An Apostichopus japonicus collagen peptide and its application

Abstract

The present invention relates to an Apostichopus japonicus collagen peptide and its application. The amino acid sequence of the Apostichopus japonicus collagen peptide is Asp-Cys-Asp-Pro-Arg-Leu (DCDPRL), which is an Apostichopus japonicus collagen peptide with both antioxidant and hypoglycemic activities. The present invention combines bioinformatics and molecular docking techniques to virtually screen novel bifunctional bioactive peptides with antioxidant and hypoglycemic activities from the collagen of Apostichopus japonicus. The Apostichopus japonicus collagen peptide DCDPRL screened in the present invention has the advantages of strong antioxidant ability and good hypoglycemic activity. The research on multi-target bioactive peptides carried out in the present invention can be applied to fields such as dietary nutrition and biomedical products.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a sea cucumber (Apostichopus japonicus) collagen peptide and its application. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a typical chronic non-communicable disease, manifested as hyperglycemia and insulin resistance. According to the IDF Diabetes Atlas (2021), it is reported that approximately 537 million people (aged 20 - 79 years) globally suffer from diabetes, and 90% of them are T2DM patients.

[0003] Bioactive peptides are considered to be one of the promising drugs for the treatment of chronic diseases, with the characteristics of comprehensive nutrition, easy absorption, and 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), and more than 70% of which is collagen. Traditional methods for preparing bioactive peptides are affected by various environmental factors such as enzyme reaction conditions, substrates, and purification methods, making it difficult to efficiently obtain the target peptide with low reproducibility. If novel antioxidant and hypoglycemic dual-functional bioactive peptides can be screened from sea cucumber (Apostichopus japonicus) collagen, it will also be of great significance for the development of biomedical products. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a sea cucumber (Apostichopus japonicus) collagen peptide and its application, and this sea cucumber (Apostichopus japonicus) collagen peptide has both antioxidant and hypoglycemic activities.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a sea cucumber (Apostichopus japonicus) collagen peptide, including 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).

[0007] The beneficial effects of adopting the above technical solution include: The bioactive peptide DCDPRL can stably bind to three targets GLP-1R, GIPR, and Keap1 through hydrogen bonds, salt bridges, hydrophobic interactions, and van der Waals forces. The bioactive peptide DCDPRL has good DPPH free radical scavenging ability (IC 50 = 206.12 μM) and ABTS free radical scavenging ability (IC 50 = 186.06 μM). At the same time, the bioactive peptide DCDPRL has good hypoglycemic ability, and the hypoglycemic ability of DCDPRL (250 μM) in the hyperglycemic zebrafish model experiment is 60.64% ± 4.82%.

[0008] The present invention provides the above-mentioned method for preparing sea cucumber collagen peptide, including the following steps: solid-phase synthesis of sea cucumber collagen peptide with the above-mentioned amino acid sequence.

[0009] 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 ability and good hypoglycemic activity.

[0010] The present invention provides the application of the above-mentioned sea cucumber collagen peptide in antioxidant and / or hypoglycemic aspects.

[0011] The above-mentioned sea cucumber collagen peptide can be used for preparing antioxidant products and / or hypoglycemic drugs.

[0012] The present invention provides a screening method for sea cucumber collagen peptide with both antioxidant and hypoglycemic activities, including the following steps:

[0013] (1) Select multiple sea cucumber collagen sequences including AYL88761.1 for homology analysis;

[0014] (2) Virtually digest AYL88761.1 and sequences with a homology analysis score lower than 50 with AYL88761.1;

[0015] (3) By comparing bioactive peptides in the BIOPEP-UWM database, screen unknown bioactive peptide segments after virtual digestion, with the peptide segment size being 2 - 20 amino acids; conduct bioactivity prediction through the Peptide Ranker website, and screen peptide fragments with a Peptide Ranker score greater than 0.8;

[0016] (4) Use the Discovery Studio software to perform molecular construction and conformational energy optimization on the peptide fragments in (3), and preprocess the target proteins in the PDB library;

[0017] (5) Select the high-throughput screening program LibDock in the Discovery Studio software, use the peptide molecules in step (4) as ligands, and the preprocessed target proteins in the PDB library as receptors for preliminary molecular docking;

[0018] (6) Select docking results with LibDock scores all greater than 150, and optimize them through the semi-flexible screening program CDOCKER with the built-in CHARMM force field;

[0019] (7) Screen the optimized results in step (6) according to the -CDOCKER ENERGY value to select the active peptides;

[0020] (8) Predict the physicochemical properties of the bioactive peptides using the ProtParam tool, evaluate the pharmacokinetics through the ADMETlab 3.0 tool, and screen the Apostichopus japonicus collagen peptides according to intestinal stability, intestinal utilization rate, and non-toxicity.

[0021] (9) Experimentally verify the antioxidant activity and hypoglycemic activity of the Apostichopus japonicus collagen peptides obtained in step (8).

[0022] Furthermore, in step (1), the IDs of multiple Apostichopus japonicus 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, PIK62546.1.

[0023] Furthermore, in step (2), the enzyme used for virtual enzymatic hydrolysis is one or several of pepsin, trypsin, and α-chymotrypsin.

[0024] Furthermore, the target proteins include GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV).

[0025] Furthermore, the Apostichopus japonicus collagen peptides obtained by the above method include the amino acid sequence shown in SEQ ID NO: 1.

[0026] The beneficial effects of the above technical solutions include: The present invention efficiently virtually screens novel antioxidant and hypoglycemic bifunctional bioactive peptides from Apostichopus japonicus collagen by combining bioinformatics and molecular docking techniques. The Apostichopus japonicus collagen peptide DCDPRL screened by the present invention has strong antioxidant ability and hypoglycemic activity, and can be used for the development of biomedical products and the like. Description of the Drawings

[0027] Figure 1 It is the alignment information of 18 Apostichopus japonicus collagen sequences.

[0028] Figure 2 It is the pharmacokinetic prediction result of the peptide. Among them, in the range of 0-1, the larger the value, the stronger the positive correlation.

[0029] Figure 3 The molecular docking results of the sea cucumber collagen peptide DCDPRL are shown as follows. From top to bottom, they are the docking results of DCDPRL with the targets GLP-1R, GIPR, and Keap1 respectively.

[0030] Figure 4 The in vitro antioxidant activity of the sea cucumber collagen peptide DCDPRL is shown. Among them, the vertical axis represents the DPPH and ABTS radical scavenging abilities respectively, and the horizontal axis represents the molar concentration of the sample. Glutathione (GSH) is used as the positive control.

[0031] Figure 5 The zebrafish embryo toxicity of the sea cucumber collagen peptide DCDPRL is shown.

[0032] Figure 6 The in vivo hypoglycemic activity of the sea cucumber collagen peptide DCDPRL is shown. Among them, the vertical axis represents the blood glucose content, and the horizontal axis represents different treatment groups. Detailed implementation manners

[0033] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain 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 meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Regarding the terms "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0034] The present invention utilizes bioinformatics and computer virtual screening to screen for sea cucumber collagen active peptides and evaluate their in vitro and in vivo activities and mechanisms of action. Through screening and experimental verification, a sea cucumber collagen peptide with both antioxidant and hypoglycemic activities is obtained. Its amino acid sequence is Asp-Cys-Asp-Pro-Arg-Leu (DCDPRL), as shown in SEQ ID NO: 1. This sea cucumber collagen 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-bonding interactions. In vitro, it has strong DPPH radical scavenging ability (IC 50 = 206.12 μM) and ABTS radical scavenging ability (IC 50 = 186.06 μM). It has a good hypoglycemic effect in the hyperglycemic zebrafish model (the hypoglycemic ability of 250 μM DCDPRL is 60.64% ± 4.82%), and has the advantages of safety, no toxic side effects, and good water solubility.

[0035] The Apostichopus japonicus collagen peptide with both antioxidant and hypoglycemic activities provided by the present invention is derived from the collagen of Apostichopus japonicus. The Apostichopus japonicus collagen peptide can be screened and obtained by the following method:

[0036] (1) Virtual screening of Apostichopus japonicus collagen peptide

[0037] First, perform a homology analysis on 18 Apostichopus japonicus collagen sequences in the PDB library, and perform virtual enzymatic hydrolysis on the selected Apostichopus japonicus collagen sequences. The enzymes used include: Pepsin (pH 1.3), Trypsin, and Chymotrypsin (A).

[0038] Further select novel enzymatically hydrolyzed peptide fragments (2 - 20 amino acids) with unknown activity for biological activity prediction, and screen peptide fragments with a Peptide Ranker score greater than 0.8. Then, perform molecular construction and conformational energy optimization on the peptide fragments through the Discovery Studio software. Select the high-throughput screening program LibDock in the Discovery Studio software, use the peptide molecule as the ligand, and the optimized target proteins GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV) in the PDB library as the receptors for preliminary molecular docking.

[0039] Further select the docking results with higher LibDock scores, optimize them through the semi-flexible precise screening program CDOCKER with the built-in CHARMM force field, screen the optimized results according to the -CDOCKER ENERGY value, select 5 - 10 superior active peptides for pharmacokinetic evaluation, and select active peptides with excellent properties such as high intestinal stability, high intestinal utilization rate, and non-toxicity. Chemically synthesize the selected active peptides by solid phase synthesis.

[0040] (2) Verification of the in vitro antioxidant activity of Apostichopus japonicus collagen peptide

[0041] Determine the DPPH free radical scavenging ability, including the following steps: Prepare a 2.0 mM 1,1-diphenyl-2-picrylhydrazine (DPPH) working solution in a 95% ethanol solution, mix it with an equal volume of the sample in triplicate, and the final volume is 200.0 μL. After incubating in the dark for 30 minutes, use glutathione (GSH) as a positive control and measure the absorbance at a wavelength of 517 nm. The calculation formula (1) for the DPPH free radical scavenging ability is as follows:

[0042]

[0043] Among them, the absorbance after mixing 100.0 μL of the sample with 100.0 μL of the working solution is As, the absorbance after mixing 100.0 μL of the sample with 100.0 μL of 95% ethanol solution is Ab, and the absorbance after mixing 100.0 μL of the working solution with 100.0 μL of water is Ac.

[0044] The determination of the ABTS free radical scavenging ability includes the following steps: Prepare stock solutions of 0.7 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2.45 mM K2S2O8 respectively. Mix the above two solutions in equal volumes and incubate overnight in the dark at 37 °C. Dilute the mixed stock solution with 80% ethanol solution until the absorbance at a wavelength of 743 nm reaches 0.700 ± 0.050, indicating the successful preparation of the working solution. Subsequently, mix 200.0 μL of the working solution with 20.0 μL of the sample and incubate in the dark for 6 minutes, in triplicate. Measure the absorbance at a wavelength of 734 nm. The calculation formula (2) of ABTS RSC is as follows:

[0045]

[0046] Among them, the absorbance after mixing 20.0 μL of the sample with 200.0 μL of the working solution is As, the absorbance after mixing 20.0 μL of the sample with 200.0 μL of 80% ethanol solution is Ab, and the absorbance after mixing 200.0 μL of the working solution with 20.0 μL of water is Ac.

[0047] (3) Verify the embryotoxicity and in vivo hypoglycemic activity of sea cucumber collagen peptides

[0048] Place zebrafish embryos (wild-type AB) at 2 days post-fertilization (2 dpf) in a 12-well plate (20 embryos per well) at a temperature of 28 °C. The blank control group (E3) and the sample group are both in triplicate. In addition, change the culture medium every day until 6 days post-fertilization (6 dpf), and immediately remove dead embryos or larvae. Use the MSHOT imaging system (MD50, Guangzhou) to observe and record the morphological changes of zebrafish after drug administration, and calculate the embryo survival rate and hatching rate 6 days after drug administration.

[0049] 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 glucose and alloxan was used to culture at 28°C for 24 hours to establish a hyperglycemic zebrafish model. The zebrafish larvae were transferred to solutions with different concentrations of peptides as the experimental group, and the solution containing metformin was the positive control group. After the culture, the zebrafish larvae were rinsed with distilled water and collected into 1.5mL centrifuge tubes (10 per tube). 200μL of ethanol was added to each tube at room temperature for 2 minutes, and then transferred to a 60°C oven for drying for 2 hours, and then 50μL of distilled water was added to each tube to homogenize the zebrafish larvae and centrifuged (8000×g) for 5 minutes. The glucose content in the zebrafish larvae tissues was detected using a glucose content detection kit (Solebo, Beijing, China) and expressed as unit protein concentration using a BCA protein detection kit (Biyuntian, Shanghai, China).

[0050] Unless otherwise specified, the reagents involved in the embodiments of the present invention are purchased from commercial channels or prepared using conventional methods in the art, and the methods not mentioned are conventional experimental methods in the art. The solutions of the present invention, unless otherwise specified, are prepared using water as the solvent.

[0051] 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.; E3 culture medium was purchased from Shanghai Feixi Biotechnology Co., Ltd.

[0052] The following is an introduction through specific embodiments.

[0053] Example 1 Virtual screening of sea cucumber collagen peptides with both antioxidant and hypoglycemic activities

[0054] (1) Sequence alignment and homology analysis

[0055] The present invention uses the Multiple Alignment Tool in BLAST tool in the 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 1As shown, through homology analysis, the alignment score between sequence AYL88761.1 and PIK62545.1 was 97.6532, and the alignment score between sequence AYL88761.1 and PIK62546.1 was 92.2705, while the homology scores between other sequences were lower than 50. This indicates that AYL88761.1 contains most of the sequences of PIK62545.1 and PIK62546.1 and is their original protein. Therefore, subsequent experiments will focus on studying the remaining 16 collagen sequences (Table 1) other than PIK62545.1 and PIK62546.1.

[0056] Table 1 18 Collagen Sequences of Apostichopus japonicus

[0057]

[0058] (2) Virtual Enzymolysis

[0059] Using the Batch Processing tool in the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), three gastrointestinal enzymes were selected to perform single and combined virtual enzymolysis on 16 collagen sequences of Apostichopus japonicus. The three enzymes were: Pepsin (pH 1.3), Trypsin, and Chymotrypsin (A). After single enzymolysis and simultaneous virtual enzymolysis with the three enzymes, the bioactive peptides in the BIOPEP-UWM database were compared, and 2,647 unknown bioactive peptide segments (2 - 20 amino acids) were screened out. Then, through the Peptide Ranker website (http: / / distilldeep.ucd.ie / PeptideRanker / ), the bioactivities of the enzymolysis peptide fragments were preliminarily predicted, and the Peptide Ranker scores of 207 enzymolysis peptide fragments were greater than 0.8.

[0060] (3) Molecular Docking

[0061] Using the Discovery Studio software, molecular construction and binding energy optimization were performed on the peptide fragments. Then, preprocessing such as dehydration, hydrogenation, and energy optimization were carried out on the target proteins GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV) in the PDB library.

[0062] The high-throughput screening program LibDock was selected. Using peptide molecules as ligands and target proteins as receptors, the target proteins were GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV) respectively, and preliminary molecular docking was carried out. The active pocket positions (X, Y, Z) for docking with GLP-1R were 126.769, 161.247, 117.843, and the radius parameter for docking with GLP-1R was 15. The active pocket positions (X, Y, Z) for docking with GIPR were 133.042, 142.472, 172.855, and the radius parameter for docking with GIPR was 10. The active pocket positions (X, Y, Z) for docking with Keap1 were -44.2235, -9.27174, 64.8752, and the radius parameter for docking with Keap1 was 10.

[0063] To further accurately screen bioactive peptides with both antioxidant and hypoglycemic activities, docking results with LibDock scores greater than 150 were selected and optimized through the semi-flexible accurate screening program CDOCKER with the 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) could successfully dock. The physicochemical properties were predicted using the ProtParam tool (http: / / web.expasy.org / protparam / ) (Table 2). Among them, DCDPRL, GPMGLK, HFR, PRGF, and WHR are all hydrophilic peptides. It was also found during the experiment that DCDPRL is soluble in water immediately. Then, pharmacokinetic evaluation was carried out using the ADMETlab 3.0 tool. The results are as Figure 2 shown. In the range of 0 - 1, the larger the value, the stronger the positive correlation. It can be seen that the peptide DCDPRL has excellent properties of high intestinal utilization rate and non-toxicity.

[0064] Table 2 Prediction results of physicochemical activities of 7 active peptides

[0065]

[0066] *: The average hydrophilicity coefficient less than 0 indicates that the molecule is hydrophilic, otherwise it is hydrophobic.

[0067] Table 3 shows the docking score table of 7 peptides with different targets. From the results of molecular docking in Table 3, it can be seen that among the 7 peptides, DCDPRL has the best binding ability score and the highest stability with multiple targets (GLP-1R / GIPR / Keap1), and the -CDOCKERENERGY values are 104.025 Kcal / mol, 90.0051 Kcal / mol, and 112.037 Kcal / mol respectively. Table 4 shows the detailed docking result description of peptide DCDPRL with the targets (GLP-1R / GIPR / Keap1). A high binding energy indicates a strong affinity between the receptor and the ligand. The polypeptide DCDPRL forms favorable bindings with GLP-1R / GIPR / Keap1 through hydrogen bonds, salt bridges, hydrophobic interactions, and van der Waals forces. Figure 3 It is the 3D and 2D interaction diagram of polypeptide DCDPRL with multiple targets.

[0068] Table 3 Docking results of 7 peptides with GLP-1R / GIPR / Keap1

[0069]

[0070]

[0071] Table 4 Docking results of peptide DCDPRL with multiple targets GLP-1R / GIPR / Keap1

[0072]

[0073] Subsequently, the active peptides screened by the chemical solid-phase synthesis of Sangon Biotech Co., Ltd. (Shanghai, China) were commissioned for the next step of in vitro and in vivo activity verification.

[0074] Example 2 Verification of the in vitro antioxidant activity of 7 synthesized Apostichopus japonicus collagen peptides

[0075] The DPPH radical scavenging ability was measured, including the following steps: Prepare a 2.0 mM working solution of 1,1-diphenyl-2-picrylhydrazine (DPPH) in a 95% ethanol solution, and mix it with an equal volume of peptide solutions with different concentrations (using water as the solvent) in triplicate, with a final volume of 200.0 μL. In the peptide solutions, the concentrations of the peptide are 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM respectively. After incubating in the dark for 30 minutes, using glutathione (GSH) as the positive control, measure the absorbance at a wavelength of 517 nm. The calculation formula (1) for the DPPH radical scavenging ability is as follows:

[0076]

[0077] Among them, the absorbance after mixing 100.0 μL of the sample with 100.0 μL of the working solution is As, the absorbance after mixing 100.0 μL of the sample with 100.0 μL of 95% ethanol solution is Ab, and the absorbance after mixing 100.0 μL of the working solution with 100.0 μL of water is Ac.

[0078] To determine the ABTS free radical scavenging ability, the following steps are included: Prepare stock solutions of 0.7 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2.45 mM K2S2O8 respectively. Mix the above two solutions in equal volumes and incubate overnight in the dark at 37 °C. Dilute the mixed stock solution with 80% ethanol solution until the absorbance at 743 nm wavelength reaches 0.700 ± 0.050, indicating the successful preparation of the working solution. Subsequently, mix 200.0 μL of the working solution and 20.0 μL of peptide solutions with different concentrations respectively, and incubate for 6 minutes in the dark, in triplicate. In the peptide solutions, the concentrations of the peptide are 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM respectively. Glutathione (GSH) is used as the positive control. Measure the absorbance at 734 nm wavelength, and the calculation formula (2) of ABTS RSC is as follows:

[0079]

[0080] Among them, the absorbance after mixing 20.0 μL of the sample with 200.0 μL of the working solution is As, the absorbance after mixing 20.0 μL of the sample with 200.0 μL of 80% ethanol solution is Ab, and the absorbance after mixing 200.0 μL of the working solution with 20.0 μL of water is Ac.

[0081] The experimental results are shown in Table 5 and Figure 4 as follows. It can be seen that the sea cucumber collagen peptide DCDPRL has strong DPPH free radical scavenging ability (IC 50 = 206.12 μM) and ABTS free radical scavenging ability (IC 50 = 186.06 μM).

[0082] Table 5 In vitro antioxidant activities of 7 sea cucumber collagen peptides

[0083]

[0084] Note: Table 5 shows the experimental results of the sea cucumber collagen peptide at a molar concentration of 4 mM.

[0085] Example 3 Detect the embryotoxicity and in vivo hypoglycemic activity of the sea cucumber collagen peptide DCDPRL

[0086] (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 a temperature of 28 °C. The blank control group (E3 medium, Normal) and the sample group (E3 medium + peptide) were both in triplicate. In addition, the medium was changed daily until 6 days post-fertilization (6 dpf), and dead embryos or larvae were immediately removed. The MSHOT imaging system (MD50, Guangzhou) was used to observe and record the morphological changes of zebrafish after drug administration, and the embryo survival rate and hatching rate were calculated 6 days after drug administration.

[0087] It can be seen that Figure 5 low concentrations of the polypeptide 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 at about 90%, indicating that zebrafish are highly sensitive to the drug and also suggesting that the polypeptide DCDPRL is non-toxic to zebrafish larvae.

[0088] (2) Hypoglycemic activity: Zebrafish larvae (wild-type AB) at 5 dpf with normal development were selected and observed under a microscope and placed in six-well plates (30 tails per well). Subsequently, using the optimal concentration combination of 10 mM glucose solution and 0.1 mM alloxan solution, they were cultured at 28 °C for 24 hours to establish a hyperglycemic zebrafish model. They were transferred to peptide solutions at different concentrations (0.05 mM, 0.1 mM, 0.25 mM) as the experimental group, and a solution containing metformin (metformin concentration of 0.25 mM) was used as the positive control group. Normal group: E3 medium; Model group: 10 mM glucose + 0.1 mM alloxan + E3 medium; Experimental group: 10 mM glucose + 0.1 mM alloxan + different concentrations of peptide + E3 medium; Positive control group: 10 mM glucose + 0.1 mM alloxan + 0.25 mM metformin + E3 medium. All the above reagents were prepared based on E3 medium.

[0089] After the culture was completed, the zebrafish larvae were rinsed with distilled water and collected into 1.5 mL centrifuge tubes (10 tails per tube). 200 μL of absolute ethanol was added to each tube and allowed to stand at room temperature for 2 minutes, then transferred to an oven at 60 °C and dried for 2 hours. Then, 50 μL of distilled water was added to each tube to homogenize the zebrafish larvae and centrifuged (8000×g) for 5 minutes. The glucose content in the zebrafish larval tissues was detected using a glucose content detection kit (Solarbio, Beijing, China), and expressed as the unit protein concentration using a BCA protein detection kit (Beyotime, Shanghai, China).

[0090] The experimental results are as Figure 6 shown. It can be seen from Figure 6It can be seen that polypeptides DCDPRL at 50 - 250 μM can all reduce blood glucose levels; after culturing with low - concentration polypeptide DCDPRL (50 μM), the blood glucose levels of hyperglycemic zebrafish larvae decreased to a certain extent. As the concentration of polypeptide DCDPRL increased, the decrease in blood glucose levels became more obvious. DCDPRL at 250 μM can significantly reduce the blood glucose levels of hyperglycemic zebrafish larvae, and the blood glucose reduction rate was 60.64% ± 4.82%.

[0091] The above - described embodiments have detailed 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 used to limit the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the spirit and scope of the present invention, various changes and modifications can be made, and these changes and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A sea cucumber collagen peptide, characterized in that: The amino acid sequence of the sea cucumber collagen-imitation peptide is shown as SEQ ID NO:

1.

2. Use of the sea cucumber collagen peptide according to claim 1 in the preparation of antioxidant and / or hypoglycemic drugs.

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