Porphyra protein source antioxidant peptide tolerant to pepsin and trypsin as well as preparation method and application of porphyra protein source antioxidant peptide

Abstract

The invention belongs to the technical field of antioxidant peptides, and particularly relates to a laver protein source antioxidant peptide tolerant to pepsin and trypsin as well as a preparation method and application of the laver protein source antioxidant peptide. According to the invention, edible seaweed is taken as a raw material, and a Keap1-targeted antioxidant peptide is screened from seaweed protein by integrating virtual enzyme digestion, molecular docking and other methods. A cell test verifies that the tripeptide can target Keap1 to activate an antioxidant system, enhance the activity of intracellular antioxidant enzyme and remove cell reactive oxygen species (ROS), and can tolerate in-vitro degradation of pepsin and trypsin, and the absorption rate in an intestinal tract model established by human colorectal adenocarcinoma cells (Caoco-2) is 1.64%. The polypeptide has the potential of being applied to oral anti-oxidation health care products and external anti-oxidation cosmetic components, and has high medicinal economic value.

Description

Technical Field

[0001] This invention belongs to the field of antioxidant peptide technology, specifically relating to a seaweed protein-derived antioxidant peptide that is resistant to pepsin and trypsin, its preparation method, and its application. Background Technology

[0002] Oxidative stress, caused by an abnormal increase in oxidative factors due to an imbalance in redox activity within the body, is a significant factor contributing to tissue damage and disease. It is closely associated with various diseases, such as neurological disorders, chronic kidney disease, obstructive pulmonary disease, atherosclerosis, and cancer. Oxidative stress is caused by excessive reactive oxygen species (ROS), including superoxide anions (O2-), hydroxyl radicals (OH-), and hydrogen peroxide (H2O2). Therefore, scavenging ROS is a crucial pathway for maintaining homeostasis.

[0003] In response to oxidative stress, the sensor cysteine ​​residue in Kelch-like ECH-associated protein-1 (Keap1) promotes a complex molecular mechanism that allows nuclear factor E2-associated factor-2 (Nrf2) to evade ubiquitination, accumulate intracellularly, and translocate to the nucleus. This facilitates its antioxidant transcriptional program, leading to the expression of antioxidant enzymes to scavenge excess ROS. Numerous studies have found that targeting the Keap1-Nrf2-antioxidant redox element (ARE) signaling pathway can activate Nrf2, thereby combating various stress and inflammation-related diseases. Therefore, the preparation or screening of small molecules that can bind to Keap1 is considered one of the strategies for screening antioxidants. Structure-based drug design has led to the development of various Keap1 inhibitors, but most of these compounds are synthetic small molecules, which may pose safety risks. Research on Keap1 inhibitors derived from natural products is relatively scarce, especially research on antioxidant peptides targeting Keap1, which is still in its early stages.

[0004] Bioactive peptides are small cationic polypeptides induced by exogenous substances, containing sequences of 2 to 50 amino acids. Many studies have found that bioactive peptides from natural products possess various physiological functions, such as antioxidant, antihypertensive, antitumor, antibacterial, and immunomodulatory properties. Antioxidant peptides typically contain specific amino acid compositions and sequence characteristics; for example, peptides rich in hydrophobic amino acids (Val, Leu, Ile, Ala, Phe, Pro), aromatic amino acids (Tyr, Trp, Phe), and basic amino acids (Lys, Arg, His) often exhibit strong antioxidant activity. In Trp-containing peptides, the spatial conformation of the indole ring enhances charge mobility and improves antioxidant capacity. However, current research mainly focuses on the direct antioxidant mechanisms of peptides (such as scavenging free radicals and chelating metal ions), with insufficient research on their effects through the regulation of antioxidant signaling pathways, particularly lacking studies on systematically screening antioxidant peptides for specific targets.

[0005] Antioxidant peptides are widely available, with seaweed being a proven edible protein resource for producing bioactive peptides. Numerous studies have shown that seaweed contains antioxidant peptides, such as *Porphyra yezoensis*, *Porphyra tenera*, *Porphyra palmatum*, and *Ulva southernis*. *Porphyra yezoensis* is an edible algae with significant nutritional and economic value. It contains various nutrients, such as protein, carbohydrates, and micronutrients, and possesses good antioxidant, anticancer, antihypertensive, and immunomodulatory effects. However, research on the preparation and mechanism of action of *Porphyra yezoensis* antioxidant peptides is still limited.

[0006] The preparation of natural bioactive peptides mainly includes direct extraction, genetic engineering, chemical synthesis, and enzymatic hydrolysis. Peptides released from natural proteins are then separated and purified using gel permeation chromatography, liquid chromatography, ion exchange chromatography, and HPLC-MS. However, the published methods for preparing bioactive peptides generally suffer from drawbacks such as long processing times and high costs. In contrast, computer-aided drug design has become a powerful tool for screening bioactive peptides. Virtual enzyme digestion and ADMET prediction, molecular docking, and molecular dynamics simulations have significant advantages and have been used for in vitro screening and identification of potential bioactive peptides. This method is efficient, inexpensive, and allows for the online release of peptides from natural proteins. Furthermore, molecular docking can explore the binding interaction between ligands and Keap1 and has been widely used in the screening of Nrf2 activators. Additionally, dynamic simulations can explore the real-time dynamic interaction between ligands and reactors.

[0007] Bioactive peptides are composed of several amino acids and are easily digested and absorbed. However, they also have the disadvantage of being easily degraded by gastrointestinal enzymes, thus losing their activity. Therefore, most peptides with antioxidant activity can only enter the body through topical application or injection, making it difficult to add them to oral health supplements, which limits their medicinal value.

[0008] Antioxidant peptides targeting the Keap1-Nrf2 signaling pathway have broad application prospects in pharmaceuticals, health products, and cosmetics. However, systematic research on Keap1-targeting antioxidant peptides derived from seaweed protein is still insufficient, and further exploration and development are urgently needed. Summary of the Invention

[0009] Based on the description in the background section above, the existing technology mainly suffers from the following drawbacks: 1. The molecular mechanisms of most antioxidant peptides are unclear, and their target genes are unknown; 2. Some synthetic antioxidant drug precursors pose potential toxicity and safety risks; 3. As a common economic algae, laver is mostly used as food and animal feed, and its medicinal value needs to be explored. 4. Most bioactive peptides have poor tolerance to gastrointestinal enzymes and cannot be administered orally. They can only be administered topically or by injection, which limits the application of antioxidant peptides.

[0010] 5. The absorption rate and absorption and transport mechanism of bioactive peptides in the intestine are unclear, which limits their development and application as oral drugs and health products.

[0011] To address the aforementioned problems in the prior art, this invention provides an antioxidant peptide derived from laver protein that is resistant to pepsin and trypsin, along with its preparation method and applications. This invention integrates methods such as virtual enzymatic hydrolysis of gastrointestinal enzymes, molecular docking, and molecular dynamics simulation to screen for antioxidant peptides targeting Keap1 from laver protein and conduct in vitro enzymatic hydrolysis experiments. This provides a new strategy for developing safe, orally administered, and highly effective antioxidant health products, and also offers new pathways and methods for the high-value utilization of laver resources.

[0012] This invention includes the following technical solutions: An antioxidant peptide derived from seaweed protein, with the amino acid sequence Trp-Trp-Arg (abbreviated as WWR).

[0013] Furthermore, the aforementioned seaweed protein-derived antioxidant peptide is resistant to degradation by pepsin and trypsin, and maintains structural integrity in an in vitro simulated gastrointestinal digestive environment.

[0014] Furthermore, the aforementioned seaweed protein-derived antioxidant peptide can specifically bind to the Keap1 protein, competitively inhibit the Keap1-Nrf2 interaction, thereby activating the Nrf2-ARE signaling pathway.

[0015] The present invention also discloses an isolated nucleic acid molecule encoding the above-mentioned seaweed protein-derived antioxidant peptide.

[0016] Furthermore, this invention discloses a method for preparing the above-mentioned seaweed protein-derived antioxidant peptides, comprising the following steps: 1) Using a solid-phase synthesis method, tryptophan Trp, tryptophan Trp and arginine Arg residues with appropriate protecting groups are sequentially coupled; 2) Purification was performed using reversed-phase high-performance liquid chromatography on a C18 column. Mobile phase A was acetonitrile containing 0.1% trifluoroacetic acid, and mobile phase B was water containing 0.1% trifluoroacetic acid. A gradient elution program was used. 3) The molecular weight and sequence correctness of the synthesized peptide were confirmed by electrospray ionization mass spectrometry (ESI-MS); 4) Freeze-dry the purified peptide solution to obtain a white or pale yellow powder or crystalline solid product.

[0017] To verify the antioxidant activity of the seaweed protein-derived antioxidant peptides screened in this invention, in vitro cell experiments were also conducted, including the following steps: The aforementioned seaweed protein-derived antioxidant peptides were dissolved in DMEM medium and applied to oxalate-pretreated HK-2 cells at a final concentration of 100 μM. The effect of the peptides on the ROS content of these cells was detected to assess their protective effect against oxalate-induced oxidative stress. Furthermore, the effects of the seaweed protein-derived antioxidant peptides on the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected in vitro using a kit. The peptides' tolerance to pepsin and trypsin was also investigated using an in vitro enzymatic digestion assay combined with mass spectrometry.

[0018] This invention also discloses a computer-aided method for screening the above-mentioned seaweed protein-derived antioxidant peptides, comprising the following steps: Step 1: Download the seaweed protein sequence from the NCBI database, upload it to Peptide Cutter (https: / / web.expasy.org / peptide_cutter / ), select pepsin and trypsin to perform virtual enzymatic digestion of seaweed protein, and screen for peptides with a length in the range of 2 to 6 amino acids; Step 2: Import the peptide sequence into the peptide bioactivity prediction website (ADMET: Interpretation-ADMElab: ADMET Prediction|ADMET Predictor|QSAR|ADMET Database) for screening bioactive peptides. Based on the bioinformatics prediction score, peptides with high bioactivity, good water solubility, and no toxicity are screened out. Step 3: Perform Lib docking between the peptides screened in Step 2 and Keap1 protein to screen for peptides with high binding scores. Step 4: Perform C-Dock and molecular dynamics simulations on the peptides screened in Step 3.

[0019] Furthermore, step 1 may include: (1) Obtain the seaweed protein sequence from the NCBI database; (2) The protein sequence was virtually digested using a Peptide Cutter, and gastrointestinal digestion was simulated using pepsin and trypsin to explore potential protein cleavage sites. (3) Limit the number of amino acids in the peptides obtained by cutting to the range of 2 to 6 to obtain a preliminary peptide library.

[0020] The reason for limiting peptide length to 2–6 amino acids is that peptides within this length range are generally more stable in vivo and easily digested and absorbed through the gastrointestinal tract. Furthermore, larger molecular weight peptides contain more amino acids and are more likely to contain affinity groups, enhancing the peptide's affinity for the target protein. However, excessively large molecular weights can restrict their entry into the active site cavity of the target protein, resulting in decreased binding affinity. The 2–6 amino acid range often makes it easier to screen for high-affinity small peptides.

[0021] Step 2 may sequentially include the following steps: (1) Use the Peptide Ranker online tool (http: / / distilldeep.ucd.ie / PeptideRanker / ) to score the bioactivity of the preliminary peptides obtained in step 1, and retain peptides with a bioactivity score > 0.5; (2) Use the Innovagen online tool (http: / / www.innovagen.com / proteomics-tools) to predict the water solubility of the above peptides and retain the peptides that show "Good water solubility"; (3) The above peptides were subjected to ADMET property detection using the admetSAR online tool, and peptides that simultaneously displayed "BBB+, HIA+, Caco²⁻" were retained; and (4) Use the ToxinPred online tool (http: / / crdd.osdd.net / raghava / toxinpred / ) to predict the toxicity of the above peptides and retain the peptides that show "Non-toxin".

[0022] The Peptide Ranker online tool, based on machine learning algorithms, can predict the bioactivity of peptides based on their amino acid composition and sequence characteristics. The scoring ranges from 0 to 1, with higher scores indicating stronger potential bioactivity.

[0023] The Innovagen online tool can predict the solubility of peptides in an aqueous environment based on characteristics such as amino acid composition, charge distribution, and hydrophobicity.

[0024] Specifically, when performing ADMET property detection, since the admetSAR database cannot directly input peptide sequences for querying, the peptide sequences need to be converted to SMILES format first. Therefore, the NovoPro online tool (https: / / www.novopro.cn / tools / peptide2smiles.html) was used to convert the peptide sequences to SMILES format before querying them in admetSAR.

[0025] The ToxinPred online tool, based on the Support Vector Machine (SVM) algorithm, can predict whether a peptide is toxic.

[0026] Furthermore, step 3 may include the following steps: Step 3-1: Keap1 receptor protein pretreatment, including: (1) downloading the crystal structure of the Keap1 receptor protein (ID: 4XMB) from the PDB (Protein Data Bank) crystal library; (2) pretreating the receptor protein in Discovery Studio software, including water molecule removal, hydrogenation treatment, and amino acid residue completion; and (3) selecting cavities including six key amino acids in the receptor protein according to actual needs, and then setting the required cavity diameter. Step 3-2: Ligand structure optimization, including: (1) using the "Build and Edit Protein" tool in Discovery Studio software to build a molecular structure containing the peptide sequence for the peptides screened in Step 2; and (2) using the "MinimizeLigands" tool, selecting the "Full Minimization" option to fully optimize the built peptide molecules. Step 3-3: Perform "Lib Dock" docking, including: (1) using the "DockLigands (Lib Dock)" function in Discovery Studio software to perform molecular docking; (2) selecting the receptor protein pretreated in step 3-1 in "InputReceptor" and the ligand optimized in step 3-2 in "Input Ligands"; and (3) saving the maximum score of "LibDockScore" to evaluate the molecular docking effect. Steps 3-4: Perform "C DOCKER" docking, including: (1) using the "DockLigands (CDOCKER)" function of Discovery Studio software to perform molecular docking; (2) saving the maximum scores of "-CDOCKER_ENERGY" and "-CDOCKER_INTERACTION_ENERGY"; and (3) evaluating the molecular docking effect based on "-CDOCKER_ENERGY".

[0027] This invention also discloses the application of the above-mentioned seaweed protein-derived antioxidant peptides in the preparation of antioxidants.

[0028] The above-mentioned seaweed protein-derived antioxidant peptides are used in the preparation of oral antioxidant drugs or functional foods.

[0029] The present invention also discloses the application of the above-mentioned seaweed protein-derived antioxidant peptides in the preparation of topical antioxidant cosmetics, preferably, the cosmetics being skin care products.

[0030] Compared with the prior art, the present invention has the following outstanding advantages: This invention is the first to screen and obtain the previously unreported antioxidant peptide WWR from laver protein, which has the following advantages: 1. Using the protein sequences of edible algae Porphyra from the NCBI database as raw materials, highly efficient antioxidant peptides were extracted, realizing the initial exploration of the medicinal development of Porphyra and increasing the added value of Porphyra applications; 2. WWR peptides have a small molecular weight, making them easily absorbed and utilized by cells; 3. Through rigorous screening, WWR has good bioactivity, water solubility, absorbability and non-toxicity, and has stable Keap1 binding ability and significant antioxidant activity.

[0031] 4. WWR can tolerate low pH environments and degradation by pepsin and trypsin.

[0032] Therefore, the seaweed protein-derived antioxidant peptides of the present invention can be used in the research and development of functional foods, pharmaceuticals, skin care products and health products, and have significant economic value and good application prospects. Attached Figure Description

[0033] Figure 1 This is a 3D diagram of the structure-optimized Keap1 receptor protein (ID: 4XMB).

[0034] Figure 2 This is a 3D diagram showing the successful docking of WWR with the Keap-1 protein molecule according to the present invention. Figure 2 -a) and 2D graphs Figure 2 -b).

[0035] Figure 3 This is a 3D diagram showing the docking of 12e, used as a control group, with the Keap-1 protein molecule. Figure 3 -a) and 2D graphs Figure 3 -b).

[0036] Figure 4 This is a reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of the WWR of the present invention.

[0037] Figure 5 This is the mass spectrometry (ESI-MS) chromatogram of the WWR of this invention.

[0038] Figure 6The effect of WWR on ROS content in HK-2 cells treated with oxalate was detected using a fluorescence microplate reader. The oxalate concentration was 1 mM, and the WWR concentration was 100 μM. #p<0.05 was compared with the control group, and *p<0.05 was compared with the oxalate group.

[0039] Figure 7 The effect of WWR on superoxide dismutase (SOD) activity in HK-2 cells treated with oxalate was detected using a total SOD activity assay kit (NBT method) (product number: S0109). The oxalate concentration was 1 mM, and the WWR concentration was 100 μM. #p<0.05 was compared with the control group, and *p<0.05 was compared with the oxalate group.

[0040] Figure 8 The effect of WWR on glutathione peroxidase (GSH-Px) activity in oxalate-treated HK-2 cells was detected using a glutathione peroxidase assay kit (product number: S0056). Oxalic acid was 1 mM, and WWR was 100 μM. #p<0.05 was compared with the control group; *p<0.05 was compared with the oxalate group.

[0041] Figure 9 The results of mass spectrometry detection of peptide tolerance to gastrointestinal enzymes are: specifically, the structure of the peptide after in vitro degradation by pepsin. Figure 10 The results of mass spectrometry detection of peptide tolerance to gastrointestinal enzymes are as follows: specifically, the structure of WWR after being degraded by pepsin and trypsin, respectively. Figure 11 The results were obtained by mass spectrometry detection of the peptide's tolerance to gastrointestinal enzymes: specifically, the control group, which had not undergone any enzyme treatment; Figure 12 The study used Caco-2 cells to establish an intestinal model to detect WWR absorption rate and absorption and transport mechanisms. Figure 12 -a is the detection of bilateral phosphatase activity in an intestinal model using an ALP activity assay kit (product number: P0321S); Figure 12 -b represents the relationship between WWR absorption rate and absorption time in the gut model; Figure 12 -c represents the effect of different transport mode inhibitors on WWR transport efficacy. ** indicates P<0.05, and different letters indicate P<0.05 between the two groups. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0043] Example 1 Virtual enzymatic hydrolysis of seaweed protein First, protein sequences of *Ulva prolifera* were searched and downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / genome). After screening, 49 proteins were selected for further research; these 64 proteins are shown in Table 1 below.

[0044] As shown in Table 1 above, the amino acid lengths of the 64 proteins range from 109 to 2751 AA, and the molecular weights range from 50.73 to 353.48 kDa, making them readily digestible and degradable by pepsin and trypsin. Forty-nine protein sequences were sequentially copied and entered into the Peptide Cutter online tool (https: / / web.expasy.org / peptide_cutter / ) for simulated digestion. Two digestive enzymes, pepsin (enzyme: pepsin, pH>2) and trypsin (enzyme: trypsin), were selected to simulate the protein digestion process in the human gastrointestinal environment, yielding a total of 2242 peptides, with lengths ranging from 2 to 6 amino acids. All peptide information was saved in an Excel spreadsheet, including protein ID, peptide sequence, peptide length, and cleavage site, to prepare for subsequent screening.

[0045] Example 2 Preliminary screening of bioactive peptides from laver 2.1 Screening based on bioactivity score The 1891 peptides obtained in Example 1 were evaluated for bioactivity using the Peptide Ranker online tool (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0046] The specific steps are as follows: 1. Input the 2242 peptide sequences into the Peptide Ranker tool in FASTA format. 2. Set the rating threshold to 0.5. 3. Submit the sequence for scoring. 4. Export the scoring results and filter out peptides with a score greater than 0.5.

[0047] The scoring results showed that 277 peptides had a bioactivity score greater than 0.5, accounting for 12.35% of the total number of peptides.

[0048] 2.2 Water solubility prediction and screening The 277 peptides were input into the Innovagen online tool (http: / / www.innovagen.com / proteomics-tools) for water solubility prediction.

[0049] The specific steps are as follows: 1. Input each of the 277 peptides into the Innovagen tool. 2. Select the "Peptide solubility and hydrophobicity" function. 3. Analyze the results and screen out peptides that show "Good water solubility".

[0050] The prediction results showed that 235 peptides were rated as "Good water solubility," accounting for 84.84% of the total number of screened peptides. These peptides typically contain a large number of polar or charged amino acids, which are beneficial for dissolution in an aqueous environment. After removing duplicate peptides, 211 unique peptides with good water solubility were retained.

[0051] 2.3 ADMET Property Prediction and Screening The ADMET properties of the above 211 peptides were predicted using the online admetSAR tool (http: / / lmmd.ecust.edu.cn / admetsar2 / ), focusing on the following three indicators: Blood-brain barrier (BBB) ​​permeability Human intestinal absorption (HIA) performance Caco 2- Cell permeability The specific steps are as follows: 1. Convert each of the 211 peptides into SMILES format. 2. Input the molecular structure in SMILES format into the admetSAR tool. 3. Analyze BBB, HIA, and Caco. 2- Permeability results 4. Filter for results that simultaneously display "BBB+", "HIA+", and high Caco. 2- Permeable peptides.

[0052] The screening results showed that 53 peptides simultaneously met the criteria of "BBB+", "HIA+", and high CaCO3 content. 2- Permeability accounted for 25.12% of the total number of screened peptides. These peptides typically have a small molecular weight (<600 Da), moderate hydrophobicity, and fewer than 5 hydrogen bond donors.

[0053] 2.4 Toxicity Prediction Screening The 53 peptides mentioned above were input into the ToxinPred online tool (http: / / crdd.osdd.net / raghava / toxinpred / ) for toxicity prediction.

[0054] The specific steps are as follows: 1. Input the 53 peptides into the ToxinPred tool in FASTA format. 2. Select the "SVM based prediction using selected features" function. 3. Set the threshold to the default value. 4. Analyze the results and screen out peptides that show "Non-toxin".

[0055] The prediction results showed that 22 peptides were rated as "Non-toxin", accounting for 41.51% of the total number of screened peptides.

[0056] The bioactivity, water solubility, ADMET properties, and toxicity evaluation results of the 22 peptides are shown in Table 2 below.

[0057] Example 3 Screening of antioxidant active peptides 3.1 Receptor protein pretreatment The crystal structure of the Keap1 receptor protein (PDB ID: 4XMB) was downloaded from the PDB (Protein Data Bank) library. This structure has a resolution of 1.9 Å, clearly showing the three-dimensional structure of Keap1. The receptor protein was preprocessed using Discovery Studio 2023 software, with the following specific steps: 1. Open the 4XMB.pdb file in Discovery Studio software. 2. Use the "PrepareProtein" tool in the "Macromolecules" section to preprocess the Keap1 receptor protein: Remove water of crystallization molecules, Add hydrogen atoms (pH=7.4). Repairing incomplete amino acid residues Optimize hydrogen bond network, Energy minimization (CHARMM force field, step size = 2000, RMS gradient = 0.01).

[0058] 3. Based on the known Keap1-Nrf2 interaction sites, define the binding pocket in the receptor protein: The cavity was selected to include six key amino acids (ARG380, ARG415, ARG483, TYR334, TYR525, ALA556). The radius of the binding site is set to 10 Å. Generate binding site spheres.

[0059] The preprocessed Keap1 receptor protein structure was saved as a 4XMB_prepared.pdb file for subsequent molecular docking.

[0060] Figure 1 A 3D diagram of the pretreated Keap1 receptor protein (ID: 4XMB) is shown.

[0061] 3.2 Ligand Structure Optimization The 3D structures of the 22 peptide ligands were constructed and optimized using Discovery Studio software. The specific steps are as follows: 1. Create a new page in Discovery Studio. 2. Using the "Build and Edit Protein" tool in the "Macromolecules" section, construct the peptide structure based on the peptide sequence. 3. In the "Small Molecules" section, use the "Minimize Ligands" tool to optimize the peptide structure: Select the "Full Minimization" option. Force field selection CHARMM, Set the maximum number of steps to 2000. The RMS gradient is set to 0.001. The dielectric constant is set to 1.

[0062] Each optimized peptide ligand was saved as a separate .mol2 file for subsequent molecular docking.

[0063] 3.3 Lib Dock Molecular Docking Use the "Dock Ligands (Lib Dock)" function under the "Receptor-LigandInteractions" section in Discovery Studio software to perform preliminary molecular docking. The specific parameter settings are as follows: 1. Input the preprocessed 4XMB_prepared.pdb file containing the receptor selection. 2. Input ligands: Select 22 optimized peptide .mol2 files. 3. Set docking parameters: The binding site is selected from the previously defined binding pocket. The Conformation Method is set to "BEST". Set Max Hits to 100 Min LibDock Score is set to 0. Keep other parameters at their default values.

[0064] After docking is complete, the Lib Dock Score value of each peptide ligand is recorded. A higher score indicates a more stable binding.

[0065] 3.4 C DOCKER molecular docking Use the "Dock Ligands (CDOCKER)" function under the "Receptor-Ligand Interactions" section in Discovery Studio software to perform more precise C DOCKER molecular docking. The specific parameter settings are as follows: 1. Input the preprocessed 4XMB_prepared.pdb file containing the receptor selection. 2. Input ligands: Select 22 optimized peptide .mol2 files. 3. Set docking parameters: The binding site is selected from the previously defined binding pocket. Set Top Hits to 10 Random Conformations is set to 20. Set Simulated Annealing to true. Keep other parameters at their default values.

[0066] After docking is completed, record the -CDOCKER_ENERGY and -CDOCKER_INTERACTION_ENERGY values ​​of each peptide ligand. The higher the value, the lower the binding energy and the more stable the binding.

[0067] The Lib Dock Score and -CDOCKER_ENERG results for the 22 peptides are shown in Table 2 below.

[0068] In Table 2 above, 12e is a known compound that targets Keap1 (see https: / / doi.org / 10.1016 / j.ejmech.2015.08.049), which has been widely used in Keap1 targeting research. In this specification, compound 12e is used as a control group to evaluate the efficacy of the antioxidant peptides of the present invention.

[0069] According to Table 2, the prediction results show that WWR has good water solubility (Good), is non-toxic (NO), can cross the blood-brain barrier (BBB+), and has good gastrointestinal absorption (HIA+).

[0070] The docking scores of Lib dock and Cdock molecules indicate that WWR docking was successful in both cases, and the highest score was obtained, suggesting that the peptide has a higher antioxidant potential.

[0071] Figure 1 A 3D diagram showing the docking of WWR with the Keap-1 protein molecule is shown. Figure 2 A 2D diagram showing the docking of WWR and Keap-1 is provided. Figure 3 A 2D diagram of the docking between the control group 12e and Keap-1 is shown; it can be seen from the above docking diagram that WWR of the present invention, as well as 12e as the control group, successfully docked with the Keap-1 protein.

[0072] Hydrogen bonds (H-bonds) are relatively strong, highly oriented, and specific non-covalent interactions present in many organic molecules. Carbon-hydrogen bonds are similar to hydrogen bonds, but with relatively lower bond energies and weaker forces. 12e is a highly efficient non-electropophilic activator of Nrf2, and it can form 8 hydrogen bonds with Keap1. Furthermore, some sites on Keap1 are important sites for the 12e-Keap1 complex (Ile461, Ser508, Arg415, Asn414, Ser363). WWR can form 7 hydrogen bonds with some key amino acid sites on Keap1, including Arg415, Ser602, Gln530, Leu365 (two hydrogen bonds), Val604, and Ile416. Additionally, Ser602 in the Keap1-Kelch region is a key site for binding to Nrf2 via hydrogen bonding. Arg415 is also a common amino acid residue in Keap1. In this study, WWR formed a hydrogen bond with Ser602 and Arg415 in Keap1. Therefore, WWR can competitively occupy key sites in Keap1 to release Nrfr2. WWR demonstrated outstanding binding ability, docking score, and good safety, and its physicochemical properties met the requirements for biological activity. Therefore, WWR was selected as the peptide for subsequent research.

[0073] Example 4 Synthesis and Characterization of WWR Based on the aforementioned virtual screening and molecular docking results, the applicant synthesized the tripeptide WWR (Trp-Trp-Arg) and performed detailed characterization. The WWR tripeptide was synthesized using solid-phase peptide synthesis technology. First, the C-terminal arginine amino acid (Arg) was immobilized on resin. Then, two tryptophans (Trp) were added sequentially, removing the protecting group of the previous amino acid before each addition. After synthesis, cleavage and deprotection operations were performed to cleave the synthesized crude peptide from the resin and remove the side-chain protecting groups.

[0074] The crude peptide was purified by high performance liquid chromatography (HPLC) using a Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 μm). Mobile phase A was acetonitrile with 0.1% TFA, and mobile phase B was water with 0.1% TFA. Gradient elution was used: 0 min (20% A, 80% B) → 20 min (45% A, 55% B) → 20.1 min (100% A, 0% B), with a flow rate of 1.0 mL / min and a detection wavelength of 220 nm.

[0075] The purified WWR tripeptide was characterized by mass spectrometry (MS) and HPLC. Figure 6 The high-performance liquid chromatography (HPLC) chromatogram of WWR is shown. Figure 7The mass spectrometry (MS) chromatogram of WWR is shown. HPLC analysis shows that the purity of WWR is as high as 98.99%, with a main peak retention time of 5.533 minutes. ESI-MS analysis shows the detection of [M+H]+ peaks (m / z=609.79) and [M+2H]2+ peaks (m / z=305.43), and the calculated molecular weight is 547.55, consistent with the theoretical molecular weight of WWR. This small molecular weight makes it easily absorbed and utilized by the human body.

[0076] The WWR tripeptide has the amino acid sequence Trp-Trp-Arg, appears as a white lyophilized powder, and has a solubility of 1 mg / mL in 17% ACN / 83% H2O. These properties indicate the successful synthesis of a high-purity WWR tripeptide, laying the foundation for subsequent bioactivity studies.

[0077] Figure 4 This is an HPLC chromatogram used for characterizing the synthesized peptide. Figure 5 Mass spectrum for peptide identification (WWR molecular weight: approximately 547 Da). Example 5 The protective effect of WWR on oxidative stress cells.

[0078] To verify the antioxidant activity of WWR, in vitro cell experiments were conducted to evaluate WWR. Human renal proximal tubular epithelial cells (HK-2) were selected as the experimental model, and oxidative stress damage was induced using oxalate.

[0079] First, WWR was dissolved in 17% ACN / 83% H2O solution at a concentration of 1 mg / ml, and then used in HK-2 cell experiments at a final concentration of 100 μM.

[0080] The experimental groups are as follows: Control group: 1×10⁶ HK-2 cells were used. 5 Each inoculum was seeded in a 96-well plate and cultured in HK-2 special medium (product number: JY-H258) at 37°C in a carbon dioxide incubator for 24 hours, then replaced with fresh medium and cultured for another 6 hours.

[0081] Damage group: 1×10⁶ HK-2 cells were collected. 5 Cells were seeded in 96-well plates using HK-2 special medium (catalog number: JY-H258) and cultured at 37°C in a CO2 incubator until the cells were confluent. Fresh medium was then added and cultured for 24 hours, followed by incubation with 1 mM oxalic acid for 6 hours.

[0082] Experimental group: 1×10⁶ HK-2 cells were used. 5Cells were seeded in 96-well plates using HK-2 dedicated medium (catalog number: JY-H258) and cultured at 37°C in a CO2 incubator until the cells were confluent. Then, the medium was replaced with fresh medium containing 100 μM WWR and cultured for 24 h. The medium was then replaced with fresh medium containing 100 μM WWR and 1 mM oxalic acid and incubated for another 6 h.

[0083] 1. Cellular ROS content detection: After the above treatment, DCFH-DA was diluted 1:1000 with probe dilution buffer to a final concentration of 10 μmol / L. The cell culture medium was removed, and 100 μL of the diluted DCFH-DA was added. The cells were incubated at 37ºC for 20 minutes. The absorbance of each group was detected using a fluorescence microplate reader (488 nm excitation wavelength, 525 nm emission wavelength).

[0084] 2. Antioxidant enzyme activity assay: The above-mentioned cells were seeded into T25 culture flasks, and other conditions were kept inhibited. After treatment, cells were scraped from the culture flasks, and the intracellular SOD activity of each treatment group was measured according to the total SOD activity assay kit (NBT method) (product number: S0109); the intracellular GSH-Px enzyme activity of each treatment group was measured according to the glutathione peroxidase assay kit (product number: S0056).

[0085] like Figure 6 As shown, after treatment with oxalic acid, the intracellular ROS content increased significantly (* P<0.05, compared with the control group), while after co-incubation with peptides (Oxalic acid-WWR), the intracellular ROS content decreased significantly (# P<0.05, compared with the oxalic acid group).

[0086] like Figure 7 As shown, after treatment with oxalic acid, the intracellular SOD enzyme activity decreased significantly (* P<0.05, compared with the control group), while after co-incubation with peptides (Oxalic acid-WWR), the intracellular SOD enzyme activity increased significantly (# P<0.05, compared with the oxalic acid group).

[0087] like Figure 8 As shown, after treatment with oxalic acid, the intracellular GSH-Px enzyme activity decreased significantly (* P < 0.05, compared to the control group), while after co-incubation with peptides (Oxalic acid-WWR), the intracellular GSH-Px enzyme activity increased significantly (# P < 0.05, compared to the oxalic acid group).

[0088] The above results indicate that the peptide WWR can activate the cellular antioxidant system, enhance the activity of antioxidant enzymes (SOD, GSH-Px), scavenge oxalate-induced ROS, and alleviate cellular oxidative damage.

[0089] Example 6 WWR's in vitro tolerance to pepsin and trypsin WWR was dissolved in deionized water to prepare a 50 μM solution, and the pH was adjusted to 2.0. Pepsin (1% E / S, w / w) was added to the peptide solution mixture, and the mixture was incubated at 37°C for 2 h. Half of the digestion solution was terminated with boiling water, and the other half was adjusted to pH 7.5, then trypsin (1% E / S, w / w) was added, and the mixture was incubated at 37°C for 2 h, with the reaction terminated by boiling water. Subsequently, the two solvents were centrifuged at 12000 rpm for 5 min, and the changes in peptide structure were analyzed using an EASY-nano LC 1200 system (Thermo Fisher Scientific, MA, USA).

[0090] like Figure 9 As shown, the WWR (approximately 547 Da) of the polypeptide was not significantly destroyed after pepsin digestion; as Figure 10 As shown, the polypeptide was not significantly degraded even after digestion with pepsin and trypsin. Figure 11 This was the control group, which did not undergo enzymatic digestion. In vitro digestion experiments showed that WWR can effectively resist degradation by pepsin and trypsin, and has the potential to be absorbed through the gastrointestinal tract.

[0091] Example 7 WWR Absorption Rate and Absorption-Transport Mechanism To investigate the maximum absorption rate of WWR in the gut, we established an intestinal model using human colorectal adenocarcinoma cells (Caco-2).

[0092] 1. Establishment of the intestinal model Caco-2 cells were taken at 1×10 5 Cells / well were seeded into 12-well Transwell plates and cultured for 21 days. 0.5 mL and 1 mL of culture medium were added to the upper and lower chambers, respectively. The culture medium was changed every two days. The final culture medium was used to detect alkaline phosphatase (ALP) levels. Detailed procedures were performed using the ALP activity assay kit (cat: P0321S).

[0093] 2. Research on peptide transport mechanisms After culturing Caco-2 cells for 21 days, the cell surface was rinsed with HBSS buffer. Then, the upper surface of the Caco-2 cell monolayer was treated with wortmannin (500 nM), cytochalasin D (0.5 μg / mL), and Gly-Pro (10 mM) for 30 min. After the transport inhibitors had finished treating the cells, 0.5 mL of HBSS containing a peptide (WWR, 100 μM) was added to the top chamber of a Transwell plate, and 1.5 mL of HBSS was added to the bottom chamber. The Transwell plate was placed in a 37°C cell culture incubator for 2 h, and the sample from the bottom chamber was aspirated for subsequent analysis. Apparent permeability coefficients (Papp, cm / s) were calculated using the following formula.

[0094] Papp=(Dq / Dt)×(1 / A)×(1 / C0) Where: Dq / Dt: the amount of peptide transported to the bottom chamber per unit time (μM / s); A: Hole surface area of ​​the Transwell plate (cm²) 2 ); C0: The initial concentration of the peptide added to the head chamber; like Figure 12 As shown in -a, after 21 days of Caco-2 cell culture, there was a significant difference in ALP activity between the upper and lower layers of the transwell plate (p<0.05). Since ALP and most other large protein molecules cannot cross the Caco-2 cell monolayer membrane, this demonstrates the differentiation and maturation of the thin monolayer membrane.

[0095] Depend on Figure 12 -b indicates that the transport rate of the *Ulva prolifera* polypeptide WWR is time-dependent, reaching a maximum of 1.63% at 60 min and decreasing at 120 min, indicating that the polypeptide is degraded.

[0096] When Caco-2 cells were treated with the transcytosis inhibitor womanpelin (Cytochalasin D), the Papp of the polypeptide WWR across the Caco-2 cell monolayer was not significantly affected (P>0.05), indicating that the uptake of multiple WWR does not penetrate the intestinal mucosa via bypass transport.

[0097] When Caco-2 cells were treated with the peptide transporter PepT1 inhibitor Gly-Pro and the endocytosis inhibitor wortmannin, the Papp value of the peptide WWR across the Caco-2 cell monolayer was significantly reduced (p<0.05), indicating that some WWRs are transported via endocytosis and peptide transporters.

[0098] Based on the results of Examples 1-7, the applicant successfully screened a highly efficient and safe antioxidant peptide, WWR, targeting Keap1 from seaweed protein. Computer-aided drug design and in vitro experiments confirmed that WWR possesses good pharmacokinetic properties, stable Keap1 binding ability, and significant antioxidant activity. Specifically, WWR exhibits good biological activity (biological activity score of 0.992998), good water solubility, high absorption (HIA+), blood-brain barrier crossing (BBB+), and non-toxicity. Molecular docking results showed a high binding score of WWR to Keap1 (LibDock Score 185.331, -CDOCKER_ENERGY 85.8911), superior to the control compound 12e. Molecular dynamics simulations showed that WWR forms 7 hydrogen bonds with Keap1, exhibiting good binding stability. Biological activity experiments confirmed that WWR can effectively scavenge oxalate-induced intracellular ROS in HK-2 cells, alleviating oxidative stress damage. It can also enhance the activity of SOD and GSH-Px enzymes, activating the cellular antioxidant system. Furthermore, WWR is resistant to degradation by pepsin and trypsin without structural damage. In an intestinal model differentiated from Caco-2, it reached a maximum absorption rate of 1.64% after 60 minutes. This polypeptide may be absorbed and transported via endocytosis and polypeptide transporters. Therefore, this invention provides a new candidate substance and theoretical basis for the development of edible laver-derived antioxidant foods, drugs, health products, and cosmetics.

Claims

1. A seaweed protein-derived antioxidant peptide, characterized in that, Its amino acid sequence is Trp-Trp-Arg.

2. The laver protein-derived antioxidant peptide according to claim 1, characterized in that, The antioxidant peptides are resistant to degradation by pepsin and trypsin and maintain structural integrity in an in vitro simulated gastrointestinal digestive environment.

3. The laver protein-derived antioxidant peptide according to claim 1, characterized in that, The antioxidant peptide can specifically bind to the Keap1 protein, competitively inhibit the Keap1-Nrf2 interaction, thereby activating the Nrf2-ARE signaling pathway.

4. An isolated nucleic acid molecule, characterized in that, The code is for the seaweed protein source antioxidant peptide as described in claim 1.

5. The method for preparing the seaweed protein-derived antioxidant peptide according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Using a solid-phase synthesis method, tryptophan Trp, tryptophan Trp and arginine Arg residues with appropriate protecting groups are sequentially coupled; 2) Purification was performed using reversed-phase high-performance liquid chromatography on a C18 column. Mobile phase A was acetonitrile containing 0.1% trifluoroacetic acid, and mobile phase B was water containing 0.1% trifluoroacetic acid. A gradient elution program was used. 3) The molecular weight and sequence correctness of the synthesized peptide were confirmed by electrospray ionization mass spectrometry (ESI-MS); 4) Freeze-dry the purified peptide solution to obtain a white or pale yellow powder or crystalline solid product.

6. A computer-aided method for screening antioxidant peptides derived from laver protein as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Obtain the seaweed protein sequence from the NCBI database, and use the Peptide Cutter tool in conjunction with pepsin and trypsin for virtual digestion to generate a peptide library with a length of 2 to 6 amino acids. S2. Bioactivity was assessed using the Peptide Ranker algorithm, and peptides with scores greater than 0.5 were retained. S3. Predict water solubility using the Innovagen tool and retain peptides with good water solubility; S4. Use admetSAR and ToxinPred tools to predict the properties and toxicity of ADMET, retaining peptides that are highly absorbable, non-toxic, and can cross the blood-brain barrier. S5. The selected peptides are molecularly docked with Keap1 protein, and high-affinity peptides are selected based on the LibDock and CDOCKER binding scores. S6. Perform molecular dynamics simulations on the selected peptides to verify their stability in binding with Keap1.

7. The method according to claim 6, characterized in that, The molecular docking step includes pretreating the Keap1 receptor protein using Discovery Studio software to remove water molecules, add hydrogen, repair residues, and define a binding pocket containing ARG380, ARG415, ARG483, TYR334, TYR525, and ALA556.

8. The use of the seaweed protein-derived antioxidant peptide as described in any one of claims 1-3 in the preparation of antioxidants.

9. The use of the seaweed protein-derived antioxidant peptide as described in any one of claims 1-3 in the preparation of oral antioxidant drugs or functional foods.

10. The application of the laver protein-derived antioxidant peptide as described in any one of claims 1-3 in the preparation of topical antioxidant cosmetics, characterized in that, The cosmetics in question are skincare products.

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