An antioxidant peptide from stichopus japonicus and a preparation method and application thereof

Abstract

The application discloses an antioxidant peptide from Apostichopus japonicus and a preparation method and application thereof, and relates to the technical field of biological medicines. The antioxidant peptide from Apostichopus japonicus has an amino acid sequence as shown in SEQ ID NO. 1. The antioxidant peptide with the amino acid sequence of LPPF is screened from the Apostichopus japonicus, and the peptide is verified to have excellent antioxidant activity and high safety, and has a good application prospect in the treatment of oxidative stress injury diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a sea cucumber-like antioxidant peptide, its preparation method, and its application. Background Technology

[0002] With the accelerated pace of modern life, changes in dietary structure, and the influence of environmental factors, imbalances in the body's oxidative stress levels have become a significant cause of various diseases. Long-term mental stress, high-fat diets, and environmental pollution can lead to excessive accumulation of reactive oxygen species in the body, causing oxidative damage to lipids, proteins, and DNA. This, in turn, can induce liver cell damage, chronic inflammation, and other oxidative stress-related diseases, seriously threatening human health.

[0003] Currently, interventions for oxidative stress damage mostly rely on antioxidants, such as vitamins and synthetic antioxidant drugs. However, these substances generally suffer from low bioavailability, single target, and side effects with long-term use, making it difficult to meet the clinical demand for safe and effective anti-oxidative stress drugs.

[0004] Studies have found that sea cucumber, a marine organism used in both medicine and food, is rich in various active ingredients such as proteins and polysaccharides. Among these, bioactive peptides produced from protein degradation exhibit outstanding antioxidant, anti-inflammatory, and cell-protective functions. Compared with traditional chemical drugs, marine-derived bioactive peptides have significant advantages such as small molecular weight, easy absorption, high bioactivity, and low toxicity, and have broad application prospects in the field of combating oxidative stress damage.

[0005] Although research on bioactive peptides derived from sea cucumber has received increasing attention, there is still a research gap in the screening and exploration of antioxidant peptides derived from sea cucumber extract, especially those that can effectively treat oxidative stress damage and have excellent biological activity.

[0006] Therefore, the inventors believe that how to screen and obtain a safe, efficient, and bioactive antioxidant peptide from sea cucumber extract is a key problem that urgently needs to be solved by those skilled in the art.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a sea cucumber-like antioxidant peptide, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0009] A sea cucumber extract-like antioxidant peptide, the amino acid sequence of which is shown in SEQ ID NO.1. SEQ ID NO.1 is LPPF.

[0010] Application of a sea cucumber extract-like antioxidant peptide as described above in the preparation of drugs for treating oxidative stress-induced diseases.

[0011] Preferably, oxidative stress injury is hepatocyte oxidative stress injury.

[0012] Preferably, hepatocellular oxidative stress-induced liver injury includes one of the following: non-alcoholic fatty liver disease, alcoholic liver disease, drug-induced liver injury, viral hepatitis, or liver fibrosis.

[0013] Preferably, it also includes pharmaceutically acceptable salts of sea cucumber extract antioxidant peptides.

[0014] Preferably, it also includes at least one of pharmaceutically acceptable carriers, excipients, excipients and mediators.

[0015] Preferably, the oxidative stress injury drug also includes other therapeutic agents; the other therapeutic agents include drugs that have a therapeutic effect on oxidative stress injury.

[0016] Preferably, the dosage form of the oxidative stress-damaging drug is an oral preparation, a mucosal delivery preparation, or an injection.

[0017] The application of the above-described sea cucumber extract antioxidant peptide in the preparation of products for treating oxidative stress damage, including functional foods or cosmetics.

[0018] A method for preparing the sea cucumber extract-based antioxidant peptide as described above includes the following steps:

[0019] S1: Thaw, wash and drain the sea cucumber extract, then grind it in a colloid mill to obtain the ground product;

[0020] S2: Add 2% papain by mass to the ground product and hydrolyze at 55℃ for 3 hours to obtain the enzymatic hydrolysate;

[0021] S3: Place the enzyme hydrolysate in a 100℃ water bath for 10 min to inactivate the enzyme, cool and centrifuge, and collect the first supernatant;

[0022] S4: Mix the first supernatant with distilled water at a mass ratio of 1:6, add 0.4% lactic acid bacteria and 3% glucose by mass, and ferment at 40℃ for 4 hours to obtain the fermentation product;

[0023] S5: After cooling the fermentation product, centrifuge it, collect the second supernatant, freeze-dry it, and obtain the sea cucumber-like peptide.

[0024] The present invention provides an antioxidant peptide derived from sea cucumber extract, its preparation method, and its application, which has the following beneficial effects: The present invention screens an antioxidant peptide with the amino acid sequence LPPF from sea cucumber extract. This peptide has been verified to have excellent antioxidant activity and high safety, and has good application prospects in the treatment of oxidative stress injury diseases. Attached Figure Description

[0025] Figure 1 a is the distribution of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the LPPF;

[0026] Figure 1 b is the distribution of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of VGFF;

[0027] Figure 1 c is the HOMO contribution rate plot of LPPF;

[0028] Figure 1 d is the HOMO contribution rate plot of VGFF;

[0029] Figure 2 These are the results of molecular docking between LPPF and VGFF and Keap1;

[0030] in, Figure 2 a is a 3D and 2D diagram of the LPPF interaction;

[0031] Figure 2 b is a 3D and 2D diagram of VGFF interactions;

[0032] Figure 2 c is the binding site of the antioxidant peptides LPPF and VGFF to the Keap1 receptor;

[0033] Figure 3 These are the results of molecular dynamics simulations (50 ns) of Keap1 and the Keap1-peptide complex.

[0034] in, Figure 3 'a' is the root mean square deviation (RMSD). Figure 3 b represents the root mean square oscillation (RMSF).

[0035] Figure 3 c is the radius of gyration (Rg); Figure 3 d is the number of hydrogen bonds;

[0036] Figure 4 a represents the scavenging activity of antioxidant peptides LPPF and VGFF against ABTS and DPPH free radicals;

[0037] Figure 4b is the median lethal concentration (LC50) of the antioxidant peptides LPPF and VGFF in zebrafish embryos. 50 );

[0038] Figure 4 c represents the effects of antioxidant peptides LPPF and VGFF on early embryonic morphological development in zebrafish.

[0039] Figure 5 a represents the cytotoxicity of LPPF on HepG2 cells;

[0040] Figure 5 b represents the repair of oxidative damage induced by H2O2;

[0041] Figure 5 c represents the effect of different treatment groups on the survival of HepG2 cells under oxidative stress;

[0042] The green fluorescence corresponds to Calcein AM, with excitation / emission at 490 nm / 515 nm; the red fluorescence corresponds to propidium iodide (PI), with excitation / emission at 535 nm / 617 nm. Scale bar: 50 μm;

[0043] Figure 5 d represents the intracellular reactive oxygen species level detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining;

[0044] The green fluorescence corresponds to 2',7'-dichlorofluorescein (DCF), with excitation / emission at 488 nm / 525 nm. Scale bar: 20 μm;

[0045] Figure 5 e indicates Figure 5 The ratio of green to red fluorescence intensity in different treatment groups in c;

[0046] Figure 5 f represents Figure 5 Quantitative analysis of fluorescence intensity in d;

[0047] In multiple samples, different lowercase letters in the column indicate significant differences between groups (p < 0.05).

[0048] Figure 6 a represents the effect of LPPF on malondialdehyde (MDA) content;

[0049] Figure 6 b represents the effect of LPPF on glutathione peroxidase (GSH-Px) activity;

[0050] Figure 6 c represents the effect of LPPF on catalase (CAT) activity;

[0051] Figure 6 d represents the effect of LPPF on superoxide dismutase (SOD) activity;

[0052] Data are expressed as mean and standard deviation (n = 3); in multiple samples, different lowercase letters in the column indicate significant differences between groups (p < 0.05). Detailed Implementation

[0053] 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.

[0054] To address the aforementioned technical problems, this invention provides a sea cucumber-like antioxidant peptide, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0055] 1. Materials and Methods

[0056] 1.1 Materials and Reagents

[0057] Sea cucumber extract was collected in Jinzhou, China. Antioxidant peptides were synthesized by Nanjing Jietai Biotechnology Co., Ltd., with a purity >95%. Wild-type AB strain adult zebrafish and E3 culture medium were purchased from Shanghai Feixi Biotechnology Co., Ltd., and experimental procedures were performed with the approval of the Academic Ethics Committee of Bohai University. HepG2 cells, MEM culture medium, and trypsin were purchased from Suzhou Haixing Biotechnology Co., Ltd. Cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester / propidium iodide (Calcein AM / PI) were purchased from Baisha Biotechnology Co., Ltd. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) kit was purchased from UELandy. Malondialdehyde (MDA) kit, glutathione peroxidase (GSH-PX) kit, catalase (CAT) kit, and total superoxide dismutase (SOD) kit were all purchased from Nanjing Jiancheng Biotechnology Institute. All chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0058] 1.2 Preparation of sea cucumber extract

[0059] The sea cucumber extract (AJSP) was thawed under running water at 4 °C, washed and drained, and then ground in a colloid mill for 5 min. Papain (2%) was added, and the mixture was hydrolyzed at 55 °C for 3 h. The hydrolysate was then inactivated by placing it in a 100 °C water bath for 10 min. After cooling, it was centrifuged (10,000 rpm, 10 min), and the supernatant was collected. The supernatant was mixed with distilled water at a ratio of 1:6 (w / w), and lactic acid bacteria (0.4%) and glucose (3%) were added. Fermentation was carried out at 40 °C for 4 h. After cooling, the mixture was centrifuged, the supernatant was collected, and freeze-dried to obtain the sea cucumber extract.

[0060] 1.3 LC-MS / MS Analysis

[0061] The sea cucumber extract was dissolved in 50 mM NH4HCO3, and then the sample was reduced, alkylated, and desalted. The processed sample was tested by LC-MS / MS to obtain the raw file of the mass spectrometry results. After de novo analysis, the peptide sequence analysis results were obtained.

[0062] 1.4 Computer Simulation Analysis

[0063] The potential bioactivity probability of identified peptides was predicted using the PeptideRanker scoring system (http: / / distilldeep.ucd.ie / PeptideRanker / ). The scoring system ranges from 0 to 1, with higher scores indicating a greater likelihood of potential bioactivity. This study only considered peptides with a PeptideRanker score threshold > 0.95. The novelty of peptides was determined using the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), selecting previously unreported peptides. ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / ) was used to predict peptide toxicity, screening for non-toxic peptides. AnOxPP (http: / / www.cqudfbp.net / AI-Tools / AnOxPP / ) was used to predict the antioxidant potential of peptides. The peptide codes (Table S2) were generated using the BIOPEP-UWM "SMILES" tool. SwissADME (http: / / www.swissadme.ch / ) was used to predict their ADME properties. The molecular structure of the peptide was predicted using the PepDraw (https: / / pepdraw.com / ) tool, and the three-dimensional structure of the antioxidant peptide was constructed using Chem 3D software.

[0064] 1.5 Quantum Chemical Calculations

[0065] Using Gaussian 16 software, structural optimization and single-point energy calculations of antioxidant peptides were performed at the B3LYP / TZVP level based on DFT / TDDFT. The optimization results were all convergent and showed no imaginary frequencies. Multiwfn 3.8 (dev) software was used to predict the electrophilic reaction sites, i.e., the antioxidant active sites, of the antioxidant peptides.

[0066] 1.6 Molecular docking

[0067] Molecular docking was performed using AutoDock Vina software to analyze the interaction between the antioxidant peptide and the Keap1 (PDB ID: 2FLU) molecule. The 3D structure was downloaded from the RCSB PDB (https: / / www.rcsb.org / ). Water molecules, hydrogen atoms, and ligands carried by the original acceptor were removed using PyMOL software, and hydrogen atoms were added. The protein-peptide interaction was visualized using Biovia Discovery Studio and PyMol software.

[0068] 1.7 Molecular Dynamics Simulation

[0069] Molecular dynamics simulations of the antioxidant peptide-Keap1 complex were performed using Gromacs 2023.2 software in an Amber99sb-IDLN force field. The complex structure was enclosed in the center of a box with a 1.0 nm distance between the box edge and the intermolecular distance. Solvation was performed using the TIP3P water model, and Na was added. + To maintain a stable neutral charge state, a two-step energy minimization process was performed before the kinetic simulation. The first step used the steepest descent method for 5000 steps, and the second step used the conjugate gradient method for 5000 steps. Afterward, 100 ps NVT and NPT equilibrations were performed. Following further equilibration, a 50 ns molecular dynamics simulation was conducted at room temperature and pressure. Finally, the trajectory file was output for analysis of root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and number of hydrogen bonds.

[0070] 1.8 In vitro antioxidant activity assay

[0071] The antioxidant activity of the antioxidant peptides was determined using ABTS and DPPH free radical scavenging assays. In the ABTS scavenging assay, an ABTS test solution was prepared using 10 mL of ABTS (7 mM) and 10 mL of potassium persulfate (2.45 mM), and stored at room temperature in the dark for 16 h. 100 μL of the sample, blank, and control solutions were each mixed with 4 mL of the ABTS test solution, incubated at room temperature in the dark for 5 min, and the absorbance was measured at 734 nm. In the DPPH scavenging assay, a 150 μM DPPH solution was prepared using 80% (v / v) anhydrous ethanol. 500 μL of the sample, blank, and control solutions were each mixed with 500 μL of the DPPH solution, incubated at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm.

[0072] 1.9 Acute toxicity test of zebrafish embryos

[0073] Fourteen days prior to the experiment, zebrafish were kept in filtered circulating water at a temperature maintained at 28.5 ± 0.5 °C, with a light-dark cycle of 14:10 h. Male and female zebrafish (1:1 ratio) were placed in the spawning tank overnight. The next morning, the partition was removed for illumination. Four hours later, healthy zebrafish embryos were collected, dead eggs and feces were removed, and the embryos were placed in E3 medium and cultured at 28 °C. Zebrafish embryos at 24 hpf (hours post-fertilization) were divided into a control group and a sample group (antioxidant peptide concentrations of 5, 10, 20, 30, 40, 50, 100, 200, 500, and 1000 mg / L). After 96 hpf, the survival rate of the zebrafish was recorded, and the median lethal concentration (LC50) was determined. 50 According to the OECD Chemicals Testing Guidelines, zebrafish embryos were exposed to a 100 mg / L antioxidant peptide solution, and the early embryonic development morphology of zebrafish was observed using a stereomicroscope (MZ101) (4–72 h) to assess the effect of antioxidant peptides on zebrafish embryonic development.

[0074] 1.10 HepG2 cell damage protection assay

[0075] HepG2 cells were cultured in MEM medium at 37 °C under a humid environment of 5% CO2. Cell damage was induced by H2O2, and cell viability was measured using CCK-8 assay. Control, model, and experimental groups were established. HepG2 cells were exposed to H2O2 for 2 h, followed by drug administration for 12 h. The protective effect of antioxidant peptides against H2O2-induced oxidative damage in HepG2 cells was evaluated. The effect of antioxidant peptides on HepG2 cell morphology was observed using Calcein-AM / PI double staining. Intracellular reactive oxygen species (ROS) levels were measured using the DCFH-DA probe. Imaging was performed using laser confocal microscopy, and image analysis was performed using ImageJ software. Intracellular MDA content, GSH-PX, CAT, and SOD activities were measured according to the kit instructions.

[0076] 1.11 Data Processing

[0077] All treatments were replicated at least three times independently. Multiple comparisons and statistical differences between samples were analyzed using Tukey's HSD test (p < 0.05). Results are expressed as mean ± standard deviation (SD). Furthermore, statistical graphs were generated using Origin2025 (OriginLab, California, USA).

[0078] 2. Results and Discussion

[0079] 2.1 Peptidomics Analysis

[0080] Peptidomics is a non-targeted approach for the rapid identification and characterization of bioactive peptides from protein hydrolysates. This study utilizes peptidomics combined with de novo sequencing to rapidly screen and identify peptide sequences of AJSP directly from MS / MS maps without relying on any sequence databases.

[0081] This experiment identified 157 peptides, composed of 4-18 amino acids with a molecular weight range of 412-1251 Da, of which peptides with a molecular weight ≤ 1000 Da accounted for 96.13%. According to relevant research, peptides with smaller molecular weights bind more readily to target molecules and exert antioxidant effects. In the AJSP prepared in this invention, tetrapeptides and pentapeptides accounted for 35.57% and 31.85%, respectively, suggesting they may have better antioxidant activity. Furthermore, all identified peptides contained one or more hydrophobic amino acids, such as alanine (Ala), glycine (Gly), proline (Pro), valine (Val), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). These amino acids provide a suitable hydrophobic environment for the peptides, increasing their lipid solubility and promoting their interaction with free radicals, thereby exerting antioxidant effects by preventing lipid peroxidation.

[0082] 2.2 Results of Computer-Aided Virtual Screening of Antioxidant Peptides

[0083] This invention used PeptideRanker to screen for nine peptides with potential biological activity (WGPGW, FPDCF, FPSF, WGPNW, WFPQ, WPLP, LPPF, TPFF, and VGFF), all of which were non-toxic (Table 1). These nine peptides were not found in the BIOPEP database and therefore can be considered novel peptides.

[0084] Table 1

[0085]

[0086] explain: 1 PeptideRanker database: Peptides with a score ≥ 0.95 are considered to be potentially bioactive peptides; 2 Predicting peptide toxicity (ToxinPred): toxic or non-toxic; 3,4 Predicting the antioxidant properties of peptides (AnOxPePred-1.0): antioxidant or non-antioxidant; 5 The search in the BIOPEP database yielded no results.

[0087] It should be noted that these peptides have a molecular weight range of 468-658 Da and are short peptides composed of 4-5 amino acids. Studies have shown that short peptides with 3-10 amino acid residues can easily cross intestinal epithelial cells, which is beneficial for human intestinal absorption. Antioxidant activity prediction indicates that all nine peptides are potential antioxidant peptides, among which LPPF exhibits the strongest antioxidant activity, with a score of 0.9996.

[0088] ADME studies are an important means of assessing pharmacokinetic properties, aiming to predict the dynamic changes of drugs in vivo and their pharmacodynamic effects (Table 2).

[0089] Table 2

[0090]

[0091] explain: 1 Lipophilicity: Five predictions (iLOGP, XLOGP3, WLOGP, MLOGP, SILLICOS-IT);

[0092] 2Water solubility: (ESOL: Topological method implemented from Delaney JS. 2004J. Chem. Inf. Model), very soluble: [-2 ≤ Log S < 0], soluble: [-4 < Log S < -2]; 3 CYP450: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4; 4 Drug-likeness prediction: Lipinski, Ghose, Veber, Egan, Muegge.

[0093] octanol-water partition coefficient (log P) o / w As a key indicator of the lipophilicity of peptides, lipophilicity has a decisive impact on their bioavailability. An excessively high lipophilicity may lead to non-specific retention of peptides in biological membranes or tissues, thereby affecting their efficacy and safety. o / w < 3 is generally considered a critical value for maintaining good lipophilicity, ensuring cell membrane permeability while reducing potential toxicity. SwissADME predictions show that, except for FPDCF, the log P values ​​for the other eight peptides are... o / w The values ​​were all less than 3, indicating that these peptides have good cell membrane permeability and low toxicological risk. Water solubility is an important physicochemical property affecting the absorption and distribution of bioactive molecules. This invention uses the ESOL calculation model to predict directly based on molecular structure in the absence of peptide melting point information. The results show that, except for WPLP, which is soluble, the other eight peptides are extremely soluble. Peptides with high solubility are generally more easily absorbed, thus having better bioavailability.

[0094] Cytochrome P450 (CYP450 enzymes) are a key enzyme system for drug metabolism, including subtypes such as CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. They dominate the oxidative metabolism of drugs, and inhibition of their activity may lead to drug interactions. The results showed that all nine peptides were predicted to be non-inhibitors of CYP450 enzymes. This indicates that the efficacy and safety of these peptides are not affected by CYP450 enzymes, and they exhibit good drug compatibility. Furthermore, they may enhance the activity of drug metabolism-related enzymes, playing a crucial role in drug metabolism pathways.

[0095] Bioavailability refers to the amount and rate at which a drug enters the systemic circulation. For peptides, the absorption process plays a decisive role in bioavailability, mainly influenced by factors such as solubility, intestinal transit time, membrane permeability, and gastric emptying rate. The results showed that FPSF, WPLP, LPPF, TPFF, and VGFF exhibited high bioavailability (0.55). Drug-likeness is an important indicator for evaluating the drug potential of peptides. Using SwissADME, the classic drug-likeness rules Lipinski, Ghose, Veber, Egan, and Muegge were used for prediction. If two or more of the five drug-likeness predictions were "Yes," the peptide was considered to have good drug-likeness. In this invention, WPLP, LPPF, and VGFF were predicted to have good drug-likeness. Through the above computer analysis, two peptides with the highest overall evaluation and potential antioxidant activity, LPPF and VGFF, were effectively screened.

[0096] 2.3 Analysis of the reaction sites of antioxidant peptides based on quantum chemical calculations

[0097] The activity of antioxidant peptides is closely related to their amino acid composition and spatial conformation. Studying the structure-activity relationship of antioxidant peptides helps to elucidate their mechanism of action. Figure 3 As shown, both LPPF and VGFF contain repeating amino acid residues, which helps enhance their antioxidant capacity. In particular, the hydrophobicity of the amino acids at both ends of the peptide chain (C-terminus and N-terminus) and the proportion of hydrophobic amino acids in the peptide chain have a significant impact on antioxidant activity. To explore the essential relationship between the structure and activity of antioxidant peptides, this study analyzed the frontier molecular orbitals of LPPF and VGFF using the DFT / TDDFT method. According to frontier orbital theory, the highest occupied molecular orbital (HOMO) determines the nucleophilic reaction capability (electron donation) of the molecule, while the lowest unoccupied molecular orbital (LUMO) is related to the electrophilic reaction (electron acceptance). The study shows that in antioxidant reactions, antioxidant peptides need to donate electrons to free radicals to reduce them, thereby exerting an antioxidant effect. Electrons in HOMOs are more easily excited or transferred; therefore, the location of HOMO distribution is considered to be the active site for reaction with free radicals. The frontier orbital energy level difference ΔE is also shown. L-H (E) LOMO With E HUMO The energy difference (ΔE) represents the energy required for a molecule to transition from its ground state to an excited state. The smaller the ΔE value, the easier it is for electrons to undergo energy level transitions, and the higher the molecular reactivity. For example... Figure 1 The frontier molecular orbital distributions of LPPF and 1b show that the HOMO orbital energy of LPPF is higher and the ΔE value is smaller than that of VGFF. This indicates that LPPF has higher electronic activity and is more likely to lose electrons, thus having higher antioxidant activity.

[0098] Furthermore, the contribution of each atom of LPPF and VGFF to the HOMO was calculated using Multiwfn 3.8 (dev) software; atoms with larger contributions were more likely to be active sites. For example... Figure 1 As shown in Figures a and 1c, the HOMO of LPPF is mainly distributed at the N-terminal Leu and the second-position Pro, with N6 contributing the most (52.20%), followed by O7 (9.68%), N12 (8.65%), C3 (6.28%), N19 (5.12%), C4 (3.65%), C5 (1.78%), H38 (1.21%), and H1 (1.13%). Therefore, the active sites are mainly concentrated on polar atoms (such as N6, O7, and N12). Studies have shown that the rigid cyclic skeleton of Pro possesses the characteristics of a hydrogen donor, capable of reacting with hydroxyl radicals and effectively enhancing antioxidant activity. Furthermore, the pyrrolidine ring structure of Pro promotes electron delocalization; when synergistically acting with the amino group of Leu, it can significantly enhance the electron-donating ability of the system, thereby enhancing the reducing power of the peptide and the free radical scavenging efficiency. Therefore, LPPF exhibits excellent antioxidant capacity. Figure 1 As shown in b and 1d, the HOMO of VGFF is mainly distributed at the C-terminus and the third-position Phe, with C33 contributing the most (22.72%), followed by C30 (21.20%), N28 (10.82%), C35 (8.30%), C31 (7.13%), O17 (6.85%), C32 (5.61%), C34 (4.18%), C26 (3.50%), and C25 (3.48%). Compared with LPPF, the atoms contributing the most in VGFF are mainly hydrophobic carbon atoms (such as C33, C30, and C35), which are concentrated in the aromatic ring of C-terminal phenylalanine and adjacent carbon chains. The π-electron system of the benzene ring forms delocalized orbitals, making C30 and C33 the core regions of electron donors. However, because carbon atoms have a weaker electron-donating ability than polar atoms such as nitrogen and oxygen, electron excitation requires higher energy, thus limiting its antioxidant activity. Based on quantum chemical calculations, we hypothesize that LPPF has higher antioxidant activity.

[0099] 2.4 Molecular docking analysis

[0100] Molecular docking helps elucidate the relationship between drugs and targets through hydrogen bonds, intermolecular forces, and interionic forces. Proteins and peptides can bind through various forces, such as hydrogen bonds, hydrophobic interactions, van der Waals forces, π bonds, and C-H bonds. Among these, hydrogen bonds, hydrophobic interactions, and van der Waals forces are crucial for protein-peptide binding. Activating the Keap1-Nrf2 pathway is an effective strategy for bioactive peptides to inhibit oxidative stress and apoptosis. Antioxidant peptides effectively block the interaction between Keap1 and Nrf2 by competitively occupying ligand-binding sites in the Keap1-Kelch domain. Free Nrf2 accumulates in the cell nucleus and binds to proteins such as Maf, activating antioxidant response elements (AREs) and initiating the transcription of downstream genes, thereby enhancing the body's antioxidant capacity. Studies have shown that a binding energy less than −5.0 kcal / mol indicates good binding affinity, and less than −7.0 kcal / mol indicates extremely strong binding affinity. Figure 2 As shown in a and 2b, the binding energies of LPPF, VGFF, and Keap1 are all below −7.0 kcal / mol (−9.4 and −8.7 kcal / mol, respectively), which is lower than the binding energy of Keap1-Nrf2 (−4.7 kcal / mol). This indicates that both peptides have extremely strong binding ability to Keap1 and can competitively occupy ligand binding sites, which is beneficial to exerting antioxidant activity. It also proves that the virtual screening based on computer analysis and quantum chemical calculations is reliable.

[0101] Studies have shown that antioxidant peptides can exert their antioxidant function by binding to five active pockets within the Kelch domain of the Keap1 protein, inhibiting the Keap1-Nrf2 interaction. Figure 2As shown in Figure c, both LPPF and VGFF can be inserted into the active pockets of Keap1. LPPF forms 14 binding sites with the active pockets of Keap1, with 5 from pocket 1, 2 from pocket 2, 3 from pocket 3, 2 from pocket 4, and 2 from pocket 5. LPPF forms one hydrogen bond with Ser602 of Keap1 and binds to Arg415, Arg483, Ile461, Ser508, Arg380, Ser363, Gly603, Phe557, and Tyr334 via van der Waals forces. VGFF forms 15 binding sites with Keap1, with 3 from pocket 1, 3 from pocket 2, 5 from pocket 3, 2 from pocket 4, and 2 from pocket 5. VGFF forms three hydrogen bonds with Ser363, Ser555, and Ser602 of Keap1, and binds to Ser508, Asn382, Asn414, Ala556, Gly509, Gly603, Tyr525, and Phe577 via van der Waals forces. Furthermore, VGFF forms an unfavorable interaction with the Arg380 residue of Keap1; studies have shown that poor linkage may lead to repulsive forces between the receptor and ligand. Therefore, the formation of unfavorable interactions may affect the stability of the Keap1-VGFF complex. Molecular docking results indicate that LPPF and VGFF mainly form complexes with Keap1 through hydrogen bonds and van der Waals forces, effectively blocking the binding of Keap1 to Nrf2 and exhibiting significant antioxidant activity.

[0102] 2.5 Molecular Dynamics Simulation Analysis

[0103] Molecular dynamics simulations study conformational changes and structural stability of complexes by simulating intermolecular motion and interactions, thereby verifying the accuracy of molecular docking predictions. This study assesses the stability of LPPF, VGFF, and Keap1 binding by analyzing RMSD, RMSF, Rg, and the number of hydrogen bonds.

[0104] RMSD is an important indicator for evaluating the stability of protein-ligand complexes; a lower RMSD value indicates higher dynamic stability. Figure 3 As shown in Figure a, the RMSD curves of Keap1, Keap1-LPPF, and Keap1-VGFF fluctuated in the range of 0.1-0.2 nm and eventually stabilized at 0.175 nm, indicating that the addition of antioxidant peptides did not affect the stability of the Keap1 structure. The complexes formed by Keap1 with LPPF and VGFF were tightly bound and had good stability.

[0105] RMSF is used to measure the overall flexibility of a protein-ligand complex. The RMSF curve represents the degree of variation in amino acid residues within the complex; a lower RMSF value indicates less residue variation and a more stable structure. Figure 3 As shown in b, the RMSF curves of Keap1, Keap1-LPPF, and Keap1-VGFF fluctuated in the range of 0.04–0.33 nm. Keap1-LPPF showed a relatively low RMSF value, indicating that the complex is relatively stable. The amino acid residues in Keap1-VGFF fluctuated significantly between 380 and 410 nm. Based on the molecular docking results, we speculate that this may be due to an unfavorable interaction between VGFF and the Arg380 residue of Keap1, leading to some fluctuations during the simulation.

[0106] The Rg value is a characteristic parameter for evaluating changes in protein structure; a smaller Rg value indicates that the complex maintains a compact and stable structure during simulation. Figure 3 As shown in Figure c, the Rg values ​​of Keap1-LPPF and Keap1-VGFF remained at a low level (1.775-1.825 nm), with fluctuations of less than 0.05 nm, indicating that the complex maintained a compact and stable conformation throughout the simulation. The trends of Keap1 and the complex were almost identical, indicating that the addition of the antioxidant peptide did not cause a significant change in the overall conformation of the Keap1 protein, and that Keap1 formed a tight and stable complex with LPPF and VGFF.

[0107] Studies have shown that significant changes in the number of hydrogen bonds can lead to alterations in molecular structure, thereby affecting the stability of the complex. For example... Figure 3 As shown in Figure d, during the first 30 ns of the simulation, the number of hydrogen bonds in Keap1-LPPF and Keap1-VGFF fluctuated relatively greatly. This may be because the peptide molecules interacted strongly with nearby water molecules in the early stages of the simulation, and the interaction with Keap1 had not yet reached a stable state. After 30 ns, the fluctuation in the number of hydrogen bonds between the peptide and Keap1 protein decreased and tended to stabilize, indicating that the complex was tightly bound and had a relatively stable conformation.

[0108] Molecular dynamics simulations showed that both Keap1-LPPF and Keap1-VGFF reached a structurally stable state during the simulation. Compared with Keap1-VGFF, Keap1-LPPF showed a relatively lower RMSF value, indicating that Keap1-LPPF has higher conformational stability.

[0109] 2.6 In vitro antioxidant activity and acute toxicity test of zebrafish embryos

[0110] In vitro antioxidant activity was evaluated by the ability of LPPF and VGFF to scavenge ABTS and DPPH free radicals, such as... Figure 4 As shown in Figure a, within the concentration range of 0.2–1 mg / mL, both LPPF and VGFF exhibited concentration-dependent effects, with their free radical scavenging ability continuously increasing with increasing concentration. At 1 mg / mL, LPPF showed inhibition rates of 49.95% and 37.86% against ABTS and DPPH, respectively, while VGFF showed inhibition rates of 44.45% and 33.01%, respectively. Among these, LPPF demonstrated better in vitro antioxidant activity.

[0111] Zebrafish embryos are characterized by rapid development, small size, ease of culture, and transparent structure, making them easy to observe and offering significant advantages in toxicity screening. Studies have shown that the zebrafish genome shares up to 87% homologous sequence similarity with the human genome, and the toxic effects observed in zebrafish embryo models can provide important references for assessing human health risks. Figure 4 As shown in b, the mortality rate of zebrafish increased with increasing LPPF and VGFF concentrations in the sample group. The LC50 of LPPF and VGFF on zebrafish was calculated. 50 The concentrations were 223.69±11.65 and 177.18±12.58 mg / L, respectively. According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) standards, the LC... 50 A concentration >100 mg / L is considered low toxicity; the higher the value, the lower the toxicity. Compared to VGFF, LPPF has lower toxicity.

[0112] like Figure 4 As shown in c, healthy zebrafish embryos exposed to 100 mg / L LPPF and VGFF exhibited normal morphological development during the blastocyst, gastrula, somites, pharyngeal pouch, and hatching stages. Within 4–72 h, the zebrafish embryo's body axis gradually straightened, the distance between the head and tail increased, and the yolk sac gradually shrank. Compared to the control group, LPPF and VGFF had no significant effect on embryonic morphological development, demonstrating high biocompatibility.

[0113] 2.7 Protective effect of antioxidant peptides against oxidative damage in HepG2 cells

[0114] Based on comprehensive bioinformatics analysis and experimental results, LPPF exhibits a lower band gap and stronger free radical reactivity compared to VGFF. It also forms a more stable complex with Keap1, demonstrating higher in vitro antioxidant activity and better in vivo safety. Therefore, LPPF was chosen for subsequent cell experiments. HepG2 cells, due to their active metabolism, are highly susceptible to oxidative stress and exhibit high sensitivity, making them an ideal cell model for studying oxidative stress-related molecular mechanisms and screening for antioxidant compounds. Therefore, this study used HepG2 cells to construct a cellular oxidative damage model and investigated the protective ability of LPPF against oxidative damage to cells.

[0115] First, the cytotoxicity of LPPF in HepG2 cells at concentrations ranging from 0.05 to 2 mg / mL was evaluated using the CCK-8 assay. Figure 5 As shown in Figure a, cell viability exceeded 90%, indicating that LPPF did not produce significant cytotoxicity to HepG2 cells. Subsequently, three concentrations (0.2, 0.5, and 1 mg / mL) were selected as the L-LPPF group, M-LPPF group, and H-LPPF group, respectively, for subsequent experiments. HepG2 cells were treated with different concentrations (0.2-2.0 mM) of H2O2 for 2 h, as shown in Figure a. Figure 4 As shown, cell viability decreased significantly with increasing H2O2 concentration, IC50... 50 Since the concentration was 0.99 mM, this study chose 1 mM H2O2 to construct the HepG2 cell oxidative damage model.

[0116] like Figure 5 As shown in b, LPPF effectively protected HepG2 cells from H2O2-induced oxidative damage. At concentrations of 0.2, 0.5, and 1 mg / mL, cell viability was 73.02%, 83.01%, and 95.75%, respectively. Compared with the model group, different concentrations of LPPF significantly improved cell viability. In particular, the H-LPPF group showed an 80.42% increase in cell viability, with no significant difference compared to the control group. This indicates that LPPF can significantly improve H2O2-induced oxidative damage in HepG2 cells.

[0117] HepG2 cells in each group were stained using a live / dead cell staining method to observe morphological changes. Calcein-AM specifically stained live cells, causing their cytoplasm to fluoresce green, while PI, as a nuclear staining dye, could not penetrate the intact plasma membrane of live cells and could only stain the nuclei of dead cells. Figure 5As shown in Figures c and 5e, compared with the control group, the HepG2 cells in the model group were shrunken and deformed, with blurred edges and reduced cell density. The number of dead cells increased significantly to 50.49%, indicating that the H2O2 damage model was successfully established. After LPPF intervention, the cell morphology showed a significant improvement. Compared with the model group, the cell outline and structure underwent positive changes. With the increase of LPPF concentration, the number of live cells increased significantly, the number of dead cells decreased significantly, and the cells were closer to a healthy and normal physiological state.

[0118] Studies have shown that ROS level is a key indicator for assessing oxidative stress, and the intensity of fluorescence signal is positively correlated with ROS concentration. Figure 5 Images 5d and 5f show the fluorescence changes in cells from different groups. The control group showed only a weak basal fluorescence signal, indicating that intracellular ROS levels are maintained at a low level under normal conditions. The model group showed significantly enhanced fluorescence signal and densely distributed fluorescent spots, indicating that intracellular ROS levels increased significantly after H2O2 treatment. After LPPF intervention, the cell fluorescence intensity decreased significantly in a concentration-dependent manner. In particular, the relative fluorescence intensity of the H-LPPF group was only 9.80%. These results indicate that LPPF can reduce ROS levels in HepG2 cells and has a protective effect against oxidative stress damage.

[0119] MDA is an end product of lipid peroxidation, and elevated levels are typically associated with cell membrane damage and oxidative stress. Higher MDA levels indicate more severe oxidative damage. Figure 6 As shown in Figure a, compared with the control group, the MDA content in the model group was significantly increased by 79.63%, which is due to the increased free radical levels caused by H2O2 leading to lipid peroxidation. After LPPF intervention, the intracellular MDA content showed a concentration-dependent decrease, indicating that LPPF can effectively inhibit MDA production. GSH-PX, CAT, and SOD, as core enzyme systems of the cellular antioxidant defense system, synergistically constitute key components of the endogenous antioxidant mechanism in organisms. They ensure redox balance by regulating intracellular ROS levels. Figure 6 As shown in b-6d, compared with the control group, the activities of GSH-PX, CAT, and SOD in the model group were significantly reduced, decreasing by 82.22%, 63.03%, and 72.46%, respectively, indicating that the antioxidant defense system of HepG2 cells was severely impaired. After LPPF intervention, the activities of antioxidant enzymes in cells gradually increased with increasing LPPF concentration. Among them, the activities of GSH-PX, CAT, and SOD in the H-LPPF group increased by 268.86%, 117.96%, and 254.93%, respectively, compared with the model group, indicating that LPPF has good antioxidant activity.

[0120] Cell viability assays showed that LPPF has high safety, significantly reduces intracellular ROS levels and MDA content, and increases the activity of antioxidant enzymes GSH-PX, CAT, and SOD. This indicates that LPPF has a strong protective effect against H2O2-induced oxidative damage to HepG2 cells, further confirming the antioxidant activity of the sea cucumber peptide.

[0121] 3. Conclusion

[0122] This study used a combination of computer simulation analysis, quantum chemical calculations, molecular docking, molecular dynamics, and in vitro evaluation to screen peptides LPPF and VGFF with potential antioxidant activity from the AJSP. It deeply elucidated the structure-activity relationship of peptides, achieving high-throughput screening of bioactive peptides. This overcomes the problems of cumbersome procedures and low efficiency in traditional methods. Furthermore, it can be extended to the structure-activity analysis and functional prediction of other food-derived bioactive peptides (such as anti-inflammatory and antihypertensive peptides), providing an efficient research paradigm for the development of multifunctional peptide drugs.

[0123] Experimental results show that the Δ of LPPF LUMO-HOMO With a smaller energy level difference, higher electronic activity, and stronger reactivity with free radicals, LPPF exhibits a stronger binding interaction with Keap1. It binds stably to Keap1 via hydrogen bonds and van der Waals forces, effectively blocking the interaction between Keap1 and Nrf2. Molecular dynamics simulations further demonstrate the enhanced stability of the Keap1-LPPF complex, revealing its potential antioxidant activity. In in vitro experiments, it demonstrated excellent ABTS and DPPH free radical scavenging capabilities and showed higher safety in zebrafish embryo acute toxicity assays. In an H2O2-induced HepG2 cell oxidative damage model, LPPF significantly reduced oxidative stress levels and improved cell viability, demonstrating a good protective effect against oxidatively damaged cells. This study is the first to identify LPPF, a peptide with significant antioxidant activity, from AJSP. Its mechanism of action involves the regulation of the Keap1-Nrf2 signaling pathway. These findings provide theoretical and technical support for the in-depth development of marine-derived antioxidant peptides and are expected to promote their widespread application in functional foods and pharmaceuticals.

[0124] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of an antioxidant peptide from Apostichopus japonicus in the preparation of an antioxidant product, characterized in that, The amino acid sequence of the antioxidant peptide from Apostichopus japonicus is shown as SEQ ID NO.

1.

2. The use of the antioxidant peptide from Apostichopus japonicus as claimed in claim 1 in the preparation of an antioxidant product, characterized in that, The product is a functional food or a cosmetic.

Images