An endogenous peptide from anglerfish with antioxidant and anti-inflammatory activities, its preparation method and application

Abstract

This invention discloses an endogenous peptide from monkfish with antioxidant and anti-inflammatory activities, its preparation method, and its applications. The endogenous peptide from monkfish includes at least one of HPLK, PRK, VLPN, LGPQ, EVL, and LGYPPQ. The preparation method includes the following steps: monkfish is skinned, the flesh is minced, and then extracted with formic acid solution overnight. After homogenization, the supernatant is collected by centrifugation to obtain a supernatant of the fish meat sample. A Fe3O4@PDA magnetic material solution is added to the supernatant of the fish meat sample, and after shaking incubation and magnetic separation, the Fe3O4@PDA sample is collected. NaCl solution is added to elute the adsorbed endogenous peptides. The eluent is collected, dialyzed to remove salt, and freeze-dried to obtain the endogenous peptide from monkfish. The invention also provides applications of the above-mentioned endogenous peptide from monkfish in the preparation of antioxidants and anti-inflammatory reagents. The advantage is that it possesses antioxidant and anti-inflammatory activities.

Description

Technical Field

[0001] This invention belongs to the field of deep processing technology of aquatic products, and in particular relates to an endogenous peptide of anglerfish with antioxidant and anti-inflammatory activity, its preparation method and application. Background Technology

[0002] Anglerfish Lophius litulo As an important marine economic fish species, the high-value utilization of its resources is of great significance. This fish belongs to the family Anglerfish, order Moniliformes, and is characterized by its large head, wide mouth, and large abdominal cavity. Current development and utilization of anglerfish mainly focus on byproducts such as skin, liver, and roe, primarily through enzymatic hydrolysis to prepare exogenous bioactive peptides. In contrast, systematic research on its naturally occurring endogenous peptides is scarce. It is noteworthy that anglerfish, living in a high-pressure, low-temperature marine environment, have evolved endogenous peptides with unique structures and specific functions, such as being rich in hydrophobic amino acids or possessing special functional groups. These naturally occurring peptides, due to their native nature within the organism, may possess superior biocompatibility and specific physiological functions compared to enzymatically hydrolyzed peptides, exhibiting unique effects in antioxidation and anti-inflammation, and have great potential as core ingredients in high-end functional foods to meet the market's demand for precise nutrition.

[0003] Endogenous peptides are a crucial class of physiologically active regulators in organisms, such as hormones and neurotransmitters. They can precisely regulate core life processes such as growth, development, metabolism, and neural activity by binding with specific receptors at extremely low physiological concentrations through high affinity. Furthermore, some endogenous peptides, particularly antimicrobial peptides (AMPs), constitute an important part of the body's innate immune defense, effectively responding to pathogen invasion by rapidly disrupting microbial membrane structures. Despite their indispensable and powerful role in life activities, the extremely low natural abundance and complex forms of endogenous peptides in organisms lead to inefficiencies and high costs in traditional extraction and enrichment methods. This technological challenge severely limits direct and systematic functional research and subsequent development and application of endogenous peptides. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an endogenous peptide from anglerfish with antioxidant and anti-inflammatory activity, its preparation method and application.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: One objective of this invention is to provide an endogenous monkfish peptide with antioxidant and anti-inflammatory activity, wherein the endogenous monkfish peptide includes at least one of HPLK, PRK, VLPN, LGPQ, EVL, and LGYPPQ.

[0006] Furthermore, the anglerfish endogenous peptide is one of EVL, VLPN, and LGPQ.

[0007] The second objective of this invention is to provide a method for preparing the above-mentioned endogenous peptides from monkfish, characterized by the following steps: skinning and removing the flesh from the monkfish, chopping the flesh and placing it in a centrifuge tube, adding 0.2-1 wt% formic acid solution at a volume ratio of 1:(1-3) at 0-4°C and extracting overnight; homogenizing with a high-speed homogenizer under ice bath conditions; equilibrating the centrifuge tube with ultrapure water, centrifuging at 0-4°C to collect the supernatant, obtaining the supernatant of the fish flesh sample; dispersing in PB... A 5 mg / mL Fe3O4@PDA magnetic material solution in S buffer was added to the supernatant of the fish sample at a volume ratio of 1:(1-3). After shaking and incubation, the Fe3O4@PDA sample was collected after magnetic separation. NaCl solution was added to the Fe3O4@PDA sample to elute the adsorbed endogenous peptides. The eluent was collected, dialyzed with a 100 Da dialysis bag to remove salts, and then freeze-dried to obtain the anglerfish endogenous peptides.

[0008] Further, the preparation method of the Fe3O4@PDA magnetic material is as follows: Ammonia water, anhydrous ethanol, deionized water and Fe3O4 solution with a concentration of 20-30 mg / mL are mixed in a volume ratio of (1-2):40:90:20 and ultrasonically treated until uniformly dispersed. Then, while stirring, a hydrochloric acid-dopamine (DA·HCl) solution with a concentration of 40-60 mg / mL is slowly added dropwise. The reaction is carried out at room temperature for 15-27 h. The product is collected by high-speed centrifugation and freeze-dried in a freeze dryer to obtain Fe3O4@PDA magnetic material, wherein the volume ratio of Fe3O4 solution to DA·HCl solution is 2:1.

[0009] Furthermore, the reaction was carried out at room temperature for 24 hours.

[0010] A third objective of this invention is to provide the application of the above-mentioned anglerfish endogenous peptides in the preparation of antioxidants.

[0011] Furthermore, the application of the aforementioned anglerfish endogenous peptides in the preparation of in vitro antioxidants, wherein the in vitro antioxidants include DPPH free radical scavengers and Fe... 2+ Chelating agents and ·OH scavengers.

[0012] Furthermore, the application of the anglerfish endogenous peptides in the preparation of in vivo antioxidants, wherein the in vivo antioxidants include SOD, CAT, and GSH-Px antioxidant enzyme activity promoters.

[0013] A third objective of this invention is to provide the application of the above-mentioned anglerfish endogenous peptides in the preparation of anti-inflammatory agents, wherein the anti-inflammatory agents include IL-1β and IL-6 inflammatory factor inhibitors.

[0014] Furthermore, in an LPS-induced RAW264.7 cell inflammation model, endogenous peptide treatment significantly reduced the release of IL-1β and IL-6 inflammatory factors.

[0015] Compared with existing technologies, the advantages of this invention are as follows: This invention provides an endogenous peptide from monkfish with antioxidant and anti-inflammatory activities, its preparation method, and its application. It utilizes a core-shell composite material of iron(III) oxide@polydopamine (Fe3O4@PDA), which, due to its superparamagnetism, enables rapid separation. Its high specific surface area provides a large adsorption capacity, and the abundant functional groups on the PDA surface, such as catechol and amino groups, endow it with excellent hydrophilicity and specific adsorption potential for peptides, enabling efficient and gentle capture of endogenous peptides from complex fish matrices. By constructing an iron(III) oxide (Fe3O4)@polydopamine (PDA) magnetic material, endogenous peptides from monkfish are adsorbed and their functions are identified. Based on the omics analysis and molecular docking of the adsorbed peptides, the endogenous peptides can bind to the oxidative stress target Keap1 protein, exhibiting potential antioxidant functions. Chemical antioxidant results show that the endogenous peptides from monkfish exhibit the lowest IC50 value in the DPPH assay. 50 The value was only 0.06 mg / ml, in the presence of ·OH and Fe. 2+ The DPPH free radical scavenging rate and Fe of endogenous peptides at concentrations of 0.12 mg / ml and 0.21 mg / ml, and 0.35 mg / ml, respectively, were observed. 2+ The chelating capacity and ·OH scavenging rate reached 93.40%, 79.06%, and 81.5%, respectively. In the H2O2-induced oxidative damage model of HepG2 cells, this peptide significantly reduced intracellular ROS levels and increased the activities of antioxidant enzymes such as SOD, CAT, and GSH-Px. Furthermore, in the LPS-induced inflammation model of RAW264.7 cells, endogenous peptide treatment significantly reduced the release of inflammatory factors such as IL-1β and IL-6. Attached Figure Description

[0016] Figure 1 shows the dispersion analysis of Fe3O4@PDA magnetic material in aqueous phase; Figure 2 The effect of different modification times on the adsorption effect of Fe3O4@PDA magnetic material on peptides; Figure 3 Transmission electron microscopy (TEM) images of PDA and Fe3O4@PDA magnetic materials with different modification times; Figure 4 shows the dynamic light scattering particle size distribution of PDA and Fe3O4@PDA magnetic materials with different modification times; Figure 5 The variation of Zeta potential values ​​of Fe3O4@PDA magnetic materials with different modification times under PDA conditions; Figure 6Fourier transform infrared spectra of PDA and Fe3O4@PDA magnetic materials with different modification times; Figure 7 IC50, an in vitro antioxidant index of peptides 50 Value analysis; Figure 8 The in vitro antioxidant activity of the peptides was analyzed, where a represents DPPH free radicals, b represents OH· free radical scavenging rate, and c represents the effect on ferrous ion chelating power. Figure 9 The effect of different H2O2 contents on the viability of HepG2 cells; Figure 10 The effect of peptide concentration on the content of antioxidant enzymes (SOD, CAT, GSH) and ROS levels; Figure 11 The effect of peptide concentration on the levels of pathway regulatory proteins Keap1 and Nrf2; Figure 12 The effect of peptide concentration on the levels of anti-inflammatory factors IL-6 and IL-1β, as well as inflammatory pathway-related proteins IKBα and NF-κB; Figure 13 Diagram showing the docking of EVL with Keap1 and TLR2 proteins; Figure 14 Diagram showing the docking of VLPN with Keap1 and TLR2 proteins; Figure 15 This diagram shows the docking of LGPQ with Keap1 and TLR2 proteins. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0018] Specific Example 1: Preparation and Structural Characterization of Magnetic Nanomaterials 1. Preparation of magnetic materials Magnetic materials were synthesized using a solution oxidation method: 1.5 ml of ammonia was added to provide an alkaline environment, followed by 40 ml of ethanol and 90 ml of deionized water. 500 mg of Fe3O4 powder was dispersed in 20 ml of deionized water and added dropwise to the above solution, followed by sonication. Then, 500 mg of dopamine hydrochloride (DA·HCl) was dissolved in 10 ml of deionized water to form a solution, which was then slowly added dropwise to the sonicated reaction solution. The stirring rate was controlled, and the reaction was stopped at room temperature for 15 h, 18 h, 21 h, 24 h, and 27 h, respectively. The product was collected by high-speed centrifugation and freeze-dried for later use. Figure 1 As shown, the synthesized magnetic material exhibits good dispersibility in the aqueous phase and can be rapidly separated under an applied magnetic field.

[0019] The monkfish was skinned and filleted, then chopped and placed in centrifuge tubes. Formic acid solution was added at 0°C and extracted overnight at a ratio of 0.5% (sample:formic acid solution = 1:2). The mixture was then homogenized for 1 min at 10000 rpm using a high-speed homogenizer under ice bath conditions. The homogenate was then centrifuged in ultrapure water at 10000 rpm for 10 min at 4°C. The supernatant was collected as the supernatant. Magnetic materials obtained from different polydopamine (PDA) modification times were used to enrich endogenous peptides in the supernatant. The content of the enriched peptides was determined using a new BCA protein quantification kit. Figure 2 As shown, the results indicate that Fe3O4@PDA modified for 24 h exhibits the best enrichment effect.

[0020] 2. Transmission electron microscopy (TEM) analysis of magnetic materials Accurately weigh the prepared Fe3O4@PDA composite material and PDA polymer powder, and disperse them in anhydrous ethanol. Then, place the sample in an ultrasonic cleaner and sonicate at 100 W for 15 minutes to ensure sufficient and uniform dispersion of nanoparticles, avoiding agglomeration that could affect observation results. A standard carbon-supported copper mesh was selected as the sample carrier. Using a pipette, 10 μL of the ultrasonically homogenized sample dispersion was carefully and slowly added to the pretreated carbon-supported copper mesh surface. The copper mesh carrying the sample was then placed in a clean petri dish and allowed to air dry at room temperature to ensure complete solvent evaporation. After the sample was completely dry, the copper mesh was placed into the sample holder of the transmission electron microscope using tweezers and inserted into the main unit. The vacuum program was started, and observation began once the vacuum level inside the microscope reached the working standard. Representative fields of view were selected at different magnifications under accelerating voltages of 80-120 kV. At least six effective images were acquired for each sample in different regions.

[0021] The results are as follows Figure 3 TEM morphology shows that too short a modification time leads to insufficient PDA polymerization, a thin shell with low functional group density, and a small specific surface area. Conversely, too long a modification time, such as 27 h, results in an excessively thick PDA layer, causing active sites to be deeply buried, severe particle aggregation, and difficulty for peptide molecules to reach effective binding sites. In contrast, the magnetic material modified for 24 h exhibits a clear core-shell structure, with dark black Fe3O4 clearly visible within the light gray PDA, forming core-shell nanospheres Fe3O4@PDA with Fe3O4 clusters as the core. These nanospheres have a less regular morphology than the PDA, a slightly larger particle size, and a shell thickness of approximately 250 nm.

[0022] 3. Particle size and potential analysis of PDAs and magnetic materials The hydrodynamic particle size of Fe3O4 and its PDA-coated composite material was determined using dynamic light scattering (DLS). First, a trace amount of the sample powder was dispersed in ultrapure water to prepare a suspension of appropriate concentration. Then, the suspension was sonicated in an ice bath to ensure sufficient dispersion of nanoparticles and prevent agglomeration. Immediately after sonication, a suitable amount of the uniformly dispersed solution was transferred into a quartz cuvette and placed in the sample cell of the DLS particle size analyzer. After setting the measurement temperature (typically 25°C) and equilibration time, the measurement was started. The instrument automatically calculated the particle size distribution by analyzing the Brownian motion rate of the particles in the solution. Each sample was measured in triplicate to obtain the average value, thereby evaluating the particle size and dispersion stability of the material. Figure 4 As shown, the Fe3O4@PDA particles after 24 hours exhibit uniform size, with a hydrated particle size of approximately 243±5 nm. Smaller diameter particles possess a relatively larger specific surface area, thus exhibiting more adsorption active sites after functionalization.

[0023] To analyze the surface charge characteristics of the material, the zeta potential was determined using dynamic light scattering. Trace amounts of Fe3O4 and Fe3O4@PDA composite material were dispersed in a buffer solution at a specific pH. The dispersion was then subjected to ultrasonic treatment in an ice bath at 100W for 10 minutes to ensure complete deagglomeration and uniform dispersion of the nanoparticles. After ultrasonic treatment, the zeta potential was measured using a zeta potential analyzer. The effect of PDA modification on the surface properties of the material was evaluated by comparing the changes in zeta potential values ​​before and after coating. Figure 5 As shown, the Zeta potential of the magnetic material polymerized for 24 hours is approximately -24.6 mV, indicating that the material has high colloidal stability.

[0024] 4. Fourier transform infrared spectral analysis of PDAs and magnetic materials like Figure 6 As shown, the infrared absorption spectra of PDA and Fe3O4@PDA are quite similar, mainly exhibiting the characteristic absorption peaks of PDA, confirming the successful PDA coating. (3337 cm⁻¹) -1 The absorption peak at 1583 cm⁻¹ is attributed to the stretching vibrations of OH and NH. -1 The absorption peak at 580 cm⁻¹ is attributed to the stretching vibration of C=O; -1 The absorption peak at that location is attributed to Fe-O.

[0025] Specific Example 2: Study on the Enrichment of Anglerfish Endogenous Peptides with Magnetic Materials and Their Functional Activities Accurately weigh 50 mg of Fe3O4@PDA polymerized for 24 h, add 10 ml of PBS buffer to disperse the material, then add 10 ml of the supernatant of each fish meat homogenate (preparation method as described in Example 1 above), and incubate with shaking for 30 min. After magnetic separation, collect the sample solution. Add 2 mL of 100 mmol / L NaCl solution to the Fe3O4@PDA obtained after magnetic separation to elute the adsorbed endogenous peptides. Collect the eluent, dialyze it through a 100 Da dialysis bag to remove salt, and freeze-dry to obtain anglerfish endogenous peptides (hereinafter referred to as polypeptides), which are then stored for later use.

[0026] 1. In vitro antioxidant activity Anglerfish endogenous peptides were prepared at four concentrations (0.15, 0.25, 0.35, and 0.45 mg / mL). One ml of each sample was mixed with one ml of 0.1 mmol / L DPPH working solution. After reacting in the dark for 30 min, the absorbance of the mixture was measured at 517 nm. A tube containing one ml of 95% ethanol mixed with DPPH served as a blank control, and a tube containing one ml of 95% ethanol mixed with the sample served as a control. The free radical scavenging capacity of DPPH was calculated using the formula.

[0027] Add four concentrations of sample solution, salicylic acid-ethanol solution (9 mmol / L), FeSO4 solution (9 mmol / L), distilled water, and H2O2 (8.8 mmol / L) to clean test tubes, respectively. After mixing, heat in a water bath at 37 ℃ for 15 min, and measure the absorbance at 510 nm. Ascorbic acid was used as a positive control, and each sample was tested in triplicate. The scavenging rate of hydroxyl radicals was calculated using the formula.

[0028] After taking 120 μL of sample, 40 μL of 0.2 mM FeCl2 solution was added. The mixture was reacted at 37°C for 20 min. After the reaction, 40 μL of 5 mM phenanthroline solution was added, and the reaction was continued at room temperature for 10 min. The absorbance of the mixture was measured at 562 nm using a UV spectrophotometer. An equal volume of distilled water was used as a blank control. The ferrous ion chelation rate was calculated according to the formula.

[0029] like Figure 7 As shown, endogenous peptides from anglerfish exhibited significant antioxidant activity in in vitro antioxidant experiments, with the endogenous peptides showing the lowest IC50 value in the DPPH assay. 50 The value was 0.06 mg / ml in ·OH and 0.12 mg / ml in Fe. 2+ The concentration was 0.21 mg / ml.

[0030] like Figure 8As shown in Figure a, with the gradual increase of peptide concentration in the range of 0.1-0.5 mg / mL, it exhibited excellent antioxidant activity against DPPH free radicals, with the 0.35 mg / mL group showing the best scavenging effect, at 94.30%. Figure 8 As shown in Figure b, the hydroxyl radical scavenging rate increases with increasing peptide concentration. Figure 8 As shown in c, Fe 2+ The chelation rate was best at 0.35 mg / mL, reaching 79.06%.

[0031] 2. Validation of antioxidant function in HepG2 cells Establishment of HepG2 cell antioxidant model: Based on the H2O2-induced oxidative damage model of HepG2 cells, the cell viability of HepG2 cells was closest to 50% after 4 hours of induction with 500 μM H2O2. This represents the IC50 of H2O2-induced damaged cells. 50 It can both induce oxidative stress and prevent excessive cell death. The results are as follows... Figure 9 As shown, HepG2 cells induced with 500 μM H2O2 for 4 hours were used as a cell oxidation model for cellular antioxidant experiments.

[0032] Log-grown HepG2 cells were prepared into a single-cell suspension and diluted with 1×10⁻⁶ cells. 6 Cells were seeded at a density of [number] cells / mL in 6-well plates and cultured in a cell culture incubator at 37°C with 5% CO2. The medium was changed after 24 h. When the adherent area of ​​the cells reached approximately 50%, the culture medium was discarded, and blank, positive control, and experimental groups were set up. The positive control group was added to medium containing H2O2, and the experimental groups were added to medium containing different concentrations of anglerfish endogenous peptides and H2O2. The cells were incubated for 4 hours. When cells are subjected to oxidative stress, reactive oxygen species (ROS) are produced, which damage the cells. Intracellular ROS were labeled with fluorescent probes to quantify intracellular ROS production. The fluorescence intensity was detected by a microplate reader after the experiment.

[0033] After treatment, discard the culture medium and wash the cells twice with PBS. Add 200 μl of pre-chilled RIPA lysis buffer (containing 1% PMSF, freshly prepared and operated on ice) to each well and lyse on ice for 10 minutes. After lysis, repeatedly pipette to ensure detachment of adherent material. Transfer the lysis buffer to pre-chilled 1.5 mL centrifuge tubes and centrifuge at 4°C and 10,000 rpm for 15 minutes. Carefully aspirate the supernatant to new pre-chilled centrifuge tubes and use an ELISA kit to measure the activities of cellular antioxidant enzymes such as SOD, CAT, and GSH-Px, and use a Keap1 and Nrf2 assay kit to measure the content of antioxidant pathway-related proteins.

[0034] like Figure 10As shown, compared with the positive control, SOD activity, CAT content, and GSH content were significantly increased within the peptide concentration range of 0.1-0.5 mg / mL. Furthermore, peptide treatment significantly reduced H2O2-induced ROS levels, indicating that the peptide has a strong protective ability against oxidative damage in HepG2 cells. The Keap1-Nrf2 pathway is an important intracellular pathway related to antioxidation. Figure 11 As shown, Keap1, a key antioxidant sensing protein, is maintained at a basal level in total cell content. However, the peptide treatment group can interfere with the inhibition of Nrf2 by Keap1, causing Nrf2 to accumulate in the cell nucleus, thereby upregulating the content of intracellular antioxidant enzymes.

[0035] 3. Validation of anti-inflammatory function in RAW cells The cell density was diluted to 2 × 10⁻⁶ using fresh nutrient solution. 5 Cells were diluted to a concentration of 10 cells / ml and added to each well of a 6-well plate at 2 mL. The plates were then incubated overnight in a cell culture incubator (5% CO2, 37℃). RAW264.7 macrophages were divided into a blank control group, an LPS-positive control group, and an endogenous peptide experimental group, with three replicates per group. The blank control group was incubated with RAW cell-specific culture medium; the LPS group was incubated with RAW cell-specific culture medium and 1 μg / mL LPS; the LPS+endogenous peptide group was incubated with cell culture medium containing 1 μg / mL LPS, and four different concentrations of endogenous peptides were added to the culture medium. The cells were incubated at ambient oxygen, 5% CO2, and 37℃ for 24 hours. Cell culture supernatants were collected. The levels of inflammatory factors IL-1β and IL-6, as well as the contents of inflammatory pathway-related proteins IKBα and NF-κB, in the cell supernatants of each group were measured according to the instructions of the corresponding ELISA kits. Figure 12 As shown, the number of intracellular inflammatory factors and inflammatory pathway proteins in the experimental group was significantly lower than that in the control group, indicating that the endogenous peptides of anglerfish have a good inhibitory effect on cellular inflammatory factors.

[0036] 4. Active fragment sequence analysis and screening Peptide sequences were identified using LC-MS / MS, and peptide sequences with high overall scores were selected. The bioactivity of endogenous peptides was assessed using the online software Peptide Ranker, water solubility was predicted using the online software Inovagen, and antioxidant activity was predicted using the online software AnOxPePred, which can predict the free radical scavenging and metal ion chelating abilities of peptides. As shown in Table 1, six peptides that met the expected criteria were finally selected.

[0037] Table 1 Active Fragment Scoring

[0038] 5. Molecular docking Molecular docking was performed using AutoDock Tools 1.5.7 to analyze protein-peptide interactions. The oxidative stress target Keap1 protein (PDB ID: 2FLU) and the anti-inflammatory protein TLR2 were selected and docked with six active fragments. PyMOL 2.5.4 software was used to remove the original ligands and all water molecules, and hydrogen atoms were added. The pretreated proteins were defined as target receptors. Chem3D 20.0 software was used to construct peptide structures and perform energy minimization to optimize their structural stability. The treated peptides were classified as ligands for subsequent molecular docking experiments. Docking parameters were set, and after docking, the most stable protein-peptide complex conformations were screened. The model with the lowest docking binding energy was selected, as lower docking energy usually indicates a more stable binding state. Finally, PyMOL software was used to visually view and plot the docking results, and to analyze the details of the protein-peptide interactions in depth. The results are shown in Table 2.

[0039] Table 2 Molecular docking binding energy

[0040] Table 2 shows the binding energies of the six active fragments to Keap1 and TLR2 proteins, respectively. It can be seen that all six active fragments can form protein-peptide complexes with low binding energies, among which EVL (… Figure 13 As shown), VLPN ( Figure 14 As shown), LGPQ ( Figure 15 (As shown) The binding energies of the three peptide segments are relatively low. Theoretically, this confirms that these peptides possess excellent antioxidant and anti-inflammatory potential. The molecular docking results show that Keap1 and TLR2 proteins bind to the peptides through hydrogen bonds, ionic bonds, etc., exerting potential antioxidant and anti-inflammatory activities.

[0041] In summary, this invention successfully constructed a high-performance Fe3O4@PDA magnetic adsorption material. Characterization results showed that when the polymerization time was 24 h, the prepared material had a uniform particle size, stable at around 246 nm. Fourier transform infrared spectroscopy, transmission electron microscopy, and Zeta potential analysis jointly confirmed that a complete and uniform polydopamine coating layer was formed on the material surface, exhibiting stable negative charge. Adsorption experiments further demonstrated that this material exhibited excellent selective adsorption capacity for endogenous peptides in monkfish meat, with a significantly improved adsorption efficiency compared to materials prepared at other polymerization times. In terms of functional evaluation, chemical antioxidant experiments showed that the isolated endogenous peptides had strong free radical scavenging ability in vitro. In cell models, this peptide effectively alleviated H2O2-induced oxidative damage in HepG2 cells, exhibiting good cellular-level antioxidant activity; simultaneously, in an LPS-induced RAW264.7 macrophage inflammation model, peptide treatment significantly reduced the release of inflammatory factors and inhibited the expression of inflammatory pathway proteins. Through peptidomics analysis, we successfully identified several endogenous peptide sequences. Molecular docking results showed that these peptides can efficiently bind to key target proteins closely related to oxidative stress and inflammatory responses, such as Keap1 and TLR2, explaining the structural basis of their significant antioxidant and anti-inflammatory activities at the molecular level.

[0042] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. An endogenous peptide from monkfish with antioxidant and anti-inflammatory activity, characterized in that: The anglerfish endogenous peptides mentioned include at least one of HPLK, PRK, VLPN, LGPQ, EVL, and LGYPPQ.

2. The anglerfish endogenous peptide with antioxidant and anti-inflammatory activity according to claim 1, characterized in that: The anglerfish endogenous peptide mentioned is one of EVL, VLPN, and LGPQ.

3. A method for preparing endogenous peptides from monkfish according to claim 1, characterized in that... The process includes the following steps: Skinning and removing the flesh from monkfish, mincing the flesh and placing it in a centrifuge tube, adding 0.2-1 wt% formic acid solution at a volume ratio of 1:(1-3) at 0-4℃ for overnight extraction, homogenizing with a high-speed homogenizer under ice bath conditions; balancing the centrifuge tube with ultrapure water, centrifuging at 0-4℃ to collect the supernatant, obtaining the supernatant of the fish flesh sample; adding a 5 mg / mL Fe3O4@PDA magnetic material solution dispersed in PBS buffer at a volume ratio of 1:(1-3) to the supernatant of the fish flesh sample, shaking and incubating, magnetically separating, and collecting the Fe3O4@PDA sample; adding NaCl solution to the Fe3O4@PDA sample to elute the adsorbed endogenous peptides, collecting the eluent, dialyzing with a 100 Da dialysis bag to remove salt, and freeze-drying to obtain monkfish endogenous peptides.

4. The method for preparing endogenous peptides from monkfish according to claim 3, characterized in that: The preparation method of the Fe3O4@PDA magnetic material is as follows: Ammonia water, anhydrous ethanol, deionized water and Fe3O4 solution with a concentration of 20-30 mg / mL are mixed in a volume ratio of (1-2):40:90:20 and ultrasonically treated until uniformly dispersed. Then, while stirring, DA·HCl solution with a concentration of 40-60 mg / mL is slowly added dropwise. The reaction is carried out at room temperature for 15-27 h. The product is collected by high-speed centrifugation and freeze-dried in a freeze dryer to obtain Fe3O4@PDA magnetic material, wherein the volume ratio of Fe3O4 solution to DA·HCl solution is 2:

1.

5. The method for preparing endogenous peptides from anglerfish according to claim 4, characterized in that: The reaction was carried out at room temperature for 24 hours.

6. The use of the anglerfish endogenous peptide of claim 1 in the preparation of an antioxidant.

7. The application according to claim 6, characterized in that: The use of the said anglerfish endogenous peptide in the preparation of an in vitro antioxidant, said in vitro antioxidant comprising a DPPH radical scavenger, a Fe 2+ chelator and a ·OH scavenger.

8. The application according to claim 6, characterized in that: The application of the aforementioned anglerfish endogenous peptides in the preparation of in vivo antioxidants, wherein the in vivo antioxidants include SOD, CAT, and GSH-Px antioxidant enzyme activity promoters.

9. The application of the anglerfish endogenous peptide according to claim 1 in the preparation of an anti-inflammatory agent, characterized in that: The anti-inflammatory agents include inhibitors of IL-1β and IL-6 inflammatory factors.

10. The application according to claim 9, characterized in that: In an LPS-induced RAW264.7 cell inflammation model, endogenous peptide treatment significantly reduced the release of IL-1β and IL-6 inflammatory factors.

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