Apostichopus japonicus collagen peptide and application thereof

Abstract

The invention relates to an apostichopus japonicus collagen peptide and application thereof. The amino acid sequence of the apostichopus japonicus collagen peptide is Asp-Cys-Asp-Pro-Arg-Leu (DCDPRL), and the apostichopus japonicus collagen peptide is the apostichopus japonicus collagen peptide with the antioxidant activity and the hypoglycemic activity. According to the invention, the bioinformatics and the molecular docking technology are combined to virtually screen the novel antioxidant and hypoglycemic bifunctional bioactive peptide from the Apostichopus japonicus collagen. The invention further discloses a preparation method of the antioxidant and hypoglycemic bifunctional bioactive peptide, and the antioxidant and hypoglycemic bifunctional bioactive peptide can be used for preparing the antioxidant and hypoglycemic bifunctional bioactive peptide. The apostichopus japonicus collagen peptide DCDPRL obtained through screening has the advantages of being high in oxidation resistance, good in hypoglycemic activity and the like. The research on the multi-target bioactive peptide can be applied to the fields of dietary nutrition, biological medicine products and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a sea cucumber-like collagen peptide and an application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a typical chronic non-communicable disease characterized by high blood sugar and insulin resistance. According to the IDF Diabetes Atlas (2021), approximately 537 million people (20-79 years old) suffer from diabetes worldwide, of which 90% are T2DM patients.

[0003] Bioactive peptides are considered to be one of the promising drugs for the treatment of chronic diseases. They are nutritionally comprehensive, easy to absorb, and have diverse functions. The functions of bioactive peptides with different amino acid sequences may be different. 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 a variety of 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 antioxidant and hypoglycemic dual-functional bioactive peptides can be screened from sea cucumber collagen, it will also be of great significance for the development of dietary nutrition and biopharmaceutical products. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a sea cucumber-like collagen peptide and application thereof, wherein the sea cucumber-like 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 collagen-like peptide, comprising an amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 can be specifically Asp-Cys-Asp-Pro-Arg-Leu (DCDPRL).

[0007] The beneficial effects of adopting the above technical solution include: the active peptide DCDPRL can stably bind to the three targets GLP-1R, G IPR 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 (IC 50 =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 the hyperglycemic zebrafish model experiment, the blood sugar lowering ability of DCDPRL (250 μM) is 60.64%±4.82%.

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

[0009] The beneficial effects of adopting the above technical solution include: the sea cucumber-like collagen peptide prepared by the above method has the advantages of strong antioxidant capacity and good blood sugar-lowering activity.

[0010] The present invention provides application of the sea cucumber collagen-imitation peptide in anti-oxidation and / or hypoglycemic effect.

[0011] The sea cucumber imitation collagen peptide can be used to prepare antioxidant products and / or blood sugar-lowering products.

[0012] Products include but are not limited to food, health products, medicines, dietary supplements, biopharmaceuticals, etc.

[0013] The present invention provides a method for screening a sea cucumber collagen peptide having both antioxidant and hypoglycemic activities, comprising the following steps:

[0014] (1) Select multiple sea cucumber collagen sequences including AYL88761.1 for homology analysis;

[0015] (2) AYL88761.1 and sequences with homology analysis scores less than 50 with AYL88761.1 were subjected to virtual enzymatic digestion;

[0016] (3) By comparing the bioactive peptides in the BIOPEP-UWM database, unknown bioactive peptides after virtual enzymatic hydrolysis were screened, with a peptide size of 2–20 amino acids; bioactivity prediction was performed through the Peptide Ranker website, and peptide fragments with a Peptide Ranker score greater than 0.8 were screened;

[0017] (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;

[0018] (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;

[0019] (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;

[0020] (7) screening the results optimized in step (6) according to the -CDOCKER ENERGY value and selecting the active peptides;

[0021] (8) The physicochemical properties of active peptides were predicted using the ProtParam tool, and pharmacokinetic evaluation was performed using the ADMETlab 3.0 tool to screen sea cucumber collagen-like peptides based on intestinal stability, intestinal availability, and non-toxicity;

[0022] (9) Experimentally verify the antioxidant activity and hypoglycemic activity of the sea cucumber collagen peptide obtained in step (8).

[0023] Further, in step (1), the IDs of the multiple sea cucumber collagen sequences are respectively: 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.

[0024] Furthermore, in step (2), the enzyme used for virtual enzymatic hydrolysis is one or more of pepsin, trypsin, and α-chymotrypsin.

[0025] Furthermore, the target proteins include GLP-1R (ID: 7LCK), GIPR (ID: 7FIY) and Keap1 (ID: 5WFV).

[0026] Furthermore, the sea cucumber collagen-mimicking peptide obtained by the above method includes the amino acid sequence shown in SEQ ID NO:1.

[0027] The beneficial effects of adopting the above technical scheme include: the present invention combines bioinformatics and molecular docking technology to effectively virtually screen new antioxidant and hypoglycemic dual-functional bioactive peptides from Apostichopus japonicus collagen. The sea cucumber collagen peptide DCDPRL screened by the present invention has strong antioxidant capacity and hypoglycemic activity, and can be used in the development of dietary nutrition and biopharmaceutical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The alignment information of 18 collagen sequences from Apostichopus japonicus.

[0029] Figure 2 It is the pharmacokinetic prediction result of the peptide, where, in the range of 0-1, the larger the value, the stronger the positive correlation.

[0030] Figure 3 This is the molecular docking result 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.

[0031] Figure 4 The in vitro antioxidant activity of sea cucumber collagen peptide DCDPRL is simulated, where 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.

[0032] Figure 5 It is a zebrafish embryo toxicity modeled after the sea cucumber collagen peptide DCDPRL.

[0033] Figure 6 It is to simulate the in vivo hypoglycemic activity of 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

[0034] 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 used 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 field of the present invention. As used herein, "including", "having", "containing", etc. are all open terms, that is, including but not limited to.

[0035] The present invention utilizes bioinformatics and computer virtual screening of sea cucumber collagen-like active peptides and evaluates 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 is obtained, and its amino acid sequence 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, acts on multiple targets (GLP-1R, GIPR and Keap1), and stably binds 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 =186.06μM). It has a good hypoglycemic effect in the hyperglycemic zebrafish model (the hypoglycemic ability of 250μM DCDPRL is 60.64%±4.82%), and has the advantages of safety, no toxic side effects, and good water solubility.

[0036] The sea cucumber-like collagen peptide with both antioxidant and hypoglycemic activity provided by the present invention is derived from the collagen of Japanese sea cucumber (Apostichopus japonicus). The sea cucumber-like collagen peptide can be obtained by screening by the following method:

[0037] (1) Virtual screening of sea cucumber collagen peptides

[0038] Firstly, the homology of 18 sea cucumber collagen sequences in PDB library was analyzed, and the screened sea cucumber collagen sequences were subjected to virtual enzymatic hydrolysis. The enzymes used included pepsin (pH 1.3), trypsin and α-chymotrypsin (A).

[0039] We further selected novel enzymatic peptide fragments (2–20 amino acids) of unknown activity for biological activity prediction, and screened peptide fragments with a Peptide Ranker score greater than 0.8. Then, we used Discovery Studio software to perform molecular construction and knot energy optimization on the peptide fragments, and selected the high-throughput screening program LibDock in Discovery Studio software, using peptide molecules as ligands and the optimized target proteins GLP-1R (ID: 7LCK), GIPR (ID: 7FIY), and Keap1 (ID: 5WFV) in the PDB library as receptors for preliminary molecular docking.

[0040] The docking results with high LibDock scores were further selected and optimized through the semi-flexible precision screening program CDOCKER with built-in CHARMM force field. The optimized results were screened according to the -CDOCKER ENERGY value, and the 5-10 best 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 screened by chemical solid phase synthesis.

[0041] (2) Verification of the in vitro antioxidant activity of sea cucumber collagen peptide

[0042] The DPPH free radical scavenging ability was determined, including the following steps: 2.0 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) working solution was prepared in 95% ethanol solution, mixed with an equal volume of sample, in triplicate, and the final volume was 200.0 μL. After incubation in the dark for 30 minutes, the absorbance was measured at a wavelength of 517 nm with glutathione (GSH) as a positive control. The calculation formula (1) for DPPH free radical scavenging ability is as follows:

[0043]

[0044] Among them, the absorbance after 100.0 μL of sample and 100.0 μL of working solution are mixed is As, the absorbance after 100.0 μL of sample and 100.0 μL of 95% ethanol solution are mixed is Ab, and the absorbance after 100.0 μL of working solution and 100.0 μL of water are mixed is Ac.

[0045] The free radical scavenging ability of ABTS 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 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 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 calculation formula (2) of ABTS RSC was as follows:

[0046]

[0047] Among them, 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.

[0048] (3) Verification of embryotoxicity and hypoglycemic activity of sea cucumber collagen peptide

[0049] Zebrafish embryos (wild-type AB) at 2 days post fertilization (2dpf) were placed in 12-well plates (20 embryos per well) at 28°C. Both the blank control group (E3) and the sample group were in triplicate. In addition, the culture medium was changed every day until the 6th day after fertilization (6dpf), 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] The following is an introduction through specific embodiments.

[0054] Example 1 Virtual screening of sea cucumber collagen peptides with both antioxidant and hypoglycemic activities

[0055] (1) Sequence alignment and homology analysis

[0056] 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 in the figure, the homology analysis found that the alignment score of the sequence AYL88761.1 and PIK62545.1 was 97.6532, the alignment score of the sequence AYL88761.1 and PIK62546.1 was 92.2705, and the homology scores between other sequences were less than 50. This shows that AYL88761.1 has most of the sequences of PIK62545.1 and PIK62546.1 and is their original protein. Therefore, subsequent experiments will focus on the remaining 16 collagen sequences except PIK62545.1 and PIK62546.1 (Table 1).

[0057] Table 1 18 collagen sequences from Apostichopus japonicus

[0058]

[0059]

[0060] (2) Virtual enzymatic hydrolysis

[0061] 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 composite virtual enzymatic digestion on 16 Apostichopus japonicus collagen sequences. The three enzymes included pepsin (pH 1.3), trypsin, and α-chymotrypsin (A). After single enzymatic digestion and simultaneous virtual enzymatic digestion with three enzymes, 2647 unknown bioactive peptides (2–20 amino acids) were screened out by comparing the bioactive peptides in the BIOPEP-UWM database. The bioactivity of the enzymatic peptide fragments was then preliminarily predicted using the Peptide Ranker website (http: / / distilldeep.ucd.ie / PeptideRanker / ), of which 207 enzymatic peptide fragments had a Peptide Ranker score greater than 0.8.

[0062] (3) Molecular docking

[0063] 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.

[0064] The high-throughput screening program LibDock was selected, and the peptide molecule was used as the ligand, the target protein was used as the receptor, and the target proteins were GLP-1R (ID: 7LCK), GIPR (ID: 7FIY) and Keap1 (ID: 5WFV) for preliminary molecular docking. The active pocket position (X, Y, Z) docked with GLP-1R was 126.769, 161.247, 117.843, and the radius parameter for docking with GLP-1R was 15. The active pocket position (X, Y, Z) docked with GIPR was 133.042, 142.472, 172.855, and the radius parameter for docking with GIPR was 10. The active pocket position (X, Y, Z) docked with Keap1 was -44.2235, -9.27174, 64.8752, and the radius parameter for docking with Keap1 was 10.

[0065] 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 by CDOCKER, a semi-flexible precision screening program with 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 be successfully docked. The ProtParam tool (http: / / web.expasy.org / protparam / ) was used to predict their physicochemical properties (Table 2). DCDPRL, GPMGLK, HFR, PRGF and WHR are all hydrophilic peptides. It was also found during the experiment that DCDPRL was soluble in water. Then the pharmacokinetic evaluation was performed using the ADMETlab 3.0 tool. 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.

[0066] Table 2 Prediction results of physicochemical activities of 7 active peptides

[0067]

[0068] *: When the average hydrophilicity coefficient is less than 0, it means that the molecule is hydrophilic, otherwise it is hydrophobic.

[0069] Table 3 is a score table for the docking 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 with multiple targets (GLP-1R / GIPR / Keap1) and the highest stability, with -CDOCKERENERGY of 104.025Kcal / mol, 90.0051Kcal / mol and 112.037Kcal / mol, respectively. Table 4 is a detailed description of the docking results of peptide DCDPRL with targets (GLP-1R / GIPR / Keap1). High binding energy indicates a strong affinity between the receptor and the ligand. Peptide DCDPRL forms a favorable binding 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 peptide DCDPRL with multiple targets.

[0070] Table 3 Docking results of 7 peptides with GLP-1R / GIPR / Keap1

[0071]

[0072]

[0073] Table 4 Docking results of peptide DCDPRL with multi-target GLP-1R / G IPR / Keap1

[0074]

[0075] Subsequently, the active peptides screened by chemical solid phase synthesis were commissioned to Sangon Biotechnology Co., Ltd. (Shanghai, China) for further in vitro and in vivo activity verification.

[0076] Example 2 Verification of the in vitro antioxidant activity of seven synthetic sea cucumber collagen peptides

[0077] The DPPH free radical scavenging ability was determined, including 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 an equal volume of peptide solutions of different concentrations (with water as the solvent) in triplicate, with a final volume of 200.0 μL. In the peptide solution, the concentrations of the peptides 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 with glutathione (GSH) as a positive control. The calculation formula (1) for the DPPH free radical scavenging ability is as follows:

[0078]

[0079] Among them, the absorbance after 100.0 μL of sample and 100.0 μL of working solution are mixed is As, the absorbance after 100.0 μL of sample and 100.0 μL of 95% ethanol solution are mixed is Ab, and the absorbance after 100.0 μL of working solution and 100.0 μL of water are mixed is Ac.

[0080] The free radical scavenging ability of ABTS 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 respectively, and the two solutions were mixed in equal volumes and incubated overnight at 37°C in the dark. 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 respectively and incubated in the dark for 6 minutes in triplicate. In the peptide solution, the concentrations of the peptides 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 calculation formula (2) of ABTS RSC was as follows:

[0081]

[0082] Among them, 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.

[0083] The experimental results are shown in Table 5 and Figure 4 As shown in the figure, 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).

[0084] Table 5 In vitro antioxidant activities of seven sea cucumber collagen peptides

[0085]

[0086] Note: Table 5 shows the experimental results when the molar concentration of the sea cucumber collagen peptide is 4 mM.

[0087] Example 3 Detection of embryotoxicity and hypoglycemic activity in vivo of sea cucumber collagen peptide DCDPRL

[0088] (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 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 were calculated 6 days after administration.

[0089] 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 at around 90%, indicating that zebrafish are highly sensitive to drugs and that the peptide DCDPRL is non-toxic to zebrafish larvae.

[0090] (2) Hypoglycemic activity: 5dpf zebrafish larvae (wild-type AB) with normal development were observed under a microscope and selected 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. The peptide solutions transferred to different concentrations (0.05mM, 0.1mM, 0.25mM) were used as the experimental group, and the solution containing metformin (metformin concentration was 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 peptides + 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.

[0091] After the incubation, the 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 at room temperature for 2 minutes, and then transferred to a 60°C oven for drying 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 larvae tissue 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).

[0092] The experimental results are as follows Figure 6 As shown. Figure 6It can be seen that 50-250μM of the peptide DCDPRL can reduce blood sugar content; after culturing with low concentration of peptide DCDPRL (50μM), the blood sugar level of hyperglycemic zebrafish larvae decreased to a certain extent. As the concentration of peptide DCDPRL increased, the decrease in blood sugar level became more obvious. 250μM DCDPRL can significantly reduce the blood sugar level of hyperglycemic zebrafish larvae, and the blood sugar reduction rate is 60.64%±4.82%.

[0093] 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 protection scope of the present invention.

Claims

1. A sea cucumber collagen peptide, characterized in that: It includes the amino acid sequence shown in SEQ ID NO:

1.

2. The method for preparing the sea cucumber collagen peptide according to claim 1, characterized in that: The following steps are involved: Solid phase synthesis of sea cucumber collagen peptide having the amino acid sequence shown in SEQ ID NO:

1.

3. Use of the sea cucumber collagen peptide according to claim 1 in anti-oxidation and / or hypoglycemic effects.

4. The use according to claim 3, characterized in that: Used for preparing antioxidant products and / or blood sugar lowering products.

5. The application according to claim 4, characterized in that: The products include food, health products, medicines, dietary supplements or biopharmaceuticals.

6. A method for screening sea cucumber collagen peptides having both antioxidant and hypoglycemic activities, characterized in that: The following steps are involved: (1) Select multiple sea cucumber collagen sequences including AYL88761.1 for homology analysis; (2) AYL88761.1 and sequences with homology analysis scores less than 50 with AYL88761.1 were subjected to virtual enzymatic digestion; (3) By comparing the bioactive peptides in the BIOPEP-UWM database, unknown bioactive peptides after virtual enzymatic hydrolysis were screened, with a peptide size of 2–20 amino acids; bioactivity prediction was performed through the Peptide Ranker website, and peptide fragments with a PeptideRanker score greater than 0.8 were screened; (4) Using Discovery Studio software, the peptide fragments in step (3) were molecularly constructed and the energy of the junction was optimized, and the target protein in the PDB library was pre-processed; (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 proteins GLP-1R, GIPR and Keap1 in the PDB library as the receptor to perform preliminary molecular docking; (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; (7) selecting active peptides according to the results of the -CDOCKER ENERGY value screening step (6); (8) The physicochemical properties of active peptides were predicted using the ProtParam tool, and pharmacokinetic evaluation was performed using the ADMETlab 3.0 tool to screen sea cucumber collagen-like peptides based on intestinal stability, intestinal availability, and non-toxicity; (9) Experimentally verify the antioxidant activity and hypoglycemic activity of the sea cucumber collagen peptide obtained in step (8).

7. The screening method according to claim 6, characterized in that: In step (1), the IDs of the multiple sea cucumber collagen sequences are respectively: 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.

8. The screening method according to claim 6 or 7, characterized in that: In step (2), the enzyme used for virtual enzymolysis is one or more of pepsin, trypsin, and α-chymotrypsin.

9. The screening method according to claim 6 or 7, characterized in that: Target proteins include GLP-1R, GIPR, and Keap1.

10. The screening method according to claim 6 or 7, characterized in that: The sea cucumber collagen-mimicking peptide comprises the amino acid sequence shown in SEQ ID NO:1.

Images