A kind of active peptide of nereis virens and the use of its preparation anticoagulant product

Abstract

The present application belongs to the technical field of polypeptide, and particularly relates to a Nereis virens active peptide and a use of the Nereis virens active peptide in preparation of an anticoagulant product. In a first aspect, a preparation method of the Nereis virens active peptide is provided, which comprises: mixing Nereis virens with water after pretreatment, and adding a protease to perform enzymolysis to obtain an enzymolysis solution; the Nereis virens active peptide is selected from the enzymolysis solution or a peptide and / or a protein separated from the enzymolysis solution; and the protease is selected from one or more of a flavor protease, an alkaline protease, a neutral protease, an acid protease, a trypsin and a papain. The Nereis virens anticoagulant active peptide is obtained through directional enzymolysis of a biological enzyme, and it is found that the active peptide obtained through flavor protease enzymolysis has the highest anticoagulant activity and can significantly prolong CT, PT, APTT and TT.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology, and particularly relates to an active peptide of *Nematostella salina* and its use in preparing anticoagulant products. Background Technology

[0002] Thrombosis is a semi-solid structure formed by the abnormal aggregation of blood components (including platelets, fibrin, erythrocytes, and leukocytes). Its formation exceeds the physiological hemostasis requirements, leading to partial or complete vascular occlusion. Thrombosis-related diseases are a significant type of cardiovascular and cerebrovascular disease that seriously threatens human health. Their occurrence and development are closely related to platelet activation, abnormal activation of the coagulation cascade, and fibrin deposition. Thrombin (THR), as a core effector enzyme in the coagulation process, can cleave fibrinogen into fibrin monomers and promote platelet aggregation and thrombus formation, making it a key target for antithrombotic drug development. Currently, commonly used anticoagulants include heparin, argatroban, and bivalirudin. These drugs inhibit the coagulation process through different mechanisms and play an important role in the prevention and treatment of thrombotic diseases. However, existing anticoagulants have drawbacks such as high bleeding risk, the need for frequent monitoring, and the potential to induce complications. Therefore, developing novel natural antithrombotic active substances that are safe, highly specific, and have a clearly defined mechanism of action has significant clinical application value.

[0003] Annelids (Phyllium of Annelids) Annelida Marine organisms are a rich source of natural antithrombotic peptides and proteins. Studies have shown that marine-derived peptides, based on their unique amino acid sequences, hydrophobicity, and binding affinity, exhibit significant biological activities in antithrombosis, blood pressure reduction, and antioxidation. Among them, polychaetes contain a variety of active substances with anticoagulant and thrombolytic activities. Polychaetes var. wartyli (… Tylorrhynchus heterochaetus Widely distributed in brackish water areas and estuaries, these organisms are rich in protein and functional active ingredients, making them both a nutritious food source and a potential bioactive resource. However, current research mainly focuses on the Japanese spiny silkworm (…). Neanthes japonica ) and double-toothed sandworm ( Perinereis aibuhitensis Systematic studies on the antithrombotic active peptides of *Nematocystis wartyri* are still limited. Summary of the Invention

[0004] To address the problems existing in the prior art, firstly, a method for preparing active peptides from *Nematocystis wartyum* is provided, comprising: After pretreatment, the warty-snout worm is mixed with water and then enzymatically hydrolyzed with protease to obtain an enzymatic hydrolysate. The active peptides of the warty-snout worm are selected from the enzymatic hydrolysate or peptides and / or proteins isolated from the enzymatic hydrolysate. The protease is selected from one or more of flavor protease, alkaline protease, neutral protease, acidic protease, trypsin, and papain.

[0005] In some embodiments, the protease is selected from flavor proteases.

[0006] In some embodiments, the active peptide of *Nematostella salina* is selected from peptides with a molecular weight <1 kDa isolated from the enzymatic hydrolysate.

[0007] In some embodiments, the active peptide of *Nematostella wartyri* comprises one or more peptides with the following amino acid sequences: GFEIPEPYKW, ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, EIPEPFKW, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, IEDHFLF, WPDGR, GFEIPEPF, EFMWN, PEPFKW.

[0008] In some embodiments, the enzymatic hydrolysis temperature is 30-70°C, for example, any one of 30, 40, 50, 60 or 70°C, or a range between any two values, preferably 40-60°C.

[0009] In some embodiments, the pH is 5.5-9.5, for example, it can be any one of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.5, or a range between any two values, which can be 5.5-7.5 or 7.0-8.0, preferably 5.5-8.0.

[0010] In some embodiments, the enzymatic hydrolysis time is 3-7 hours, for example, any value among 3, 4, 5, 6 or 7 hours, or a range between any two values, preferably 3-5 hours.

[0011] In some embodiments, the enzyme dosage is 3000-7000 U / g, for example, it can be any one of 3000, 4000, 5000, 6000 or 7000 U / g, or a range between any two values, preferably 3000-5000 U / g.

[0012] In some embodiments, the material-to-liquid ratio is 1:30-70 g / mL, for example, it can be any one of 1:30, 1:40, 1:50, 1:60 or 1:70 g / mL, or a range between any two values, preferably 1:30-40 g / mL.

[0013] Secondly, an active peptide of *Nematostella salina* prepared by the method described in the first aspect is provided.

[0014] Thirdly, a polypeptide is provided, the amino acid sequence of which is selected from one or more of the following: GFEIPEPYKW, ARDWPDGRG, WDESFKVF, DWPDGRGIWH, EIPEPFKW, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, IEDHFLF, WPDGR, GFEIPEPF, EFMWN, PEPFKW.

[0015] In some embodiments of the third aspect, the amino acid sequence of the polypeptide is selected from one or more of the following peptides: ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, WPDGR, GFEIPEPF, EFMWN.

[0016] In some embodiments of the third aspect, the amino acid sequence of the polypeptide is selected from one or more of the following peptides: ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, WPDGR, EFMWN.

[0017] Fourthly, a polypeptide composition is provided, comprising one or more peptides with the following amino acid sequence: GFEIPEPYKW, ARDWPDGRG, WDESFKVF, DWPDGRGIWH, EIPEPFKW, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, IEDHFLF, WPDGR, GFEIPEPF, EFMWN, PEPFKW.

[0018] In some embodiments of the fourth aspect, the polypeptide composition comprises one or more peptides with the following amino acid sequences: ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, WPDGR, GFEIPEPF, EFMWN.

[0019] In some embodiments of the fourth aspect, the polypeptide composition comprises one or more peptides with the following amino acid sequences: ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, WPDGR, EFMWN.

[0020] Fifthly, the invention provides the use of the active peptide of *Nematostella spp.* prepared by the method of the first aspect, or the active peptide of the second aspect, or the polypeptide of the third aspect, or the peptide-containing composition of the fourth aspect, in the preparation of anticoagulant products and / or antithrombotic products and / or products that improve coagulation function indicators.

[0021] In some embodiments, the product is selected from one or more of the following: general food, functional food, dietary supplement, health product, and pharmaceutical.

[0022] Terminology Definition As used in this article, the term "room temperature" refers to 20-25°C.

[0023] As used herein, the term “coagulation function index” includes clotting time (CT), activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT), and the term “improvement of coagulation function index” refers to the ability to alter at least one of CT, APTT, TT, and PT.

[0024] As used herein, the terms “anticoagulation” and “anticoagulant” have the same meaning, referring to the ability to delay or prevent blood clotting both in vivo and / or in vitro.

[0025] As used herein, the term "antithrombotic" refers to a substance that has the ability to prevent or reduce the formation of abnormal thrombi in the blood, and / or to treat and / or dissolve existing thrombi, and / or to have antiplatelet activity, and / or to restore or maintain normal blood flow in humans and / or animals.

[0026] As used herein, the term "ordinary food" means any substance intended for human consumption, whether processed, semi-processed or unprocessed, and any substance used in the manufacture, preparation or processing of food.

[0027] As used in this article, the term "functional food" refers to food with specific nutritional and health benefits, that is, food suitable for specific groups of people, which has the function of regulating bodily functions and is not intended for treatment.

[0028] As used in this article, the term "dietary supplement" is also known as a nutritional supplement, nutritional product, nutrient, or dietary supplement. It is used as an adjunct to diet to supplement the amino acids, trace elements, vitamins, minerals, etc. required by the human body.

[0029] As used in this article, the term "Mw" refers to molecular weight.

[0030] As used herein, the term "aminolysis" refers to the process by which a molecule (usually a compound containing an acyl group) reacts with ammonia (NH3) or an amine, resulting in the breaking of existing chemical bonds and the formation of one or more new amide bonds or amine compounds.

[0031] Compared with the prior art, a certain embodiment of the present invention includes at least one of the following beneficial effects: Anticoagulant active peptides from *Nereidum wartyum* were obtained by targeted enzymatic hydrolysis. It was found that the active peptides obtained by enzymatic hydrolysis with flavor protease had the highest anticoagulant activity and could significantly prolong CT, PT, APTT, and TT.

[0032] Through single-factor experiments, the optimal production conditions for producing anticoagulant active peptides from *Nereis spp.* using flavored protease were identified.

[0033] Further experiments demonstrated that peptides with a molecular weight <1 kDa isolated from the flavor protease hydrolysate by membrane filtration exhibited high anticoagulant activity. At least 15 peptide fragments with certain anticoagulant activity were identified, providing novel anticoagulant active peptides and novel functional peptides of *Nereis wartyus* for existing technologies. This enhances the added value of the *Nereis wartyus* industry and provides new ideas for the comprehensive utilization of *Nereis wartyus*. Attached Figure Description

[0034] Figure 1 To investigate the anticoagulant activity of different enzyme-treated *Nereidum wartyum* hydrolysates.

[0035] Figure 2 The effects of different enzymatic hydrolysis conditions (temperature (A), pH (B), feed-to-liquid ratio (C), time (D), and enzyme dosage (E)) on the anticoagulant activity of the enzymatic hydrolysate under single-factor experiments were investigated.

[0036] Figure 3 The effects of THACP on TT, APTT, and PT.

[0037] Figure 4 Anticoagulant activity of flavor proteases of different molecular weights from *Nephropus glomeratus* hydrolysate powders.

[0038] Figure 5 Screening for thrombin inhibitory activity of candidate peptides.

[0039] In the above figures, anticoagulant activity refers to anticoagulant activity, absorbance refers to absorbance, time refers to time, temperature refers to temperature, material to liquid ratio refers to the material-to-liquid ratio, and enzyme dosage refers to the amount of enzyme used. Detailed Implementation

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise stated below, the terms used herein have the meanings conventionally understood by those skilled in the art. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0041] Experimental materials: Flavor protease, alkaline protease, neutral protease, acidic protease, trypsin, papain, and heparin were purchased from Beijing Solarbio Science & Technology Co., Ltd.; thrombin, bovine fibrinogen, activated partial thromboplastin time assay kit, thrombin time assay kit, and prothrombin time assay kit were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; PH1904 sodium citrate anticoagulant was purchased from Fuzhou Feijing Biotechnology Co., Ltd.; Tris(hydroxymethyl)aminomethane (Tris) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; HCl (0.1 mol / L) was purchased from Shenzhen Bolinda Technology Co., Ltd.; NaCl (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.; Thrombin chromogenic substrate S-2238 was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0042] Each embodiment's experimental determination was performed four times, and the final data are expressed as mean ± standard deviation. GraphPad Prism software was used to analyze and plot the significance of each experimental result; p < 0.05 indicates a significant difference between the two groups. In the following embodiments, the statistical significance test results are labeled using letters. In the relevant figures, if the same indicator contains only one letter, the difference is not significant (P > 0.05), and different letters represent significant differences between groups.

[0043] Example 1: Preparation and activity analysis of anticoagulant active peptides from *Nereidum wartyum* (1) After the warty sandworm is crushed and homogenized, it is subjected to vacuum freeze drying. The specific parameters are (-40 ℃, 3 min; -30 ℃, 12 h; -20 ℃, 12 h; -10 ℃, 12 h; 0 ℃) to obtain powdered sandworm powder, which is stored at -20 ℃ for later use.

[0044] (2) Add powdered *Nereis spp.* powder to ultrapure water at a material-to-liquid ratio of 1:50 g / mL and shake thoroughly to mix. Preheat the mixture in a constant-temperature water bath at the corresponding protease temperature (Table 1). Adjust the pH of the system according to the applicable pH value of each protease (Table 1), and then add the corresponding protease at a dosage of 5000 U / g. Hydrolyze continuously at the appropriate temperature (Table 1) for 5 h, then transfer the mixture to a 95–100℃ water bath and heat for 20 min to terminate enzyme activity. After the liquid cools naturally to room temperature, centrifuge at 4℃ and 8000 rpm for 20 min and collect the supernatant. In this study, pepsin (PEP), acidic protease (ACP), flavor protease (FLA), papain (PAP), alkaline protease (ALP), and neutral protease (NP) were selected as hydrolytic enzymes. After enzymatic hydrolysis, the anticoagulant activity of different hydrolysates was determined by titration to screen for the optimal protease type for hydrolyzing *Nereis spp.*.

[0045] Table 1 Optimal enzymatic hydrolysis conditions for different proteases

[0046] (3) Anticoagulant activity assay: Referring to the research of Zhong Chao (Zhong Chao. Enzymatic preparation and activity study of anticoagulant peptides from leeches [D]. South China University of Technology, 2012.), the antithrombotic activity was determined by titration. A blank control experiment was first conducted. 100 μL of 0.9% (w / w) sodium chloride solution and 200 μL of 0.5% (w / w) bovine fibrinogen Tris-HCl buffer solution (Tris-HCl buffer concentration was 50 mM) were mixed, and 5 μL of THR (150 U / mL) solution was added dropwise to observe whether coagulation occurred. The result was coagulation, that is, the fibrinogen and thrombin activities were normal.

[0047] Formal Experiment: Take 1 g of the lyophilized sample after enzymatic hydrolysis, prepare a sample solution of 0.2 g / mL with 0.9% (w / w) sodium chloride solution, centrifuge at 8000 rpm for 10 min, and take 100 μL of the supernatant into a test tube. Add 200 μL of 0.5% (w / w) bovine fibrinogen Tris-HCl buffer, mix thoroughly, and incubate in a 37.0℃ water bath for 5 min. Subsequently, add 5 μL of THR (150 U / mL) solution dropwise every 1 min, while gently mixing, until the mixture forms a clot, and record the required number of THR additions. Calculate the anticoagulant activity of the sample according to the following formula:

[0048] In the formula, U is the anticoagulant activity (U / g), c is the THR activity concentration (U / mL), ρ is the sample solution mass concentration (g / mL), V1 is the volume of THR consumed (μL), and V2 is the sample solution volume (μL).

[0049] Experimental results: such as Figure 1 As shown, all enzymatic hydrolysates exhibited certain anticoagulant activity. Among them, the hydrolysates of alkaline protease and flavor protease showed stronger anticoagulant activity, at 810 U / g and 795 U / g, respectively, with no significant difference in the significance analysis. Therefore, considering factors such as the anticoagulant activity of the hydrolysates, peptide characteristics, and reaction conditions, flavor protease was ultimately selected for the subsequent preparation of anticoagulant peptides from *Nereis wartyus*.

[0050] Example 2: Single-factor experiment on the preparation of anticoagulant active peptides from *Nereis spp.* using flavor protease. The preparation process was optimized through single-factor experiments. The effects of four factors—enzymatic hydrolysis temperature, pH value, enzymatic hydrolysis time, and enzyme dosage—on the anticoagulation activity of the enzymatic hydrolysate (the determination method is the same as in Example 1 (3)) were investigated by setting different levels.

[0051] 1. Enzymatic hydrolysis temperature: Under the conditions of pH 7.5, hydrolysis time of 5 h, enzyme dosage of 5000 U / g, and material-to-liquid ratio of 1:50 g / mL, isothermal enzymatic hydrolysis was performed at temperatures of 30, 40, 50, 60, and 70 °C. The effect of different temperatures on the anticoagulant activity of the protein hydrolysate was analyzed using the anticoagulant activity of the hydrolysate as an indicator.

[0052] 2. Enzymatic hydrolysis pH: Under the conditions of enzymatic hydrolysis temperature of 50 ℃, enzymatic hydrolysis time of 5 h, enzyme dosage of 5000 U / g, and material-to-liquid ratio of 1:50 g / mL, five systems with pH values ​​of 5.5, 6.5, 7.5, 8.5, and 9.5 were set up, and the anticoagulant activity of the enzymatic hydrolysate was measured to analyze the effect of pH change on the anticoagulant effect.

[0053] 3. Enzymatic hydrolysis time: Under the conditions of enzymatic hydrolysis temperature of 50 ℃, pH value of 7.5, enzyme dosage of 5000 U / g, and material-liquid ratio of 1:50 g / mL, the anticoagulant activity was measured at enzymatic hydrolysis times of 3, 4, 5, 6 and 7 h to determine the optimal enzymatic hydrolysis time and analyze its effect on the anticoagulant properties of the enzymatic hydrolysate.

[0054] 4. Enzyme dosage: Under the conditions of enzymatic hydrolysis temperature of 50 ℃, pH value of 7.5, enzymatic hydrolysis time of 5 h, and material-to-liquid ratio of 1:50 g / mL, the enzyme dosage was set to 3000, 4000, 5000, 6000 and 7000 U / g, and the trend of anticoagulant activity was measured to analyze the effect of enzyme dosage on the anticoagulant activity of the enzymatic hydrolysate.

[0055] 5. Material-to-liquid ratio: Under the conditions of enzymatic hydrolysis temperature of 50 ℃, pH value of 7.5, enzymatic hydrolysis time of 5 h, and enzyme dosage of 5000 U / g, the material-to-liquid ratio was set to 1:30, 1:40, 1:50, 1:60 and 1:70 g / mL, respectively, and the trend of anticoagulant activity was measured to evaluate the effect of material-to-liquid ratio on the anticoagulant activity of the enzymatic hydrolysate.

[0056] Experimental results: Depend on Figure 2 As shown in Figure A, the temperature during enzymatic hydrolysis has a significant impact on the anticoagulant activity of the *Nereidum spp.* hydrolysate. The results indicate that the anticoagulant activity first increases and then decreases with increasing temperature, reaching its highest value between 40 and 60 °C. The inhibition rate increases rapidly with rising temperature, reaching a maximum of 796.88 U / g at 50 °C, and then shows a decreasing trend.

[0057] like Figure 2 As shown in Figure B, pH value has a significant impact on the THR inhibitory activity of the enzymatic hydrolysate. When the pH of the protease hydrolysate is between 5.5 and 7.5, the anticoagulant activity gradually increases, reaching a maximum of 812.5 U / g at pH 7.5, and then gradually decreases. The optimal pH range for flavor proteases is generally between 7.0 and 8.0; deviations from this range will lead to a significant decrease in enzyme activity or even complete inactivation.

[0058] like Figure 2 As shown in Figure C, the substrate-to-liquid ratio has a certain influence on the anticoagulant activity of the enzymatic hydrolysate. With the increase of the substrate-to-liquid ratio, the anticoagulant activity of the enzymatic hydrolysate first increases and then tends to stabilize; the anticoagulant activity reaches its highest value of 834.38 U / g when the substrate-to-liquid ratio is 1 / 40 g / mL; at this point, further increasing the substrate concentration actually leads to a decrease in anticoagulant activity.

[0059] like Figure 2 As shown in Figure D, the effect of enzymatic hydrolysis time on the anticoagulant activity of the hydrolysate is relatively limited. The anticoagulant activity of the hydrolysate first increases steadily with time and then decreases slowly. At 5 h of hydrolysis, the anticoagulant activity reaches its highest value of 787.5 U / g and gradually decreases, showing no significant difference compared to the anticoagulant activity of 775 U / g at 6 h (P > 0.05). Further extension of the reaction time does not lead to a further increase in anticoagulant activity. The results indicate that the enzymatic hydrolysis reaction has essentially reached equilibrium at 5 h.

[0060] Figure 2 E shows the effect of enzyme dosage on the anticoagulant activity of the enzyme hydrolysate. When the enzyme dosage is 3000-5000 U / g, the anticoagulant activity increases slowly, reaching a maximum of 800 U / g at an enzyme dosage of 5000 U / g, at which point there is no significant difference compared to other enzyme dosages.

[0061] Example 3: Response surface methodology optimization for the preparation conditions of anticoagulant active peptides from *Nematocystis wartyum* using flavored protease. Based on the single-factor experiment results of Example 2, when the enzymatic hydrolysis time reached 5 h, there was no significant difference in the degree of hydrolysis at different time points (P<0.05), therefore the enzymatic hydrolysis time was determined to be 5 h. Simultaneously, considering both the enzymatic hydrolysis effect and cost factors, the optimal enzyme dosage was determined to be 5000 U / g. Using the Box-Behnken Design response surface methodology and Design-Expert software, the anticoagulant activity of the *Nematocystis jirovecii* enzymatic hydrolysate was used as the response value (Y). Three factors affecting the anticoagulant activity—temperature (A), pH (B), and feed-to-liquid ratio (C)—were selected as independent variables. A three-factor, three-level response surface analysis method was used for the response surface experimental design. The analysis factors and level design are shown in Table 2. Specific experimental schemes and results are shown in Table 3.

[0062] Table 2 Response Surface Experimental Factor Level Design

[0063] Table 3 Response Surface Analysis Scheme and Results

[0064] The data in Table 3 were fitted and analyzed to obtain the multiple regression equation:

[0065] The results of the analysis of variance for the regression model are shown in Table 4. The F-value of this model is 137.59, corresponding to... p <0.0001 indicates that the model as a whole reaches a highly significant level, the established equation fits the actual data well, and can accurately reflect the effects of enzymatic hydrolysis temperature, pH value, and material-to-liquid ratio on the enzymatic hydrolysis process of *Nereidium warta*. (Misfit term) p The value is 0.8387 ( p The coefficient of determination (R²) is greater than 0.05, indicating that the model did not exhibit significant systematic bias and could reflect the true variation of the experimental data well. 2 =0.9944, indicating that the model can explain 99.44% of the response value changes, has good predictive ability, and is suitable for the optimization analysis of the enzymatic hydrolysis process of *Nematostella spp.*

[0066] Table 4. Analysis of Variance of Secondary Response Surface

[0067] The optimal enzymatic hydrolysis conditions predicted by the model are: hydrolysis temperature 50.87 ℃, pH 7.46, and feed-to-liquid ratio 1:41.5 g / mL. Under these conditions, the theoretical anticoagulant activity is 806.76 U / g. Further, four parallel validation experiments were conducted at a hydrolysis temperature of 51 ℃, pH 7.5, feed-to-liquid ratio of 1:41.5 g / mL, enzyme dosage of 5000 U / g, and hydrolysis time of 5 h. The actual anticoagulant activity was measured to be 808.13 U / g. The measured value is close to the theoretical prediction, and the data fluctuation is small, indicating that the established model is reliable, the process is stable, and it can be used to guide actual enzymatic hydrolysis production. Therefore, in the following examples, the preparation conditions for the preparation of anticoagulant active peptides of *Nereis spp.* (hereinafter referred to as THACP) using flavor protease were 51.0°C, pH 7.5, enzyme dosage 5000 U / g, material-to-liquid ratio 1:41.5 g / mL, and time 5 h. Other preparation operations are as described in Example 1.

[0068] Example 4: THACP in vitro coagulation time analysis In vitro blood coagulation test: Using the THACP-containing enzymatic hydrolysate obtained by hydrolyzing powdered *Nephropus spp.* according to the THACP preparation conditions obtained in Example 2 as the sample, the in vitro anticoagulant activity was verified according to the methods of Lü Binfei (Lü Binfei. Preparation and product development of egg yolk high-phosphorus protein anticoagulant peptide [D]. Tianjin University of Science and Technology, 2024.) and Wei Wei (Wei Wei. Isolation, purification, structural identification and antithrombotic activity study of earthworm protein peptide [D]. Jiangsu University, 2022.). Specifically, 5 mL of rabbit venous blood was centrifuged at 800 rpm for 10 min, and the supernatant was collected to obtain platelet-rich plasma; subsequently, the lower layer of blood was centrifuged at 3000 rpm for 15 min, and the supernatant was collected to obtain anemic platelet-rich plasma (PPP). Physiological saline, trisodium citrate (4% by mass), sample (40 mg / mL, high-dose group), sample (20 mg / mL, medium-dose group), and sample (10 mg / mL, low-dose group) solutions were added to the anemic platelet-rich plasma, respectively. The coagulation time of each group was measured, with each group repeated 5 times. The experimental method was performed in accordance with the kit instructions. CT: Add 8 mL of sterile saline solution to a centrifuge tube, slowly add 1 mL of fresh rabbit blood along the tube wall, and then add 1 mL of the sample to be tested. Place in a 37 ℃ water bath and record the time required for complete blood coagulation.

[0069] PT: Take 0.1 mL of PPP into a centrifuge tube, add 0.1 mL of the sample to be tested, mix well, and incubate at 37 ℃ for 2 min. Then add PT thromboplastin solution, start timing immediately, and gently tilt the test tube to record the time required for the liquid to stop flowing. Repeat 2–3 times and take the average value.

[0070] APTT: Add 0.1 mL of PPP and 0.1 mL of the sample to be tested to each tube. After equilibrating the APTT ellagic acid solution to room temperature, add 0.1 mL to each tube, mix well, and place in a 37 ℃ water bath for 5 min, gently mixing several times during this period. Then add 0.1 mL of CaCl2 (25 mM) incubated at 37 ℃, start timing immediately, and continue to gently shake. After about 30 s, remove the tube and observe the fibrin filament formation time. Repeat twice and take the average value.

[0071] TT: Add 0.1 mL of PPP and 0.1 mL of the sample to be tested to each tube, and incubate in a 37 ℃ water bath for 5 min. Then add 0.1 mL of THR solution and record the solidification time. Repeat 2-3 times and take the average value.

[0072] Experimental results: The coagulation process encompasses intrinsic, extrinsic, and common pathways, and involves multiple coagulation factors. CT, APTT, PT, and TT are commonly used indicators for assessing anticoagulant activity, reflecting the whole blood coagulation status, intrinsic coagulation pathway, extrinsic coagulation pathway, and THR function in the common pathway, respectively.

[0073] like Figure 3 As shown, after standing for 5 minutes, both the medium- and high-dose THACP groups significantly prolonged the CT value (P<0.05 compared to the control group), and the thrombus morphology was small, with increased release of red blood cells in the supernatant, exhibiting anticoagulant and thrombolytic effects similar to those of sodium citrate (positive control). Further analysis of coagulation pathways showed that THACP significantly prolonged PT, APTT, and TT, with significant differences between the medium- and high-dose groups and the control group (P<0.05). Specifically, the high-dose group prolonged PT to 357.05 s, TT to 88.1 s, and APTT to 88.05 s. These results indicate that THACP can effectively inhibit the extrinsic coagulation pathway and has a certain inhibitory effect on the intrinsic pathway, with its mechanism of action mainly manifested in the potent inhibition of THR activity. Compared to sodium citrate (positive control), which non-specifically blocks the coagulation process by chelating calcium ions, THACP, as a bioactive peptide, exhibits broad-spectrum inhibitory activity, likely due to its specific binding to specific targets. This typically implies better biocompatibility and a potentially lower bleeding risk. Compared to the egg yolk high-phosphorus protein anticoagulant peptide prepared by Lü Binfei (see reference above) (APTT 94.67 s, TT 51.67 s, PT 12.33 s), THACP comprehensively surpasses it in terms of the prolongation of PT, APTT, and TT, directly demonstrating its stronger in vitro anticoagulant potency.

[0074] Example 5: Ultrafiltration Separation and Activity Analysis of THACP (1) The THACP-containing enzymatic hydrolysate obtained by hydrolyzing powdered worm powder according to the THACP preparation conditions obtained in Example 2 was first passed through a filtration system equipped with a 200 nm ceramic membrane, and then through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to separate two anticoagulant components with different molecular weights, THACP-L (Mw<1 kDa) and THACP-H (Mw>1 kDa). After lyophilization, 1 g of lyophilized sample was taken and prepared into a sample solution of 0.2 g / mL with 0.9% sodium chloride solution for anticoagulant activity determination.

[0075] (2) Determination of anticoagulant activity: Same as in Example 1 (3).

[0076] Experimental results: The results are as follows Figure 4 As shown, at the same mass concentration, the anticoagulant activity of THACP in different molecular weight ranges varies significantly. Among them, THACP-L exhibits the highest anticoagulant activity, reaching 1012.5 U / g. THACP-L has a relatively simple structure and a small size, making it easier to penetrate and reach the target site, or possessing specific key active sequences, thus exhibiting better anticoagulant performance. Therefore, THACP-L was selected to continue the experiments in the following examples.

[0077] Example 6: Screening of THACP-L based on thrombin inhibitory activity (1) THACP-L Peptide Omics Identification: Samples were analyzed using an LC-MS / MS system equipped with an online nano-spray ionization source. The system consisted of a tandem EASY-nanoLC 1200 liquid chromatograph and an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA). Each sample loading was 5 μL, using a C18 column (20 cm × 75 μm id, 1.9 μm particle size) at a flow rate of 300 nL / min and a column temperature of 40 °C for 60 min gradient separation. The gradient program was: initial B-phase content 4%, increased to 50% within 53 min 40 s, then increased to 95% within 40 s and maintained for 5 min 40 s. The mass spectrometer operated in data-dependent acquisition (DDA) mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: MS scan: Scan range m / z 100–1500; resolution 120,000; normalized AGC target 200%; maximum injection time 100 ms.

[0078] HCD-MS / MS: Resolution 50,000; Normalized AGC target 200%; Maximum injection time 86 ms; Collision energy 25%, 30%, 35%; Dynamic exclusion time 30 s.

[0079] (2) Molecular docking screening based on THR targets Based on mass spectrometry analysis, the potential biological activity of peptides was predicted using the Peptide Ranker program, and peptides with activity values ​​higher than 0.60 were screened. ToxinPred was used to predict the toxicity of peptide compounds, and toxic peptides were removed. The three-dimensional crystal structure of THR (PDB: 2BVR) was downloaded from the PDB database, and the water of crystallization and co-crystallization ligands were removed using the YInfotek platform (https: / / www.yinfotek.com / ), and hydrogen atoms were added to the protein. The original ligand hirudin was used as the active site to determine the docking site. The active site was enclosed in a 3D 30.00 Å grid, and the YInfotek platform was used to optimize the ligands and minimize energy for the screened peptides. DOCK 6.9 was used to calculate the docking score; the lower the score, the stronger the binding force. The docking was considered rigid.

[0080] (3) Solid-phase synthesis of peptides: Based on the results of molecular docking, THR inhibitory peptides were selected according to the grid score and binding mode. The THR inhibitory peptides were entrusted to GenScript for solid-phase synthesis. The purity and molecular weight were identified by RP-HPLC and LC / MS.

[0081] (4) Detection of in vitro anticoagulant activity based on S-2238 chromogenic substrate method Blank group (NC): 50 μL of buffer (50 mM Tris-HCl, 100 mM sodium chloride, pH 7.4); Experimental group: 50 μL of buffer solutions of different THR inhibitory peptides (i.e., buffer solution of the blank group, with the concentration of THR inhibitory peptide relative to the buffer solution being 1 mg / mL); Positive control (PC) group: 50 μL of heparin buffer solution (i.e., the buffer solution of the blank group, with a heparin concentration of 1 mg / mL relative to the buffer solution); Each group was incubated at 37 °C for 2 min, followed by the addition of THR (20 μL, 10 U) and incubation at 37 °C for 1 min. The reaction was initiated by adding S-2238 chromogenic substrate (20 μL, 1.5 mM), and the release of the colored product p-nitroaniline (p-NA) was monitored by absorbance at 405 nm. The results are as follows: Figure 3 As shown.

[0082] Experimental Results: Based on the mass spectrometry results, peptides with a Peptide Ranker activity value higher than 0.60 and no toxicity were screened, ultimately resulting in 100 peptide sequences. These 100 peptides were molecularly docked with THR receptor proteins (DOCK6.9), and 15 THR repressive peptide sequences were selected based on docking scores and binding modes. The peptide sequence fragments are shown in Table 5.

[0083] Table 5. Peptide sequences and THR docking scores

[0084] The above 15 peptides were synthesized in a solid phase, and the THR inhibitory activity of the candidate peptides was determined by the S-2238 chromogenic substrate method, and the THR inhibitory peptides were preliminarily screened. Figure 5 The results showed that the amination activity of THR was inhibited in a time-dependent manner under different protein peptides. Since the coagulation process involves the formation of peptide bonds (amide bonds) catalyzed by THR, the amination activity of THR in this experiment represents the enzymatic activity of thrombin. Figure 5 This indicates that the inhibition of thrombin activity decreases over time. Figure 5 Of the curves corresponding to these 15 peptides, only those below NC (blank group) indicate significant THR inhibitory activity. Clearly, most of these 15 peptides (except for peptides 11, 7, 5, and 15) exhibit some THR inhibitory activity.

[0085] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing an active peptide from *Nematocystis wartyri*, characterized in that, include: After pretreatment, the warty-snout worm is mixed with water and then enzymatically hydrolyzed with protease to obtain an enzymatic hydrolysate. The active peptides of the warty-snout worm are selected from the enzymatic hydrolysate or peptides and / or proteins isolated from the enzymatic hydrolysate. The protease is selected from one or more of flavor protease, alkaline protease, neutral protease, acidic protease, trypsin, and papain.

2. The method according to claim 1, characterized in that: The protease is selected from flavor proteases; and / or the active peptide of *Nematostella styracifolium* is selected from peptides with a molecular weight <1 kDa isolated from the enzymatic hydrolysate.

3. The method according to claim 2, characterized in that: The active peptides of *Nematostella wartyri* include one or more peptides with the following amino acid sequences: GFEIPEPYKW, ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, EIPEPFKW, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, IEDHFLF, WPDGR, GFEIPEPF, EFMWN, PEPFKW.

4. The method according to claim 2 or 3, characterized in that: The conditions for the enzymatic hydrolysis are as follows: The enzymatic hydrolysis temperature is 30-70℃, and / or the pH is 5.5-9.5, and / or the enzymatic hydrolysis time is 3-7h, and / or the enzyme dosage is 3000-7000U / g, and / or the material-to-liquid ratio is 1:30-70 g / mL.

5. The method according to claim 4, characterized in that: The conditions for the enzymatic hydrolysis are as follows: The enzymatic hydrolysis temperature is 40-60℃, and / or the pH is 5.5-8.0, and / or the enzymatic hydrolysis time is 3-5 h, and / or the enzyme dosage is 3000-5000 U / g, and / or the material-to-liquid ratio is 1:30-40 g / mL.

6. An active peptide of *Nematostella spp.* prepared by the method of any one of claims 1-5.

7. A polypeptide, characterized in that, Its amino acid sequence is selected from one or more of the following: GFEIPEPYKW, ARDWPDGRG, WDESFKVF, DWPDGRGIWH, EIPEPFKW, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, IEDHFLF, WPDGR, GFEIPEPF, EFMWN, PEPFKW.

8. A peptide-containing composition, characterized in that: Including one or more peptides with the following amino acid sequences: GFEIPEPYKW, ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, EIPEPFKW, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, IEDHFLF, WPDGR, GFEIPEPF, EFMWN, PEPFKW; Or include one or more peptides with the following amino acid sequences: ARDWPDGRGIW, WDESFKVF, DWPDGRGIWH, DWPDGRGIW, GFEIPEPFKW, GVDNPGHPFIM, RDWPDGRG, DWPDGRG, WPDGR, GFEIPEPF, EFMWN.

9. Use of a *Nereis globosum* active peptide prepared by the method of any one of claims 1-5, or an active peptide of claim 6, or a polypeptide of claim 7, or a peptide-containing composition of claim 8, in the preparation of anticoagulant products and / or antithrombotic and / or products that improve coagulation function indicators.

10. The use according to claim 9, characterized in that, The product is selected from one or more of the following: general food, functional food, dietary supplement, health product, and medicine.

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