Sea cucumber gonad-derived anti-inflammatory peptide and its preparation method and application

Abstract

This invention belongs to the field of food biotechnology and specifically relates to an anti-inflammatory peptide derived from the gonads of sea cucumbers, its preparation method, and its use. The anti-inflammatory peptide is obtained by enzymatic hydrolysis and molecular docking screening of sea cucumber gonads and comprises at least one of the peptides with amino acid sequences shown in SEQ ID NOs. 1-3. Experimental verification has demonstrated that the anti-inflammatory peptide exhibits high anti-inflammatory activity, providing a foundation for the production of functional foods from natural food sources and having important implications for the development of novel anti-inflammatory products.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology, and in particular relates to a sea cucumber gonad-derived anti-inflammatory peptide and a preparation method and application thereof. Background Art

[0002] Inflammation is a critical, ongoing process that occurs after the body is damaged by inflammatory factors, primarily as a defensive response. When chronic inflammation occurs, the body's immune tolerance is compromised, activating immune cells and causing them to release inflammatory mediators. Inflammatory mediators activate inflammatory signaling pathways inside and outside cells, stimulating immune cells to secrete excessive amounts of proinflammatory cytokines, which in turn promote cellular inflammatory responses. An excessive immune response can ultimately lead to tissue damage and cause significant harm to the body.

[0003] Anti-inflammatory peptides are a class of small molecule peptides with anti-inflammatory activity that can alleviate inflammatory symptoms by regulating inflammatory responses. They are easy to absorb, stable, and non-immunoreactive. They also have nutritional properties and multiple biologically active functions, such as regulating immunity and promoting tissue repair. Natural anti-inflammatory peptides have application prospects in the fields of medicine, cosmetics, and food due to their natural, safe, non-toxic, and high nutritional value. They have become one of the important natural products in the fields of functional foods and biomedicine. The use of low-cost biological resources to develop anti-inflammatory peptides with high anti-inflammatory activity and safety has always been a research hotspot in the field of peptides. In the existing technology, natural anti-inflammatory peptides are usually isolated from plant foods such as soybeans, citrus, and amaranth by chromatography and ultrafiltration, or anti-inflammatory active peptides are prepared by enzymatic methods or microbial fermentation methods.

[0004] A large number of by-products are produced during the processing of sea cucumbers, such as viscera and gonads. Studies have found that sea cucumber gonads have a high protein content and are rich in active ingredients such as sea cucumber polysaccharides. The development and utilization of sea cucumber gonads are low. At present, no commercial products developed with sea cucumber gonads as raw materials have been found in the domestic market. In recent years, homology modeling and molecular docking have been used to screen active peptides, providing an effective way to discover active peptides. Therefore, exploring the interaction between sea cucumber gonad-derived anti-inflammatory peptides and anti-inflammatory receptors TLR2 and TLR4, and finding their mechanism, can provide a new way to high-value utilization of sea cucumber gonads. Summary of the Invention

[0005] The present invention provides a sea cucumber gonad-derived anti-inflammatory peptide and its preparation method and application. The sea cucumber gonad-derived anti-inflammatory peptide of the present invention is prepared and screened from sea cucumber gonad tissue and has significant anti-inflammatory activity.

[0006] The specific technical solutions are as follows:

[0007] One of the purposes of the present invention is to provide a sea cucumber gonad-derived anti-inflammatory peptide, wherein the sea cucumber gonad-derived anti-inflammatory peptide comprises at least one of the peptides whose amino acid sequences are shown in SEQ.ID.NO.1~3.

[0008] Among them, SEQ.ID.NO.1 is PSNLGTGLR, and its docking energy with the anti-inflammatory receptor TLR2 is -7.3 kcal / mol, and its docking energy with the anti-inflammatory receptor TLR4 is -7.6 kcal / mol.

[0009] Among them, SEQ.ID.NO.2 is GDRGF, and its docking energy with the anti-inflammatory receptor TLR2 is -8.0 kcal / mol, and its docking energy with the anti-inflammatory receptor TLR4 is -7.0 kcal / mol.

[0010] Among them, SEQ.ID.NO.3 is FDGPEGPRGPPGSEGRQG, and its docking energy with the anti-inflammatory receptor TLR2 is -7.5 kcal / mol, and its docking energy with the anti-inflammatory receptor TLR4 is -7.3 kcal / mol.

[0011] Specifically, the sea cucumber gonad is a sea cucumber ( Apostichopus japonicu ) of mixed male and female gonads.

[0012] A second object of the present invention is to provide a method for preparing the above-mentioned anti-inflammatory peptide, comprising the following steps:

[0013] S1. Obtaining sea cucumber gonadal peptides;

[0014] S2. Sequence identification of sea cucumber gonadal peptides;

[0015] S3. Molecular docking of sea cucumber gonadal peptides with receptors TLR2 and / or TLR4 was performed to screen for anti-inflammatory peptides.

[0016] Furthermore, in step S1: sea cucumber gonad peptides are obtained by enzymatically hydrolyzing the sea cucumber gonad.

[0017] Specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include: adding pepsin to the raw material to be processed for enzymatic hydrolysis, and then adding flavor protease for enzymatic hydrolysis.

[0018] More specifically, in step S1, the enzymatic hydrolysis working conditions preferably include: adding pepsin to the raw material to be processed, adjusting the pH to 1.5-3.5, and enzymatic hydrolysis at 36-38°C for 2-5 hours; then adding flavor protease, adjusting the pH to 6.0-8.0, and enzymatic hydrolysis at 45-55°C for 1-3 hours, and then inactivating the enzyme.

[0019] The amount of pepsin used is preferably 1000-3000 U / g based on the raw material to be processed.

[0020] The amount of flavor protease used is preferably 1000-2000 U / g based on the raw material to be processed.

[0021] Specifically, in step S1, before enzymatic hydrolysis of the sea cucumber gonads, the sea cucumber gonads are preferably pretreated. The pretreatment comprises homogenizing the sea cucumber gonads and heating them in a boiling water bath for 10 to 30 minutes.

[0022] Furthermore, in step S1: after enzymatic hydrolysis, the enzymatic hydrolyzate is separated and purified.

[0023] Furthermore, in step S1, the separation and purification includes fractionating the enzymatic hydrolysate using nanofiltration and ultrafiltration. Nanofiltration can remove salts and free amino acids, while ultrafiltration can remove macromolecular substances. Specifically, the enzymatic hydrolysate is preferably subjected to nanofiltration and ultrafiltration to obtain a fraction with a molecular weight of 200 to 3000 Da.

[0024] Furthermore, in step S2: peptide sequence analysis was performed using LC-MS / MS, and all peptide sequences were obtained by comparison analysis using a database. The mass spectrometry database search software was MaxQuant 2.4.14.0, and the sample database used was the Uniprot protein database.

[0025] Specifically, in step S2, the product obtained in step S1 is preferably desalted before performing polypeptide sequence analysis, preferably using a C18 StageTip column for desalting.

[0026] Furthermore, in step S3: preferably, molecular docking is performed using VINA-2.0 within the Pyrx software to screen antioxidant peptides. The affinity value represents the binding ability between the two. The lower the docking energy, the more stable the binding between the ligand and the receptor.

[0027] The third object of the present invention is to provide the use of the above-mentioned sea cucumber gonad-derived anti-inflammatory peptide in cosmetics.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention obtained three anti-inflammatory peptides from sea cucumber gonads through enzymatic hydrolysis and molecular docking screening. Among them, SEQ.ID.NO.1 has a docking energy of -7.3 kcal / mol with the anti-inflammatory receptor TLR2 and a docking energy of -7.6 kcal / mol with the anti-inflammatory receptor TLR4; SEQ.ID.NO.2 has a docking energy of -8.0 kcal / mol with the anti-inflammatory receptor TLR2 and a docking energy of -7.0 kcal / mol with the anti-inflammatory receptor TLR4; SEQ.ID.NO.3 has a docking energy of -7.5 kcal / mol with the anti-inflammatory receptor TLR2 and a docking energy of -7.3 kcal / mol with the anti-inflammatory receptor TLR4. Experimental verification shows that the above anti-inflammatory peptides have high anti-inflammatory activity and are of great significance for the development of new anti-inflammatory products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a molecular docking diagram of the sea cucumber gonad-derived anti-inflammatory peptide with the amino acid sequence shown in SEQ.ID.NO.1 and the receptor TLR2;

[0031] Figure 2 It is a molecular docking diagram of the sea cucumber gonad-derived anti-inflammatory peptide with the amino acid sequence shown in SEQ.ID.NO.2 and the receptor TLR2;

[0032] Figure 3 It is a molecular docking diagram of the sea cucumber gonad-derived anti-inflammatory peptide with the amino acid sequence shown in SEQ.ID.NO.3 and the receptor TLR2;

[0033] Figure 4 It is a molecular docking diagram of the sea cucumber gonad-derived anti-inflammatory peptide with the amino acid sequence shown in SEQ.ID.NO.1 and the receptor TLR4;

[0034] Figure 5 It is a molecular docking diagram of the sea cucumber gonad-derived anti-inflammatory peptide with the amino acid sequence shown in SEQ.ID.NO.2 and the receptor TLR4;

[0035] Figure 6 It is a molecular docking diagram of the sea cucumber gonad-derived anti-inflammatory peptide with the amino acid sequence shown in SEQ.ID.NO.3 and the receptor TLR4;

[0036] Figure 7 To test the effects of the three peptides on the relative proliferation rate of RAW264.7 cells;

[0037] Figure 8 To test the effects of the three peptides on NO secretion in RAW264.7 cells. DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0039] In a specific embodiment, the pepsin used was purchased from Shenggong Bioengineering; the flavor protease used was purchased from Solebao Biotechnology.

[0040] In a specific embodiment: the sea cucumber gonad used is a sea cucumber ( Apostichopus japonicu ) of mixed male and female gonads. Example

[0041] The steps for preparing sea cucumber gonad-derived anti-inflammatory peptide are as follows:

[0042] S1. Preparation of sea cucumber gonadal peptides:

[0043] (1) Raw material pretreatment: Homogenize the sea cucumber gonadal tissue, heat in a boiling water bath for 15 min to obtain the homogenate, and let it cool for later use;

[0044] (2) Enzymatic hydrolysis of raw materials: add 2000 U / g of pepsin based on the mass of the homogenate obtained in step (1), adjust the pH to 2.5, and perform enzymatic hydrolysis at 37°C for 4 h; then add 1200 U / g of flavor protease based on the mass of the homogenate, adjust the pH to 7.0, perform enzymatic hydrolysis at 50°C for 2 h, and then boil at 100°C to inactivate the enzyme; then centrifuge at 5000 r / min for 15 min, collect the supernatant, and obtain the enzymatic hydrolyzate;

[0045] (3) Purification: The enzymatic hydrolysate obtained in step (2) was subjected to nanofiltration and ultrafiltration classification. A 200 Da nanofiltration membrane was first used to remove salt and free amino acids, and then a 3000 Da spiral membrane was selected for ultrafiltration. The obtained 200-3000 Da components were freeze-dried to obtain sea cucumber gonadal peptide powder, which was stored at -20°C for future use.

[0046] S2. Sequence identification of sea cucumber gonadal peptides:

[0047] The peptide powder obtained in step S1 was desalted using a C18 StageTip column, and peptide sequence analysis was performed using LC-MS / MS. The complete peptide sequence was obtained by comparison analysis using the database. The mass spectrometry database retrieval software was MaxQuant 2.4.14.0, and the sample database used was the Uniprot protein database.

[0048] S3. Molecular docking of sea cucumber gonadal peptide with anti-inflammatory receptors TLR2 and TLR4:

[0049] The SDF format files of the main active ingredients of the core drugs were obtained from the Pubchem database, and the key target protein structures were collected from the PDB database. The targets were optimized by removing water molecules and small molecule ligands using Pymol-2.1.0 software, and hydrogenation and charge processing were performed using AutoDock Tools-1.5.6 and saved in pdbqt format.

[0050] Molecular docking was performed using the key target receptor and the corresponding active ingredient as the ligand using VINA-2.0 within the PyRx software. Binding energies were calculated and output as result files. The results were visualized using PyMol. The affinity (kcal / mol) value represents the binding affinity between the two. The lower the docking energy, the more stable the ligand-receptor binding. Visualization analysis was performed using PyMol, and 2D plots were visualized using the Discovery Studio 2020 client.

[0051] Three anti-inflammatory peptides and their docking energies were screened and obtained. The amino acid sequences of the three anti-inflammatory peptides are shown in SEQ ID NOs. 1 to 3. The amino acid sequences of the three anti-inflammatory peptides and their corresponding docking energies are shown in Table 1.

[0052] Table 1 Amino acid sequence and docking energy of sea cucumber gonad-derived anti-inflammatory peptides

[0053] anti-inflammatory peptides sequence TLR2 docking energy (kcal / mol) TLR4 docking energy (kcal / mol) SEQ.ID.NO.1 PSNLGTGLR -7.3 -7.6 SEQ.ID.NO.2 GDRGF -8.0 -7.0 SEQ.ID.NO.3 FDGPEGPRGPPGSEGRQG -7.5 -7.3

[0054] The molecular docking results of the peptides with amino acid sequences as shown in SEQ.ID.NO.1~3 and receptor TLR2 are shown in the following order. Figures 1-3 The molecular docking results of the peptides with amino acid sequences as shown in SEQ.ID.NO.1~3 and the receptor TLR4 are shown in the following order. Figures 4-6 .pass Figures 1 to 6 The binding patterns and binding sites of anti-inflammatory peptides and anti-inflammatory receptors shown in the figure show that the binding of anti-inflammatory peptides to TLR2 and TLR4 is mainly through hydrogen bonds, carbon-hydrogen bonds, alkyl groups, π-alkyl groups and other interactions with the amino acid residues of the receptors.

[0055] like Figure 1 As shown, the binding sites of the peptide shown in SEQ.ID.NO.1 to TLR2 are SER-424, SER-445, ASN-467, ARG-486, TRP-535, THR-532, LYS-561, ARG-508, ARG-447, and LYS-422. Figure 2As shown, the binding sites of the peptide shown in SEQ.ID.NO.2 to TLR2 are TYR-326, PHE-325, PHE-349, SER-346, LYS-347, VAL-348, LEU-266, PHE-284, LEU-289, and LEU-317. Figure 3 As shown, the binding sites of the peptide shown in SEQ.ID.NO.3 to TLR2 are LYS-561, ASN-533, THR-532, ASN-487, HIS-398, ASN-397, GLN-396, LYS-422, SER-445, ARG-447, SER-424, GLY-566, SER-563, ARG-508, ASN-370, ASN-466, LEU-371, and ARG-486. Figure 4 As shown, the binding sites of the peptide shown in SEQ.ID.NO.1 to TLR4 are ASP-354, HIS-305, ASN-330, SER-334, LEU-249, ASN-248, LEU-216, ILE-378, and ALA-301. Figure 5 As shown, the binding sites of the peptide shown in SEQ.ID.NO.2 to TLR4 are ASN-330, LEU-302, SER-303, HIS-305, TYR-275, GLU-217, THR-250, LEU-249, and ALA-301. Figure 6 As shown, the binding sites of the peptide shown in the amino acid sequence of SEQ.ID.NO.3 and TLR4 are THR-332, ILE-278, SER-277, SER-219, HIS-193, SER-279, LYS-166, ASN-280, GLU-217, HIS-305, ASN-253, VAL-195, PHE-123, ASP-354, and MET-381.

[0056] test

[0057] According to the mass spectrometry sequencing results, the three peptide sequences were chemically synthesized and tested, and their effects on the relative proliferation rate of RAW264.7 cells and the NO secretion of RAW264.7 cells were tested.

[0058] 1. Determination of relative proliferation rate of RAW264.7 cells

[0059] When the RAW264.7 cell density reached 80%, the cells were resuspended in complete medium, mixed, and counted to adjust the cell density to 6 × 10 5Cell suspension (100 μL / well) was added to a 96-well cell culture plate at a concentration of 1 μg / mL. 100 μL of complete medium was added to the blank control group and the cells were cultured at 37°C in a 5% CO2 incubator. After 24 hours, the supernatant was removed and 100 μL of complete medium was added to the blank and control groups. In the experimental groups, 100 μL of complete medium containing various concentrations of PSNLGTGLR, GDRGF, and FDGPEGPRGPPGSEGRQG (25, 50, 100, 200, and 400 μg / mL) was added to each well. To prevent evaporation of the cell supernatant and affect the experimental results, 200 μL of PBS was added to the outermost well of the 96-well plate. After 24 hours of incubation in the incubator, follow the CCK-8 kit instructions and add 100 μL of 10% CCK-8 culture medium to each well. Incubate in a 37°C incubator for 1 hour. Measure the absorbance (OD) at 450 nm using a microplate reader. Five replicate wells were set up in the experiment to calculate the relative proliferation rate of RAW264.7 cells and screen the experimental concentration of the sample. The relative cell proliferation rate was calculated using the following formula:

[0060] Relative cell proliferation rate (%) = (OD experimental group - OD blank group) / (OD normal group - OD blank group) × 100%.

[0061] The effects of three synthetic peptides on the relative proliferation rate of RAW264.7 cells are shown in Figure 7 Synthetic peptides at different concentrations (50-400 µg / mL) all exhibited a proliferative effect on RAW264.7 cells, with the proliferative effect increasing with increasing peptide concentration. GDRGF exhibited the greatest cell proliferation-promoting effect, with a relative cell proliferation rate reaching 151.45±2.33% at a concentration of 400 µg / mL. These results indicate that the three synthetic peptides exhibited no cytotoxic effects within the 50-400 µg / mL concentration range. A concentration range of 100-400 µg / mL was selected for subsequent experiments.

[0062] 2. Determination of NO Secretion in RAW264.7 Cells

[0063] The NO kit was used for determination. The experimental grouping and operation steps were the same as those for the relative proliferation rate determination of RAW264.7 cells. After culturing for 24 hours, the cell culture medium was collected and the NO kit instructions were followed. 50 μL of the supernatant of each group of cells after centrifugation was added to each well of a 96-well plate. 50 μL of Griess Reagent I and Griess Reagent II were added in sequence, and the absorbance was measured at 540 nm. The standard curve of nitrite was determined and drawn. According to the standard curve equation Y=0.0051X-0.0067, R 2=0.9991 to calculate the sodium nitrite content in the cell culture medium, thereby reflecting the level of NO secretion by the cells.

[0064] The effects of different active peptides on inflammation were evaluated by detecting the NO content in RAW264.7 macrophages induced by LPS (lipopolysaccharide). The effects of three synthetic peptides on NO secretion in RAW264.7 cells are shown in Figure 8 .like Figure 8 As shown, LPS stimulation significantly increased NO production (28.36±0.856 μmol / L), while intervention with PSNLGTGLR, GDRGF, and FDGPEGPRGPPGSEGRQG significantly decreased NO levels (P < 0.05), indicating that these three peptides can effectively inhibit LPS-induced inflammatory responses. Based on these test results, the three anti-inflammatory peptides can be used in the preparation of anti-inflammatory drugs.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sea cucumber gonad-derived anti-inflammatory peptide, characterized in that: It is a peptide whose amino acid sequence is shown in SEQ.ID.NO.2 or / and SEQ.ID.NO.

3.

2. A method for preparing the sea cucumber gonad-derived anti-inflammatory peptide according to claim 1, characterized in that: The steps include: S1. Sea cucumber gonadal peptides were obtained by enzymatic hydrolysis of sea cucumber gonads. The enzymatic hydrolysis conditions included adding pepsin to the raw material for 2-5 hours, followed by adding flavor protease for 1-3 hours. S2. Sequence identification of sea cucumber gonadal peptides; S3. Molecular docking of sea cucumber gonadal peptides with receptors TLR2 and / or TLR4 was performed to screen anti-inflammatory peptides. Molecular docking was performed using VINA-2.0 within the Pyrx software.

3. The preparation method according to claim 2, characterized in that In step S1: after enzymatic hydrolysis, the enzymatic hydrolyzate is separated and purified.

4. The preparation method according to claim 3, characterized in that In step S1: the separation and purification includes classifying the enzymatic hydrolysate using nanofiltration and ultrafiltration.

5. The preparation method according to claim 2, characterized in that In step S2: peptide sequence analysis is performed using LC-MS / MS.

6. Use of the sea cucumber gonad-derived anti-inflammatory peptide as claimed in claim 1 in cosmetics.

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