table 1. Anti-photoaging peptide sequences 6. Preparation and characterization of the above-mentioned polypeptides Peptide synthesis was performed using the Fmoc solid-phase peptide synthesis (SPPS) method. Taking TiP5:WIGGE (Trp-Ile-Gly-Gly-Glu) as an example, all other peptides were synthesized using the same principle. Using 2-chlorotriphenylmethyl chloride resin with a substitution degree of approximately 1.1 mmol / g as the starting resin, after swelling with DMF, Fmoc-Glu(OtBu)-OH was first linked, and unreacted sites were blocked with methanol. Subsequently, the Fmoc protecting group was removed using a 20% piperidine / DMF solution. Using HBTU as the coupling agent and DIEA as the base, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Fmoc-Trp(Boc)-OH were sequentially coupled from the C-terminus to the N-terminus. After each round of coupling, the degree of reaction completion was monitored using Kaiser's reagent, and the deprotection, washing, and condensation steps were repeated until the target peptide chain was assembled.

[0026] After synthesis, the resin was cleaved and side-chain protecting groups were removed using a TFA / water / EDT / TIS (95:2:2:1, v / v / v / v) reaction. After reacting for 180 min, the crude peptide was precipitated with cold anhydrous diethyl ether. The crude product was purified by C18 reversed-phase HPLC using a water-acetonitrile system containing 0.1% TFA as the mobile phase. The target main peak was collected and lyophilized to obtain the purified peptide. Finally, HPLC and mass spectrometry verified that its purity reached over 95%, and the measured molecular weight was consistent with the theoretical value (e.g., ...). Figures 3-7 (As shown).

[0027] 7. Cytotoxicity test The cell safety of TiP1-5 cells was evaluated using the human spontaneously immortalized keratinocyte cell line HaCaT. Logarithmic growth phase cells were harvested, digested with trypsin, and observed and counted under a microscope. Cells were then prepared into 3×10⁻⁶ cells. 5 A cell suspension of 3 × 10⁶ cells / mL was prepared. 100 μL of the cell suspension was seeded into 96-well plates, with approximately 3 × 10⁶ cells per well. 4Cells were cultured at 37℃ and 5% CO2 for 24 h to allow for full cell adhesion. After discarding the original culture medium, 100 μL of culture medium containing different concentrations of the sample (<1 kDa active component 0.1-2 mg / mL, candidate peptides TiP1-TiP5 25-100 μg / mL) was added to each well, while the control group received an equal volume of culture medium. After culturing for another 24 h, the culture medium was discarded, and 100 μL of serum-free culture medium containing 10% CCK-8 was added to each well. The cells were incubated at 37℃ for 2 h, and the absorbance (OD) value was measured at 450 nm using a microplate reader. Cell viability was calculated from the OD value to evaluate the effect of different concentrations of TiP1-5 on HaCaT cell viability. The calculation method is as follows: (3) In equation (3), OD sample The absorbance of the sample group, OD control The absorbance value of the untreated control group is shown in Figure OD. blank The absorbance value is for the blank group, which contains no cells.

[0028] like Figure 8 As shown, the <1 kDa molecular weight fraction exhibited near 100% cell viability within the concentration range of 0.1–2.0 mg / mL without significant cytotoxicity. Within the concentration range of 25–100 μg / mL, the HaCaT cell viability in the TiP1-TiP5 treatment group was not significantly lower than that in the blank control group, indicating that TiP1-TiP5 did not exhibit significant cytotoxicity within the tested concentration range. This demonstrates that the <1 kDa active fraction of the mealworm protein hydrolysate and its key peptide TiP1-TiP5 both possess good cellular safety.

[0029] 8. The preventive and therapeutic effects of <1 kDa active components and their key peptides TiP1-TiP5 on UVB-damaged HaCaT cells. HaCaT cells were divided into 3×10 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. In preventative mode, the culture medium was then discarded, and different concentrations of sample were added for further 24 h of culture. After culture, the culture medium was discarded, 30 μL of PBS was added to each well, and the cells were then irradiated with a UVB lamp to 200 mJ / cm². 2 After irradiation, the PBS in the wells was removed, and complete culture medium was added for 24 h of further culture. Cell viability was then measured. In the treatment mode, the culture medium was discarded 24 h after inoculation, followed by the addition of 30 μL of PBS and irradiation with a UVB lamp to 200 mJ / cm². 2 After removing PBS, samples of different concentrations were added and cultured for 24 h, followed by measurement of cell viability.

[0030] like Figure 9 As shown, the <1 kDa active component at doses of 0.1–1.0 mg / mL significantly improved HaCaT cell survival rates in both preventative and therapeutic modes compared to the UVB irradiation group (p < 0.05), indicating that the <1 kDa active component possesses anti-photoaging potential in both preventative and therapeutic modes. In the therapeutic mode, among TiP1–TiP5, TiP1, TiP3, and TiP4 significantly improved cell survival rates compared to the UVB irradiation group at doses ranging from 25–100 μg / mL (p < 0.05), while TiP2 significantly improved cell survival rates compared to the model group at 100 μg / mL (p < 0.05). In the preventative mode, TiP1 significantly improved HaCaT cell survival rates after UVB damage in the range of 25–100 μg / mL, while TiP2 only showed a significant protective effect at 100 μg / mL. These results indicate that TiP1 has cytoprotective effects in both pre-irradiation and post-irradiation treatment modes.

[0031] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mealworm-derived anti-UV-induced skin photoaging polypeptide, characterized in that: The amino acid sequence of the anti-UV-induced skin photoaging peptide is: Trp-Ile-Gly-Gly-Glu.

2. A method for obtaining the anti-UV-induced skin photoaging polypeptide derived from yellow mealworms as described in claim 1, characterized in that, The acquisition method includes the following steps: S1. Yellow mealworm protein was obtained by alkaline extraction and acid precipitation using defatted yellow mealworm powder as raw material. S2. The mealworm protein was enzymatically hydrolyzed with alkaline protease for 3 hours and then the enzyme was inactivated to obtain the mealworm protein hydrolysate for later use. S3. The mealworm protein hydrolysate was fractionated using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to obtain components with a molecular weight of <1 kDa. S4. Peptide identification and analysis of <1 kDa molecular weight components of yellow mealworm protein hydrolysate were performed using LC-MS / MS peptidomics method. S5. The identified peptides were input into Toxinpred 3.0, plm4Alg, Innovagen, Peptide Ranker, and BIOPEP_UWM for toxicity prediction, sensitization prediction, water solubility prediction, bioactivity scoring, and comparison with activity databases. Finally, the peptides were molecularly docked with Keap1 protein to screen candidate peptides. The candidate peptides were then synthesized in a solid phase chemical process and subjected to a UVB-induced damage prevention experiment on HaCaT cells. The anti-UV-induced skin photoaging peptide was identified as Trp-Ile-Gly-Gly-Glu.

3. The acquisition method according to claim 2, characterized in that: The specific screening method for candidate peptides in step S5 is as follows: peptides with a Peptide Ranker score greater than 0.5 are classified as potential bioactive peptides. Combined with the non-toxic, non-allergenic, and soluble peptides predicted by the online website, the final score is ranked by the molecular docking binding energy with Keap1 protein. The top-ranked peptides are selected as candidate peptides for solid-phase chemical synthesis.

4. The use of the mealworm-derived anti-UV-induced skin photoaging polypeptide as described in claim 1 in the preparation of drugs for the prevention and / or treatment of UVB irradiation-induced skin damage.

5. A peptide composition for combating skin photoaging, characterized in that: The composition contains at least one of a polypeptide with the amino acid sequence Trp-Ile-Gly-Gly-Glu, or a pharmaceutically acceptable salt, derivative, modification, isotope label, and conjugate thereof with a carrier.

6. The composition according to claim 4, characterized in that: The composition is used to prepare peptide powders, skin care products, or pharmaceutical preparations that protect against UV-induced skin damage and aging.

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