A sea cucumber active polypeptide and a preparation method and application thereof

Abstract

The application discloses a sea cucumber active polypeptide as well as a preparation method and application thereof, and belongs to the field of marine biological technology. The sea cucumber active polypeptide provided by the application has an amino acid sequence from an N terminal to a C terminal as follows: Ile-Gly-Tyr-Thr-Pro-Arg-Pro-Ile-Leu-Phe; or Ile-Gly-Gly-Arg-Asp-Phe-Leu-Leu; or Asp-Phe-Leu-Leu-Gln-Pro-Ile-Met-Met. The anti-inflammatory polypeptide is prepared by enzymolysis from the sea cucumber, is purified by adopting ultrafiltration grading, is identified by LC-MS / MS, and is screened by using an anti-inflammatory peptide prediction website and molecular docking technology to select an anti-inflammatory active fragment. The polypeptide with a higher score is synthesized to verify the anti-inflammatory activity in an in-vitro cell experiment. The active peptide is green and safe, and has a good development and application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of marine biotechnology, and particularly relates to a sea cucumber active polypeptide and a preparation method and application thereof. BACKGROUND

[0002] Inflammation is one of the main causes of various chronic diseases such as arthritis, type II diabetes, inflammatory bowel disease, and atherosclerosis. The prevention and treatment of inflammation are mainly based on inhibiting cellular immune response, reducing pro-inflammatory cytokine secretion, and intervening in inflammatory signal transduction. However, the commonly used anti-inflammatory drugs in clinical practice have certain side effects, and long-term use may cause cardiovascular diseases and gastrointestinal bleeding. In view of the concern about the side effects of traditional anti-inflammatory drugs, the demand for using natural compounds and their derivatives to prevent and relieve chronic diseases is increasingly urgent. Bioactive peptides are extremely potential ingredients with high safety, low cost and easy absorption and other health benefits. Food-derived anti-inflammatory peptides have been shown to exhibit anti-inflammatory activity by inhibiting or reducing the expression of inflammatory markers or by regulating the activity of transcription factors. At present, anti-inflammatory peptides are mainly obtained by enzyme digestion, however, the separation and purification of anti-inflammatory peptides need to be achieved through many tedious steps, and some high-activity peptides may be lost in the screening and identification process, making it difficult to carry out large-scale screening and identification.

[0003] Therefore, it is urgent to develop a method for targeted and rapid screening of active peptides. SUMMARY

[0004] The application aims to provide a sea cucumber anti-inflammatory active peptide and a preparation method and application thereof to make up for the deficiencies of the prior art.

[0005] To achieve the above-mentioned purpose, the specific technical scheme provided by the application is:

[0006] A sea cucumber active polypeptide, whose amino acid sequence from N-terminal to C-terminal is:

[0007] Ile-Gly-Tyr-Thr-Pro-Arg-Pro-Ile-Leu-Phe, hereinafter referred to as IGYTPRPILF;

[0008] or Ile-Gly-Gly-Arg-Asp-Phe-Leu-Leu, hereinafter referred to as IGGRDFLL;

[0009] or Asp-Phe-Leu-Leu-Gln-Pro-Ile-Met-Met, hereinafter referred to as DFLLQPIMM.

[0010] A preparation method of a sea cucumber active polypeptide: first, polypeptide crude extract is extracted from sea cucumber, and then the component with anti-inflammatory activity is purified by ultrafiltration fractionation.

[0011] The specific steps of the preparation method include:

[0012] (1) Liquid nitrogen bath grinding of sea cucumber, adding water, low temperature stirring, centrifugation to obtain supernatant, dialysis and freeze-drying to obtain sea cucumber crude protein sample, and then enzymolysis to obtain sea cucumber protein hydrolysate;

[0013] (2) The sea cucumber protein hydrolysate is subjected to fractionation treatment using an ultrafiltration membrane to obtain sea cucumber peptide components with different molecular weights.

[0014] Further, in the step (1), the enzyme used for enzymolysis includes one or more combinations of flavor protease, neutral protease, complex protease, trypsin, papain and alkaline protease, and the alkaline protease is preferred; the enzymolysis time is 3-9 hours.

[0015] Further, in the step (2), the cut-off molecular weight of the ultrafiltration membrane is 3kDa, 5kDa and 10kDa, respectively; through continuous fractionation treatment, sea cucumber peptides with molecular weights of 0-3kDa, 3-5kDa and 5-10kDa are obtained, respectively.

[0016] The sea cucumber active polypeptide components with different molecular weights are identified, and the identification specifically includes:

[0017] (1) The peptide segments identified by mass spectrometry are scored and predicted using the Pre-AIP prediction website;

[0018] (2) Molecular docking is performed using Autodock Vina.

[0019] Further, in the step (1), high-confidence sea cucumber peptides, i.e., peptides with scores greater than or equal to 0.45, are screened.

[0020] Further, in the step (2), the receptors are selected as COX-2 and iNOS.

[0021] The sea cucumber active polypeptide IGYTPRPILF is applied to the preparation of an anti-inflammatory drug.

[0022] The sea cucumber active polypeptide IGGRDFLL is applied to the preparation of an anti-inflammatory drug.

[0023] The sea cucumber active polypeptide DFLLQPIMM is applied to the preparation of an anti-inflammatory drug.

[0024] The advantages and beneficial effects of the present application are that:

[0025] The application prepares anti-inflammatory polypeptides from sea cucumber by enzymatic hydrolysis, purifies by ultrafiltration fractionation, identifies polypeptide sequences by LC-MS / MS, screens anti-inflammatory active fragments by using anti-inflammatory peptide prediction website and molecular docking technology, and verifies the anti-inflammatory activity of polypeptides with higher scores by in vitro cell experiments. The obtained sea cucumber peptides can effectively inhibit the generation of inflammatory factors such as NO, IL-6, IL-1β and TNF-α, and therefore have good anti-inflammatory activity and can be applied to drug development.

[0026] The anti-inflammatory peptides provided by the application are derived from sea cucumber and have significant anti-inflammatory activity and no cytotoxicity. Therefore, the active peptides are derived from green and safe sources and have good development and application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The superoxide anion radical scavenging rate and DPPH radical scavenging rate of each component in Example 1 ultrafiltration are shown in the table.

[0028] Figure 2 The influence of different concentrations of sea cucumber active polypeptides on cell survival rate in Example 2 is shown in the table.

[0029] Figure 3 The influence of different concentrations of sea cucumber active polypeptides on the NO secretion amount of cells in Example 2 is shown in the table.

[0030] Figure 4 The influence of different concentrations of sea cucumber active polypeptides on the IL-6 secretion amount of cells in Example 2 is shown in the table.

[0031] Figure 5 The influence of different concentrations of sea cucumber active polypeptides on the IL-1β secretion amount of cells in Example 2 is shown in the table.

[0032] Figure 6 The influence of different concentrations of sea cucumber active polypeptides on the TNF-α secretion amount of cells in Example 2 is shown in the table.

[0033] Figure 7 The interaction diagram of sea cucumber active polypeptides and COX-2 in Example 2 is shown in the figure.

[0034] Figure 8 The interaction diagram of sea cucumber active polypeptides and iNOS in Example 2 is shown in the figure. DETAILED DESCRIPTION

[0035] To make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0036] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Part of the experimental methods used in the present application are as follows:

[0038] (1) Cell culture

[0039] Mouse macrophage RAW264.7 was cultured in DMEM medium containing 10% fetal bovine serum, and placed in a 37℃, 5% CO2 incubator for culture.

[0040] (2) Determination of cell viability

[0041] CCK-8 colorimetric method was used to evaluate the toxicity of sea cucumber polypeptide to RAW264.7 cells. Macrophage RAW264.7 was inoculated into 96-well plates at a concentration of 1×10 5 6 / mL, and cultured at 37℃, 5% CO2 for 24h. Then, 25, 50, 100, 200, 500 and 1000 μg / mL peptide segments and LPS (1 μg / mL) solutions were added respectively and cultured for 24h. 10 μL of CCK-8 solution was added to each well, and incubated in a 37℃ incubator for 2h. The absorbance value at 450nm was measured, and the cell viability was calculated according to formula (1-1).

[0042] Cell viability = [OD (experimental) - OD (blank)] / [OD (normal) - OD (blank)] × 100% (1-1)

[0043] In formula 1-1, OD (experimental) represents the absorbance value of polypeptide cell group added with CCK-8; OD (blank) represents the blank absorbance value of CCK-8; OD (normal) represents the absorbance value of normal cell group added with CCK-8.

[0044] (3) Determination of NO production

[0045] Macrophage RAW264.7 was inoculated into 96-well plates at a concentration of 1×10 5 6 / mL, and cultured for 24h. Then, 25 and 100 μg / mL peptide segments and LPS (1 μg / mL) solutions were added respectively and cultured for 24h. 50 μL of cell culture supernatant was taken, and 50 μL of Griess I and 50 μL of Griess II were added at room temperature. The absorbance value was measured at 540nm.

[0046] (4) Determination of cytokines TNF-α, IL-1β and IL-6

[0047] Plating, administration were performed according to the determination of (3) NO production. After the culture was completed, the supernatant was collected. The effects of different concentrations of sea cucumber anti-inflammatory peptide on the production of inflammatory factors (TNF-α, IL-1β, IL-6) of cells after induction were determined by ELISA kit.

[0048] (5) Molecular docking

[0049] ① Treatment of receptors

[0050] Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are common inflammation-related proteins. The 3D structures of COX-2 (PDB ID: 1CX2) and iNOS (PDB ID: 3NW2) were downloaded from the protein database (https: / / www1.rcsb.org / ). Using PyMol software, water molecules and original ligands were removed from the receptor protein molecules, and then saved in PDB format.

[0051] ② Preparation of ligands

[0052] The Auto Dock was used to adjust the torsion key of the peptide, and saved in PDBQT format.

[0053] ③ Docking process

[0054] The AutoDock Vina was used to dock the above-mentioned receptors and ligands, and the docking results were expressed as binding energy. The binding energy is generally negative, and the smaller the value, the higher the docking result score, that is, the tighter the combination between the receptor and the ligand, the stronger the interaction, and the greater the potential of anti-inflammatory activity. Finally, the LigPlus software was used to visualize and analyze the molecular docking results.

[0055] The present application will be further explained and described below through specific embodiments.

[0056] Example 1:

[0057] A sea cucumber active polypeptide preparation, structure identification and molecular docking, comprising the following steps:

[0058] 1. Preparation of sea cucumber active polypeptide

[0059] (1) The sea cucumber was ground into powder in a liquid nitrogen bath, and a certain amount of sea cucumber powder was mixed with distilled water at a ratio of 1:2 (w / v). Stirring at 4℃ for 12h, centrifugation, collection of supernatant, dialysis of supernatant in 10kDa dialysis bag for 2 days, freeze-drying of dialyzed supernatant, and obtaining sea cucumber crude protein sample.

[0060] (2) Mix sea cucumber crude protein with distilled water at a ratio of 1:40, add alkaline protease, react in a 50°C water bath for 9 hours, inactivate the enzyme in a boiling water bath, cool to room temperature, centrifuge, collect the supernatant, and dry to obtain a mixture of sea cucumber peptides.

[0061] (3) The protein content, total sugar content, DPPH free radical scavenging rate, and superoxide anion free radical scavenging rate of the sea cucumber peptide mixture were determined. The protein content was 47.91%, the total sugar content was 16.83%, the DPPH free radical scavenging rate was 61.90%, and the superoxide anion free radical scavenging rate was 62.27%.

[0062] 2. Preparation of sea cucumber polypeptides with different molecular weights and determination of their free radical scavenging rate

[0063] (1) The mixture of sea cucumber peptides was separated stepwise using ultrafiltration tubes with molecular weight cutoffs of 3kDa, 5kDa and 10kDa. The resulting peptide fractions had molecular weights of 0-3kDa, 3-5kDa and 5-10kDa, respectively. The peptide fractions were collected, freeze-dried and stored at 4°C for later use.

[0064] (2) The DPPH free radical scavenging rate and superoxide anion free radical scavenging rate of each component peptide were determined. For example... Figure 1 As shown, the free radical scavenging rates of components with molecular weights in the 0-3 kDa and 3-5 kDa ranges are significantly higher than those of other components. The DPPH free radical scavenging rate of the 0-3 kDa peptide component is 42.15%, and the superoxide anion free radical scavenging rate is 48.12%; while the DPPH free radical scavenging rate of the 3-5 kDa peptide component is 66.93%, and the superoxide anion free radical scavenging rate is 41.66%.

[0065] 3. Identification of polypeptide structure

[0066] (1) Dissolve the sample in 0.1% formic acid aqueous solution, centrifuge, collect the supernatant, filter through a 0.22 μm membrane, and perform LC-MS / MS analysis. Separation was performed using an Agilent Zorbax Eclipse Plus C18 RRHD (1.8 μm, 2.1 × 100 mm, Agilent, USA). Each sample was run at a flow rate of 0.3 mL / min, with an injection volume of 2 μL. Mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile solution containing 0.1% formic acid. Mass spectrometry conditions were: ESI ion source, positive ion mode, MS scan range 100-1700 m / z. Data processing was performed using MaxQuant 2.0 software, with a mother ion mass tolerance of 10 ppm and a daughter ion mass tolerance of 0.02 Da.

[0067] (2) According to database retrieval, 63 and 107 peptides were identified from 0-3 kDa and 3-5 kDa peptide components, respectively, mainly from the main yolk protein of sea cucumber. Using the PreAIP website to predict the anti-inflammatory activity of the identified peptides, 96 peptides with potential anti-inflammatory activity were obtained, of which the number of high confidence anti-inflammatory peptides was 12 peptides, as shown in Table 1.

[0068] Table 1 Anti-inflammatory activity prediction results of 12 anti-inflammatory peptides

[0069]

[0070] 4、Molecular docking

[0071] (1) Based on high confidence anti-inflammatory peptides, a total of 12 peptide models were constructed as ligands for 3 repeated molecular docking assays, and the average binding energy was calculated. In the activation process of inflammatory response, COX-2 and iNOS play a key role in amplifying inflammatory response. Many studies have shown that inhibiting COX-2 and iNOS activity can reduce the expression of pro-inflammatory factors downstream of the inflammatory pathway, hinder the pathogenesis of inflammatory diseases, and effectively alleviate the symptoms of inflammatory diseases. Anti-inflammatory substances that bind to the active sites of COX-2 and iNOS can effectively inhibit COX-2 and iNOS. Moreover, the lower the binding energy, the stronger the binding ability, and the more effective the inhibition of receptor expression.

[0072] (2) IGYTPRPILF (peptide 1), IGGRDFLL (peptide 2) and DFLLQPIMM (peptide 3) with the lowest binding energy to iNOS and COX-2 were selected for further analysis of their anti-inflammatory activity and mechanism. The binding energy of IGYTPRPILF, IGGRDFLL and DFLLQPIMM to COX-2 receptor protein was -9.0, -8.6 and -8.3 kcal / mol, respectively, and the binding energy to iNOS receptor protein was -10.2, -9.6 and -9.4 kcal / mol, respectively, indicating that the three anti-inflammatory peptides can spontaneously bind to COX-2 and iNOS receptor proteins at key active sites, achieving stable binding.

[0073] Example 2:

[0074] The anti-inflammatory activity of the active polypeptide of sea cucumber includes the following steps:

[0075] In order to further obtain anti-inflammatory activity peptides, the higher scoring peptide 1, peptide 2 and peptide 3 were artificially synthesized to verify their anti-inflammatory activity.

[0076] The three polypeptides were synthesized by Shanghai Shengong Bioengineering Co., Ltd. with a purity of more than 95%, meeting the requirements of related experiments for activity detection.

[0077] (1) Cytotoxicity

[0078] First, the safety of the three peptides was investigated separately, such as... Figure 2 As shown, peptides 1, 2, and 3 were not cytotoxic and could enhance macrophage activity within a concentration range of 25–200 μg / mL.

[0079] (2) Effects on the secretion of NO, TNF-α, IL-1β and IL-6 in cells

[0080] Cells were used with 100 μg / mL as the high-dose peptide concentration and 25 μg / mL as the low-dose peptide concentration to assess NO production and inflammatory factor levels.

[0081] like Figure 3 As shown, the NO secretion level in the LPS group was significantly higher than that in the normal group and the test substance group. The NO secretion level of different concentrations of sea cucumber peptides was significantly higher than that in the normal group (p<0.05) but lower than that in the LPS group, indicating that the peptides can stimulate macrophages, reduce NO synthesis, and thus exert an anti-inflammatory effect. In the blank control group, macrophages secreted very little NO (2.44±0.86 μM), while LPS stimulation significantly increased NO production by macrophages, reaching 47.07±2.57 μM. All peptides significantly inhibited NO production, especially peptides 1 and 2. High doses of peptides 1 and 2 reduced NO production by 45.29% and 45.08%, respectively, while 100 μg / mL peptide 3 showed a NO inhibition rate of 38.41%.

[0082] like Figure 4 As shown, LPS stimulation significantly increased IL-6 secretion in the induced group, increasing 2.81 times compared to the control group. High doses of the three peptides significantly inhibited IL-6 production. Specifically, peptide 1 and peptide 2 showed inhibition rates of 61.36% and 54.72% for IL-6 at 100 μg / mL, respectively.

[0083] like Figure 5 As shown, LPS stimulation significantly increased macrophage secretion of IL-1β, with the secretion level being 3.05 times that of the control group. Peptides 1, 2, and 3 at 100 μg / mL all significantly inhibited IL-1β secretion. Among them, peptide 1 showed the most significant inhibitory effect on IL-1β, reaching 50.57%.

[0084] like Figure 6 As shown, LPS stimulation significantly increased TNF-α secretion in the induced group, approximately 1.47 times that of the control group. Peptides 1, 2, and 3 at 100 μg / mL all significantly inhibited TNF-α secretion. Among them, peptide 1 showed the most significant inhibitory effect on TNF-α, reaching 58.52%.

[0085] (3) Molecular docking of peptides with iNOS

[0086] To further verify the anti-inflammatory mechanism of the anti-inflammatory peptides, they were docked with the receptor proteins COX-2 and iNOS, and the docking results are shown in Figure 7 and Figure 8 .

[0087] As shown in Table 2, peptide 1 forms 10 hydrophobic bonds with the COX-2 protein residues, and forms a hydrogen bond with Arg44 with a bond length of . Peptide 2 binds to 16 COX-2 protein residues, with a bond length of with Lys546, a bond length of with Arg44, and a bond length of with Lys532. Peptide 3 forms hydrophobic bonds with 21 amino acid residues of the COX-2 protein, and forms hydrogen bonds with Ser49, Asn34 and Tyr147, with a distance of 3.03, 3.08 and In addition, the oxygen atom of Asp672 on the peptide 3 and the nitrogen atom of His133 on the COX-2 are bonded by a salt bridge. Among them, peptide 1 and peptide 2 share 7 same hydrophobic sites (Phe64, Lys79, Arg61, Tyr122, Thr118, Ser119 and Ser121) and one same hydrogen bond site (Arg44) with the COX-2 receptor.

[0088] Table 2 Analysis of interaction force of anti-inflammatory peptides with receptor protein COX-2

[0089]

[0090] As shown in Table 3, peptide 1 forms hydrophobic bonds with 21 iNOS protein residues, forms a hydrogen bond with Arg375 with a bond length of , and forms a hydrogen bond with Arg260 with a bond length of . Peptide 2 forms 25 hydrophobic bonds, 2 hydrogen bonds and 1 salt bridge with the iNOS receptor protein. Peptide 3 interacts with the iNOS receptor protein through 27 hydrophobic bonds and 3 hydrogen bonds. Among them, the three peptides share 14 same iNOS active sites, peptide 1 and peptide 3 have 4 same sites, including Arg260, Tyr485, Ser256 and Asn115, and peptide 2 and peptide 3 have 6 same sites (Glu371, Pro344, Leu203, Met368, Gly196 and Ala191).

[0091] Table 3 Analysis of interaction force of anti-inflammatory peptides with receptor protein iNOS

[0092]

[0093]

[0094] From the above, the three active polypeptides of sea cucumber prepared by the present application can bind to the active sites of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), inhibit the activities of COX-2 and iNOS, and also can significantly inhibit the secretion of NO and pro-inflammatory cytokines, thereby effectively regulating the inflammatory response pathway of macrophage RAW264.7, and having significant anti-inflammatory activity.

[0095] The specific embodiments of the present application are described above. It needs to be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which does not affect the essential content of the present application.

[0096] Finally, although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A sea cucumber active polypeptide, characterized in that, The amino acid sequence of the sea cucumber active polypeptide is from N-terminal to C-terminal: Ile-Gly-Tyr-Thr-Pro-Arg-Pro-Ile-Leu-Phe, abbreviated as IGYTPRPILF; Or Ile-Gly-Gly-Arg-Asp-Phe-Leu-Leu, abbreviated as IGGRDFLL; Or Asp-Phe-Leu-Leu-Gln-Pro-Ile-Met-Met, abbreviated as DFLLQPIMM.

2. The method for preparing the active polypeptide of sea cucumber according to claim 1, characterized in that, The method comprises the following specific steps: (1) sea cucumber liquid nitrogen bath grinding, adding water, low temperature stirring, centrifugation to obtain supernatant, dialysis freeze-drying to obtain sea cucumber crude protein sample, then enzyme hydrolysis to obtain sea cucumber protein hydrolysate; the enzyme selected for enzyme hydrolysis is alkaline protease, and the enzyme hydrolysis time is 3-9 hours; (2) the sea cucumber protein hydrolysate is subjected to fractionation treatment using ultrafiltration membrane to obtain sea cucumber peptide segment components with different molecular weights; the molecular weight cut-off of the ultrafiltration membrane is 3 kDa, 5 kDa and 10 kDa respectively; Through continuous fractionation treatment, sea cucumber peptides with molecular weights of 0-3 kDa, 3-5 kDa and 5-10 kDa are obtained respectively.

3. The sea cucumber active polypeptide IGYTPRPILF in claim 1 is used for preparing an anti-inflammatory drug.

4. The sea cucumber active polypeptide IGGRDFLL in claim 1 is used for preparing an anti-inflammatory drug.

5. The sea cucumber active polypeptide DFLLQPIMM in claim 1 is used for preparing an anti-inflammatory drug.

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