Apostichopus japonicus collagen peptide and application thereof in preparation of antioxidant product

Abstract

The invention relates to an apostichopus japonicus collagen peptide and application of the apostichopus japonicus collagen peptide to preparation of an antioxidant product, and the method comprises the following steps: solid-phase synthesis of the apostichopus japonicus collagen peptide is carried out, and the amino acid sequence of the apostichopus japonicus collagen peptide is shown as SEQ ID NO: 1; the apostichopus japonicus collagen peptide prepared by the method disclosed by the invention is an apostichopus japonicus collagen peptide with antioxidant activity.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number is 202411847105.0, the application date is December 16, 2024, and the patent name at the time of application is: A sea cucumber-like collagen peptide and its application. Technical Field

[0002] The present invention belongs to the technical field of biomedicine, and in particular relates to a sea cucumber-like collagen peptide and an application thereof in preparing antioxidant products. Background Art

[0003] Type 2 diabetes mellitus (T2DM) is a typical chronic non-communicable disease characterized by hyperglycemia and insulin resistance. According to the IDF Diabetes Atlas (2021), approximately 537 million people (aged 20-79) worldwide suffer from diabetes, of which 90% suffer from T2DM.

[0004] Bioactive peptides are considered to be one of the promising drugs for the treatment of chronic diseases. They are nutritionally comprehensive, easily absorbed, and have diverse functions. The functions of bioactive peptides with different amino acid sequences may vary. The body wall of sea cucumbers contains 40–60% protein (dry weight), of which collagen accounts for more than 70%. Traditional methods for preparing bioactive peptides are affected by various environmental factors such as enzyme reaction conditions, substrates, and purification methods. It is difficult to obtain target peptides efficiently and the reproducibility is low. If new bioactive peptides with antioxidant and hypoglycemic dual functions can be screened and prepared from sea cucumber collagen, it will also be of great significance for the development of dietary nutrition and biopharmaceutical products. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a sea cucumber-like collagen peptide and its application in preparing antioxidant products. The sea cucumber-like collagen peptide prepared by the present invention has both antioxidant and hypoglycemic activities.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a sea cucumber collagen-mimicking peptide, comprising the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 can specifically be Asp-Cys-Asp-Pro-Arg-Leu (DCDPRL).

[0008] The beneficial effects of adopting the above technical solution include: the active peptide DCDPRL can stably bind to the three targets GLP-1R, GIPR and Keap1 through hydrogen bonds, salt bridges, hydrophobic interactions and van der Waals forces. The active peptide DCDPRL has a good DPPH free radical scavenging ability (IC50 =206.12 μM) and ABTS free radical scavenging ability (IC 50 =186.06 μM). At the same time, the active peptide DCDPRL has a good blood sugar lowering ability. In a hyperglycemic zebrafish model experiment, the blood sugar lowering ability of DCDPRL (250 μM) was 60.64%±4.82%.

[0009] The present invention provides a method for preparing the above-mentioned sea cucumber collagen-mimicking peptide, comprising the following method: solid-phase synthesis of the sea cucumber collagen-mimicking peptide having the above-mentioned amino acid sequence.

[0010] The beneficial effects of adopting the above technical solution include: the sea cucumber collagen peptide prepared by the above method has the advantages of strong antioxidant capacity and good blood sugar lowering activity.

[0011] The present invention provides the use of the sea cucumber collagen peptide in anti-oxidation and / or blood sugar reduction.

[0012] The sea cucumber collagen peptide can be used to prepare antioxidant products and / or blood sugar-lowering products.

[0013] Application of the above-mentioned sea cucumber imitation collagen peptide in the preparation of antioxidant health food.

[0014] Application of the above-mentioned sea cucumber imitation collagen peptide in the preparation of health food for assisting in lowering blood sugar.

[0015] Products include but are not limited to food, health products, medicines, dietary supplements, biopharmaceutical products, etc.

[0016] The present invention provides a method for screening sea cucumber collagen-like peptides having both antioxidant and hypoglycemic activities, comprising the following steps:

[0017] (1) Multiple sea cucumber collagen sequences, including AYL88761.1, were selected for homology analysis;

[0018] (2) AYL88761.1 and sequences with homology analysis scores lower than 50 with AYL88761.1 were subjected to virtual enzymatic digestion;

[0019] (3) By comparing the bioactive peptides in the BIOPEP-UWM database, unknown bioactive peptides after virtual enzymatic hydrolysis were screened, with the peptide size ranging from 2 to 20 amino acids; bioactivity prediction was performed using the Peptide Ranker website, and peptide fragments with a Peptide Ranker score greater than 0.8 were screened;

[0020] (4) Using Discovery Studio software, the peptide fragments in (3) were molecularly constructed and the energy of the junction was optimized, and the target protein in the PDB library was pre-processed;

[0021] (5) Select the high-throughput screening program LibDock in the Discovery Studio software, use the peptide molecule in step (4) as the ligand, and the pre-treated target protein in the PDB library as the receptor to perform preliminary molecular docking;

[0022] (6) The docking results with LibDock scores greater than 150 were selected and optimized using the semi-flexible screening program CDOCKER with built-in CHARMM force field;

[0023] (7) screening the results optimized in step (6) according to the -CDOCKER ENERGY value and selecting the active peptide;

[0024] (8) The physicochemical properties of active peptides were predicted using the ProtParam tool, and pharmacokinetic evaluation was performed using the ADMETlab 3.0 tool. Sea cucumber collagen-like peptides were screened based on intestinal stability, intestinal utilization, and non-toxicity;

[0025] (9) Experimental verification of the antioxidant activity and hypoglycemic activity of the sea cucumber collagen peptide obtained in step (8).

[0026] Furthermore, in step (1), the IDs of the multiple sea cucumber collagen sequences are: AYL88761.1, PIK36494.1, PIK37246.1, PIK38678.1, PIK42190.1, PIK42317.1, PIK42491.1, PIK44932.1, PIK50307.1, PIK55424.1, PIK56377.1, PIK59223.1, PIK60691.1, PIK60692.1, PIK60693.1, PIK60696.1, PIK62545.1, and PIK62546.1.

[0027] Furthermore, in step (2), the enzyme used for virtual enzymatic hydrolysis is one or more of pepsin, trypsin, and α-chymotrypsin.

[0028] Furthermore, the target proteins include GLP-1R (ID: 7LCK), GIPR (ID: 7FIY) and Keap1 (ID: 5WFV).

[0029] Furthermore, the sea cucumber collagen-mimicking peptide obtained by the above method includes the amino acid sequence shown in SEQ ID NO: 1.

[0030] The beneficial effects of the above technical solution include: combining bioinformatics and molecular docking techniques to efficiently screen novel bioactive peptides with dual antioxidant and hypoglycemic functions from Apostichopus japonicus collagen. The resulting sea cucumber collagen peptide DCDPRL exhibits strong antioxidant and hypoglycemic activity and can be used in the development of dietary nutrition and biopharmaceutical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the alignment information of 18 collagen sequences from Apostichopus japonicus.

[0032] Figure 2 is the pharmacokinetic prediction result of the peptide, where, within the range of 0-1, the larger the value, the stronger the positive correlation.

[0033] Figure 3 These are the molecular docking results of the sea cucumber collagen peptide DCDPRL. From top to bottom, they are the docking results of DCDPRL with the targets GLP-1R, GIPR, and Keap1.

[0034] Figure 4 The in vitro antioxidant activity of sea cucumber collagen peptide DCDPRL is shown in Figure 2. The ordinates represent the DPPH and ABTS free radical scavenging abilities, the abscissa represents the molar concentration of the sample, and glutathione (GSH) is used as the positive control.

[0035] Figure 5 It is a zebrafish embryo toxicity modeled after the sea cucumber collagen peptide DCDPRL.

[0036] Figure 6 This is the in vivo hypoglycemic activity of the sea cucumber collagen peptide DCDPRL, where the vertical axis is the blood glucose content and the horizontal axis is the different treatment groups. DETAILED DESCRIPTION

[0037] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the art to which the present invention pertains. As used herein, the terms "including," "having," "containing," etc. are open-ended terms, meaning including but not limited to.

[0038] The present invention utilizes bioinformatics and computer virtual screening to screen sea cucumber collagen-like active peptides and evaluate their in vivo and in vitro activities and mechanisms of action. Through screening and experimental verification, a sea cucumber collagen-like peptide with both antioxidant and hypoglycemic activities was obtained, the amino acid sequence of which is Asp-Cys-Asp-Pro-Arg-Leu (DCDPRL), as shown in SEQ ID NO: 1. The sea cucumber collagen-like peptide is an active peptide with both antioxidant and hypoglycemic activities, acting on multiple targets (GLP-1R, GIPR and Keap1), and stably binding to multiple targets through non-bonded interactions. In vitro, it has a strong DPPH free radical scavenging ability (IC 50 =206.12 μM) and ABTS free radical scavenging ability (IC 50 =186.06 μM). It has a good hypoglycemic effect in a hyperglycemic zebrafish model (the hypoglycemic capacity of 250 μM DCDPRL is 60.64%±4.82%), and has the advantages of being safe, having no toxic side effects, and having good water solubility.

[0039] The sea cucumber-like collagen peptide provided by the present invention has both antioxidant and hypoglycemic activities, and its raw material is derived from the Japanese sea cucumber (Apostichopus japonicus) collagen. The sea cucumber-like collagen peptide can be obtained by screening using the following method:

[0040] (1) Virtual screening of sea cucumber collagen peptides

[0041] Firstly, homology analysis was performed on 18 sea cucumber-like collagen sequences in the PDB library, and the screened sea cucumber-like collagen sequences were subjected to virtual enzymatic hydrolysis using the following enzymes: pepsin (pH 1.3), trypsin, and α-chymotrypsin (A).

[0042] Novel enzymatic peptide fragments (2–20 amino acids) with unknown activity were further selected for bioactivity prediction, and peptide fragments with a Peptide Ranker score greater than 0.8 were screened. Molecular construction and energy optimization of the peptide fragments were then performed using Discovery Studio software. Preliminary molecular docking was performed using the high-throughput screening program LibDock within Discovery Studio, using the peptide molecules as ligands and the optimized target proteins GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV) from the PDB library as receptors.

[0043] We then selected docking results with high LibDock scores and optimized them using CDOCKER, a semi-flexible precision screening program with a built-in CHARMM force field. The optimized results were screened based on the -CDOCKER ENERGY value, and the top 5-10 active peptides were selected for pharmacokinetic evaluation. Active peptides with excellent properties such as high intestinal stability, high intestinal utilization, and non-toxicity were selected. Active peptides were screened by chemical solid-phase synthesis.

[0044] (2) Verification of the in vitro antioxidant activity of sea cucumber collagen peptide

[0045] The DPPH free radical scavenging ability was determined by the following steps: a 2.0 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) working solution was prepared in 95% ethanol solution and mixed with an equal volume of sample in triplicate to a final volume of 200.0 μL. After incubation in the dark for 30 minutes, the absorbance was measured at a wavelength of 517 nm using glutathione (GSH) as a positive control. The calculation formula (1) for DPPH free radical scavenging ability is as follows:

[0046]

[0047] The absorbance of 100.0 μL of sample mixed with 100.0 μL of working solution is As, the absorbance of 100.0 μL of sample mixed with 100.0 μL of 95% ethanol solution is Ab, and the absorbance of 100.0 μL of working solution mixed with 100.0 μL of water is Ac.

[0048] The ABTS free radical scavenging ability was determined, including the following steps: 0.7 mM 2,2'-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2.45 mM K2S2O8 stock solutions were prepared, the two solutions were mixed in equal volumes, and the mixture was incubated overnight at 37°C in the dark. The mixed stock solution was diluted with 80% ethanol solution until the absorbance at 743 nm reached 0.700 ± 0.050, indicating that the working solution was successfully prepared. Subsequently, 200.0 μL of the working solution and 20.0 μL of the sample were mixed and incubated in the dark for 6 minutes in triplicate. The absorbance was measured at a wavelength of 734 nm, and the ABTS RSC was calculated using the following formula (2):

[0049]

[0050] The absorbance after mixing 20.0 μL of sample with 200.0 μL of working solution is As, the absorbance after mixing 20.0 μL of sample with 200.0 μL of 80% ethanol solution is Ab, and the absorbance after mixing 200.0 μL of working solution with 20.0 μL of water is Ac.

[0051] (3) Verification of embryotoxicity and hypoglycemic activity of sea cucumber collagen peptide

[0052] Zebrafish embryos (wild-type AB) at 2 days post fertilization (2 dpf) were placed in 12-well plates (20 embryos per well) at 28°C. Both the blank control group (E3) and the sample group were cultured in triplicate. In addition, the culture medium was changed every day until the 6th day after fertilization (6 dpf), and dead embryos or larvae were immediately removed. The morphological changes of zebrafish after drug administration were observed and recorded using the MSHOT imaging system (MD50, Guangzhou), and the embryo survival rate and hatching rate were calculated 6 days after drug administration.

[0053] 5-dpf zebrafish larvae (wild-type AB) were selected and placed in six-well plates (30 per well) after microscopic observation and normal development. Subsequently, the zebrafish were cultured at 28°C for 24 hours using an optimal combination of glucose and alloxan concentrations to establish a hyperglycemic zebrafish model. The larvae were then transferred to solutions containing different peptide concentrations as experimental groups, while a solution containing metformin served as a positive control. Following the incubation period, the larvae were rinsed with distilled water and collected into 1.5 mL centrifuge tubes (10 per tube). 200 μL of ethanol was added to each tube at room temperature for 2 minutes, followed by drying in a 60°C oven for 2 hours. The larvae were then homogenized with 50 μL of distilled water per tube and centrifuged at 8000 × g for 5 minutes. Glucose content in zebrafish larvae tissues was measured using a glucose assay kit (Solarbio, Beijing, China) and expressed as unit protein concentration using a BCA protein assay kit (Biyuntian, Shanghai, China).

[0054] Unless otherwise specified, the reagents involved in the examples of the present invention were purchased from commercial channels or prepared using conventional methods in the field. All methods not mentioned are conventional experimental methods in the field. The solutions of the present invention, unless otherwise specified, were prepared using water as the solvent.

[0055] Zebrafish embryos (wild-type AB) were purchased from Shanghai Feixi Biotechnology Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 2,2'-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and E3 culture medium was purchased from Shanghai Feixi Biotechnology Co., Ltd.

[0056] The following is an introduction through specific embodiments.

[0057] Example 1 Virtual screening of sea cucumber collagen peptides with both antioxidant and hypoglycemic activities

[0058] (1) Sequence alignment and homology analysis

[0059] The present invention used the Multiple Alignment Tool in BLAST tool in NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to perform sequence alignment and homology analysis on 18 sea cucumber collagen proteins (Table 1). The experimental results are as follows: Figure 1 As shown, homology analysis revealed that the alignment scores for sequences AYL88761.1 and PIK62545.1 were 97.6532, and for sequences AYL88761.1 and PIK62546.1 were 92.2705. Homology scores between the other sequences were less than 50. This indicates that AYL88761.1 shares most of the sequences of PIK62545.1 and PIK62546.1 and is their protoprotein. Therefore, subsequent experiments will focus on the remaining 16 collagen sequences (Table 1), excluding PIK62545.1 and PIK62546.1.

[0060] Table 1 18 collagen sequences from Apostichopus japonicus

[0061]

[0062]

[0063] (2) Virtual enzymatic hydrolysis

[0064] Using the Batch Processing tool in the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), 16 sea cucumber (Apostichopus japonicus) collagen sequences were subjected to single and combined virtual enzymatic digestion with three gastrointestinal enzymes: pepsin (pH 1.3), trypsin, and α-chymotrypsin (A). Following single and combined virtual digestion with three enzymes, 2,647 unknown bioactive peptides (2–20 amino acids) were identified by comparison with bioactive peptides in the BIOPEP-UWM database. The bioactivities of these peptide fragments were initially predicted using the Peptide Ranker website (http: / / distilldeep.ucd.ie / PeptideRanker / ). 207 of these peptide fragments achieved Peptide Ranker scores greater than 0.8.

[0065] (3) Molecular docking

[0066] The peptide fragments were molecularly constructed and the junction energy was optimized using Discovery Studio software, and then the target proteins GLP-1R (ID: 7LCK), GIPR (ID: 7FIY) and Keap1 (ID: 5WFV) in the PDB library were pre-processed by dehydration, hydrogenation and energy optimization.

[0067] Preliminary molecular docking was performed using the high-throughput screening program LibDock, using the peptide as the ligand and the target proteins as the receptor. The target proteins were GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV), respectively. The active pocket positions (X, Y, Z) for GLP-1R docking were 126.769, 161.247, and 117.843, with a radius parameter of 15. The active pocket positions (X, Y, Z) for GIPR docking were 133.042, 142.472, and 172.855, with a radius parameter of 10. The active pocket positions (X, Y, Z) for Keap1 docking were -44.2235, -9.27174, and 64.8752, with a radius parameter of 10.

[0068] In order to further accurately screen bioactive peptides with both antioxidant and hypoglycemic properties, docking results with LibDock scores greater than 150 were selected and optimized using the semi-flexible precision screening program CDOCKER with a built-in CHARMM force field. The optimized results were screened according to the -CDOCKER ENERGY value. Only 7 active peptides (DCDPRL, DPWVF, GPMGLK, HFR, PRGF, RSIPF, and WHR) were successfully docked. The ProtParam tool (http: / / web.expasy.org / protparam / ) was used to predict their physicochemical properties (Table 2). Among them, DCDPRL, GPMGLK, HFR, PRGF, and WHR are all hydrophilic peptides. During the experiment, it was also found that DCDPRL dissolves in water. Pharmacokinetic evaluation was then performed using the ADMETlab 3.0 tool, and the results are as follows: Figure 2 As shown, within the range of 0-1, the larger the value, the stronger the positive correlation. It can be seen that the peptide DCDPRL has the excellent characteristics of high intestinal utilization and non-toxicity.

[0069] Table 2 Prediction results of physicochemical activities of 7 active peptides

[0070]

[0071] *: If the average hydrophilicity coefficient is less than 0, it means the molecule is hydrophilic, otherwise it is hydrophobic.

[0072] Table 3 shows the docking scores of seven peptides with different targets. The molecular docking results in Table 3 indicate that among the seven peptides, DCDPRL has the best binding ability scores with multiple targets (GLP-1R / GIPR / Keap1) and the highest stability, with -CDOCKERENERGY values ​​of 104.025 kcal / mol, 90.0051 kcal / mol, and 112.037 kcal / mol, respectively. Table 4 provides a detailed description of the docking results of the peptide DCDPRL with the targets (GLP-1R / GIPR / Keap1). High binding energy indicates strong affinity between the receptor and ligand. The peptide DCDPRL forms favorable binding with GLP-1R / GIPR / Keap1 through hydrogen bonds, salt bridges, hydrophobic interactions, and van der Waals forces. Figure 3 These are the 3D and 2D interaction diagrams of the peptide DCDPRL with multiple targets.

[0073] Table 3 Docking results of 7 peptides with GLP-1R / GIPR / Keap1

[0074]

[0075] Table 4 Docking results of peptide DCDPRL with multi-target GLP-1R / GIPR / Keap1

[0076]

[0077]

[0078] Subsequently, the active peptides screened by chemical solid-phase synthesis were commissioned to Sangon Biotechnology Co., Ltd. (Shanghai, China) for the next step of in vitro and in vivo activity verification.

[0079] Example 2 Verification of the in vitro antioxidant activity of seven synthesized sea cucumber collagen peptides

[0080] The DPPH free radical scavenging ability was determined by the following steps: a 2.0 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) working solution was prepared in a 95% ethanol solution and mixed with equal volumes of peptide solutions of different concentrations (using water as the solvent) in triplicate, with a final volume of 200.0 μL. The peptide concentrations in the peptide solutions were 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM, respectively. After incubation in the dark for 30 minutes, the absorbance was measured at a wavelength of 517 nm using glutathione (GSH) as a positive control. The calculation formula (1) for the DPPH free radical scavenging ability is as follows:

[0081]

[0082] The absorbance of 100.0 μL of sample mixed with 100.0 μL of working solution is As, the absorbance of 100.0 μL of sample mixed with 100.0 μL of 95% ethanol solution is Ab, and the absorbance of 100.0 μL of working solution mixed with 100.0 μL of water is Ac.

[0083] The ABTS free radical scavenging ability was determined by the following steps: 0.7 mM 2,2'-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2.45 mM K2S2O8 stock solutions were prepared respectively, and the two solutions were mixed in equal volumes and incubated overnight in the dark at 37°C. The mixed stock solution was diluted with 80% ethanol solution until the absorbance at a wavelength of 743 nm reached 0.700±0.050, indicating that the working solution was successfully prepared. Subsequently, 200.0 μL of the working solution and 20.0 μL of peptide solutions of different concentrations were mixed and incubated in the dark for 6 minutes in triplicate. The peptide concentrations in the peptide solutions were 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM, respectively. Glutathione (GSH) was used as a positive control. The absorbance was measured at a wavelength of 734 nm, and the ABTS RSC was calculated using the following formula (2):

[0084]

[0085] The absorbance after mixing 20.0 μL of sample with 200.0 μL of working solution is As, the absorbance after mixing 20.0 μL of sample with 200.0 μL of 80% ethanol solution is Ab, and the absorbance after mixing 200.0 μL of working solution with 20.0 μL of water is Ac.

[0086] The experimental results are shown in Table 5 and Figure 4 As shown in Figure 2, it can be seen that the sea cucumber collagen peptide DCDPRL has a strong DPPH free radical scavenging ability (IC 50 =206.12 μM) and ABTS free radical scavenging ability (IC 50 =186.06 μM).

[0087] Table 5 In vitro antioxidant activities of seven sea cucumber collagen peptides

[0088]

[0089] Note: Table 5 shows the experimental results when the molar concentration of the sea cucumber collagen peptide is 4 mM.

[0090] Example 3 Detection of Embryotoxicity and In Vivo Hypoglycemic Activity of Sea Cucumber Collagen-Imitating Peptide DCDPRL

[0091] (1) Embryotoxicity: Zebrafish embryos (wild-type AB) at 2 days post fertilization (2 dpf) were placed in 12-well plates (20 embryos per well) at 28°C. The blank control group (E3 culture medium, Normal) and the sample group (E3 culture medium + peptide) were all performed in triplicate. In addition, the culture medium was changed every day until the 6th day after fertilization (6 dpf), and dead embryos or larvae were immediately removed. The morphological changes of zebrafish after administration were observed and recorded using the MSHOT imaging system (MD50, Guangzhou), and the embryo survival rate and hatching rate after 6 days of administration were calculated.

[0092] from Figure 5 It can be seen that low concentrations of the peptide DCDPRL can gradually promote the hatching process of zebrafish embryos without affecting their survival. The survival curve shows that even after 96 hours of culture, the survival rate of zebrafish exposed to high concentrations of DCDPRL remains around 90%, indicating that zebrafish are highly sensitive to the drug and that the peptide DCDPRL is non-toxic to zebrafish larvae.

[0093] (2) Hypoglycemic activity: 5dpf zebrafish larvae (wild-type AB) with normal development were selected under a microscope and placed in a six-well plate (30 per well). Subsequently, the optimal concentration combination of 10mM glucose solution and 0.1mM alloxan solution was used and cultured at 28°C for 24 hours to establish a hyperglycemic zebrafish model. Peptide solutions of different concentrations (0.05mM, 0.1mM, 0.25mM) were transferred as the experimental group, and the solution containing metformin (metformin concentration of 0.25mM) was used as the positive control group. Normal group: E3 culture medium; Model group: 10mM glucose + 0.1mM alloxan + E3 culture medium; Experimental group: 10mM glucose + 0.1mM alloxan + different concentrations of peptide + E3 culture medium; Positive control group: 10mM glucose + 0.1mM alloxan + 0.25mM metformin + E3 culture medium. All the above reagents were prepared based on E3 culture medium.

[0094] After incubation, zebrafish larvae were rinsed with distilled water and collected into 1.5 mL centrifuge tubes (10 per tube). 200 μL of anhydrous ethanol was added to each tube and allowed to stand at room temperature for 2 minutes. The tubes were then transferred to a 60°C oven and dried for 2 hours. The larvae were then homogenized by adding 50 μL of distilled water to each tube and centrifuged (8000 × g) for 5 minutes. Glucose levels in zebrafish larvae tissues were measured using a glucose assay kit (Solar Bio, Beijing, China) and expressed as unit protein concentration using a BCA protein assay kit (Biyuntian, Shanghai, China).

[0095] The experimental results are as follows Figure 6 As shown. Figure 6As can be seen, the DCDPRL peptide at concentrations ranging from 50 to 250 μM can lower blood glucose levels. Incubation with a low concentration of DCDPRL (50 μM) resulted in a moderate decrease in blood glucose levels in hyperglycemic zebrafish larvae. The decrease in blood glucose levels became more pronounced with increasing DCDPRL concentrations. DCDPRL at 250 μM significantly lowered blood glucose levels in hyperglycemic zebrafish larvae, with a blood glucose reduction rate of 60.64% ± 4.82%.

[0096] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that for ordinary technicians in this technical field, various changes and modifications can be made without departing from the spirit and scope of the present invention. These changes and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing sea cucumber-like collagen peptide, characterized in that: The following steps are involved: The sea cucumber collagen-like peptide was synthesized in solid phase. The amino acid sequence of the sea cucumber collagen-like peptide is shown in SEQ ID NO:

1.

2. The application of sea cucumber collagen peptide in the preparation of antioxidant health food is characterized in that: The amino acid sequence of the sea cucumber collagen-mimicking peptide is shown in SEQ ID NO:

1.

3. The application of sea cucumber collagen peptide in the preparation of auxiliary blood sugar lowering health food, characterized in that: The amino acid sequence of the sea cucumber collagen-mimicking peptide is shown in SEQ ID NO: 1.

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