An anticoagulant peptide, its preparation method and application

Abstract

The present invention discloses an anticoagulant peptide, its preparation method and application. The present invention optimizes the extraction and separation method of anticoagulant activity of Eupolyphaga sinensis, thereby detecting ultra-trace anticoagulant peptide RF7 from complex crude peptide extracts, and using the NanoLC-Q-Orbitrap-MS / MS method to perform high-throughput analysis and identification of the peptide sequences of the screened active anticoagulant peptides. Through the search and comparison of the intelligent data processing system and the polypeptide database, the biological information and pharmaceutical components of the anticoagulant peptide are comprehensively analyzed. Finally, by constructing molecular level, cell and animal models, pharmacokinetic and pharmacodynamic studies are carried out on the anticoagulant peptides with obvious activity to evaluate the safety and effectiveness of the drug, laying a research foundation for the development and drug-likeness evaluation of new anticoagulant drugs.

Description

Technical Field

[0001] The present invention relates to a peptide, a preparation method and an application thereof, and particularly relates to an anticoagulant peptide, a preparation method and an application thereof, belonging to the field of bioactive peptides. Background Art

[0002] Thrombotic diseases are a type of diseases with relatively high incidence, mortality and clinical disability rates in cardiovascular diseases. Therefore, antithrombotic therapy is crucial for the prevention and treatment of cardiovascular diseases. Currently, commonly used anticoagulant drugs such as heparin and warfarin have good curative effects in clinical practice. However, due to disturbing the delicate balance between procoagulation and anticoagulation in the body, they have potential bleeding risks. Therefore, finding new anticoagulant drugs with good anticoagulant effects and low bleeding risks has become a research hotspot in cardiovascular diseases.

[0003] Bioactive peptides are compounds between proteins and amino acids, with a molecular weight less than 6000 Da and composed of two or more amino acid residues. Due to their unique structures and sequences, they have good biocompatibility, can specifically interact with target proteins, and have a low incidence of adverse reactions. They have gradually become popular candidate drugs in current drug development. Traditional Chinese animal medicines are rich in a large amount of proteins and are important sources of bioactive peptides. Hirudin, a polypeptide extracted from leeches, is the strongest natural thrombin inhibitor discovered so far. The analgesic ziconotide is extracted from the venom of sea snails and exerts analgesic effects by effectively and selectively blocking neuronal N-type voltage-gated calcium channels (N-VSCCs). Traditional Chinese medicine Eupolyphaga seu Steleophaga, as a traditional blood-activating and stasis-dispelling medicine, is widely used in clinical practice due to its strong effect of breaking blood stasis. Modern pharmacological studies have shown that Eupolyphaga seu Steleophaga has pharmacological effects such as dissolving thrombi, anticoagulation, and regulating blood lipids. Eupolyphaga seu Steleophaga contains various active proteins (enzymes), amino acids, unsaturated fatty acids, trace elements, alkaloids, fat-soluble vitamins and other components, among which protein polypeptide components account for more than 60%. Currently, the main components reported in the literature to exert anticoagulant activity are protein polypeptides. However, currently, few anticoagulant active components have been obtained, and most of them are still in the in vitro and animal experiment stages, and the effective components and action mechanisms are not yet clear. Therefore, it is still necessary to continue to study the extraction and isolation of new anticoagulant peptides with low bleeding risks from Eupolyphaga seu Steleophaga. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide an anticoagulant peptide with good anticoagulant effect; another object of the present invention is to provide a preparation method of an anticoagulant peptide; another object of the present invention is to provide an application of an anticoagulant peptide.

[0005] Technical Solution: An anticoagulant peptide of the present invention has the following structural formula:

[0006]

[0007] On the other hand, the present invention provides a method for preparing the above-mentioned anticoagulant peptide, comprising the following steps:

[0008]

[0009] Activate the Fmoc-Phe-Wang-Resin resin, remove the protecting group Fmoc, and then connect Fmoc-Arg-OH; block the activation site to obtain Compound 1;

[0010] Remove the protecting group Fmoc in Compound 1, and connect Fmoc-Ile-OH to obtain Compound 2;

[0011] Remove the protecting group Fmoc in Compound 2, and connect Fmoc-Asp-OH to obtain Compound 3;

[0012] Remove the protecting group Fmoc in Compound 3, and connect Fmoc-Ser-OH to obtain Compound 4;

[0013] Remove the protecting group Fmoc in Compound 4, and connect Fmoc-Ser-OH to obtain Compound 5;

[0014] Remove the protecting group Fmoc in Compound 5, and connect Fmoc-Arg-OH to obtain Compound 6;

[0015] Remove the protecting group Fmoc in Compound 6, cleave the resin to obtain the target product RF7.

[0016] Further, the reagent used for activating the Fmoc-Phe-Wang-Resin resin is dichloromethane.

[0017] Further, the reagent used for cleaving the resin is trifluoroacetic acid.

[0018] Further, the reagent used for blocking the activation site is a mixture of dichloromethane, methanol and diisopropylethylamine (80:15:5).

[0019] Further, the reagent used for removing the protecting group Fmoc in each compound is diisobutyl ketone.

[0020] Further, after removing the protecting group Fmoc in Compound 6, wash the obtained product, cleave the resin, filter, and precipitate to obtain purified RF7.

[0021] Further, after removing the protecting group Fmoc in Compound 6, wash the product with N,N-dimethylformamide (DMF), dichloromethane (DCM) and methanol respectively.

[0022] Further, after removing the protecting group Fmoc in compound 6, the reagent used for resin cleavage is trifluoroacetic acid.

[0023] Further, after removing the protecting group Fmoc in compound 6, the reagent used for precipitation is ice-cold diethyl ether.

[0024] On the other hand, the present invention provides an application of the above anticoagulant peptide in the preparation of anticoagulant drugs.

[0025] On the other hand, the present invention provides a method for extracting a crude peptide extract from Eupolyphaga sinensis, comprising the following steps:

[0026] Take the powder of Eupolyphaga sinensis, dissolve it in hydrochloric acid solution, shake at 37 °C, add 2% pepsin, and shake at 37 °C. Adjust the pH to 8.0, add 4% trypsin, and shake at 37 °C. Take out the crude extract and inactivate trypsin by boiling water bath. Wait for the solution to cool, centrifuge, take the supernatant and freeze-thaw it three times at -20 °C, remove the precipitate formed during the freeze-thaw process, and freeze-dry the supernatant.

[0027] On the other hand, the present invention provides a method for isolating and screening the anticoagulant peptide RF7 from the crude peptide extract of Eupolyphaga sinensis, comprising the following steps:

[0028] Identification: After dialysis and desalting the crude peptide extract of Eupolyphaga sinensis, perform mass spectrometry to obtain the original mass spectrometry data of several polypeptides. Match this original mass spectrometry data with the data in the Eupolyphaga sinensis polypeptide database, and find the matching protein and polypeptide sequences in the database through the masses of the first- and second-stage fragments of the peptide segments to achieve the identification of the polypeptide sequences in the sample;

[0029] Computer-aided activity screening: Perform bioactivity analysis on the identified polypeptides, predict peptide toxicity, and screen out peptides with good bioactivity, good water solubility, non-toxicity and not included in the Eupolyphaga sinensis database for molecular docking;

[0030] Molecular docking: Use the Auto Dock Vina program to conduct docking studies, obtain the predicted binding free energy and binding constant of the docking ligand, and determine the affinity of the best docking site of the ligand and protein target complex by the E value (kcal / mol). Screen out the anticoagulant peptide RF7 according to the above results.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: By combining the polypeptide group technology of multi-dimensional triple quadrupole mass spectrometry and searching and comparing through an intelligent data processing system and a polypeptide database, the biological information and pharmaceutical components of anticoagulant peptides are comprehensively analyzed, and a novel anticoagulant peptide RF7 with good anticoagulant effect and low bleeding risk is screened from the complex crude peptide extract of Eupolyphaga sinensis Walker. Finally, by constructing molecular level, cellular and animal models, pharmacokinetic and pharmacodynamic studies are carried out on the anticoagulant peptides with obvious activity to evaluate the safety and effectiveness of the drug, laying a research foundation for the development and drug-likeness evaluation of novel anticoagulant drugs. Description of the Drawings

[0032] Figure 1 It is a result graph for comparing the anticoagulant effects of different crude peptide extracts. Among them, (A) is the effect on activated partial thromboplastin time (APTT); (B) is the effect on prothrombin time (PT); (C) is the effect on thrombin time (TT); (D) is the effect on fibrinogen clotting time (FCT); in the figure, IM is extraction by impregnation method, EHM is extraction by enzymatic hydrolysis method, FC is freeze-thaw process, DT is defatting process; in the figure, a is P <0.05, b is P <0.05, c is P <0.01, d is P <0.001.

[0033] Figure 2 It is a result graph for investigating the anticoagulant activity of the crude peptide extracted by the enzymatic hydrolysis freeze-thaw method. Among them, (A) is the effect of the crude peptide on activated partial thromboplastin time (APTT); (B) is the effect of the crude peptide on prothrombin time (PT); (C) is the effect of the crude peptide on thrombin time (TT); (D) is the effect of the crude peptide on fibrinogen clotting time (FCT); (E) is the effect of the crude peptide on thrombin clotting time (TCT); (F) is the volume of thrombin consumed by the crude peptide in the thrombin titration method; (G) is the effect of the crude peptide on plasma recalcification time (PRT); (H) is the effect of the crude peptide on fibrinogen in the fibrinogen plate method; (I) is the effect of the crude peptide on fibrin in the fibrin plate method; Note: In the fibrinogen plate and fibrin plate, the labels 1-3 are blank controls: PBS buffer, 4-6 are urokinase (0.1 mg / mL), 7-9 are crude peptide (30 mg / mL), 10-12 are crude peptide (60 mg / mL), 13-15 are crude peptide (90 mg / mL), 16 is the control without adding any sample; in the figure, ns is P >0.05, is P <0.05, is P <0.01, isP <0.001, For P <0.0001.

[0034] Figure 3 It is the result graph of the anticoagulant activity and bleeding risk investigation of the crude peptide in rats. Among them, (A) is the effect of the crude peptide administration on the activated partial thromboplastin time (APTT) of rats; (B) is the effect of the crude peptide administration on the prothrombin time (PT) of rats; (C) is the effect of the crude peptide administration on the thrombin time (TT) of rats; (D) is the effect of the crude peptide administration on the normal coagulation system of mice; Note: ns in the figure is P >0.05, For P <0.05, For P <0.01, For P <0.0001.

[0035] Figure 4 It is the molecular docking result graph of the anticoagulant peptide RF7 with thrombin and fibrinogen. Among them, (A) is the 3D view of the interaction between the anticoagulant peptide RF7 and thrombin and the formation of hydrogen bonds by its amino acid residues; (B) is the 3D view of the interaction between the anticoagulant peptide RF7 and fibrinogen and the formation of hydrogen bonds by its amino acid residues.

[0036] Figure 5 It is the molecular docking result graph of the anticoagulant peptide RF7 with FXIa, FXIIa and FXIIIa. Among them, (A) is the 3D view of the interaction between the anticoagulant peptide RF7 and FXIa and the formation of hydrogen bonds by its amino acid residues; (B) is the 3D view of the interaction between the anticoagulant peptide RF7 and FXIIa and the formation of hydrogen bonds by its amino acid residues; (C) is the 3D view of the interaction between the anticoagulant peptide RF7 and FXIIIa and the formation of hydrogen bonds by its amino acid residues.

[0037] Figure 6 It is the related characterization graph of the polypeptide RF7. Among them, (A) is the ultraviolet spectrum graph of the polypeptide RF7 (0.02 mg / mL); (B) is the circular dichroism spectrum graph of the polypeptide RF7 (0.5 mg / mL); (C) is the infrared spectrum graph of the polypeptide RF7; (D) is the potential characterization of the polypeptide RF7 (5 mg / mL).

[0038] Figure 7Results graph of the in vitro anticoagulant activity of RF7. Among them, (A) shows the effect of RF7 on activated partial thromboplastin time (APTT); (B) shows the effect of RF7 on prothrombin time (PT); (C) shows the effect of RF7 on thrombin time (TT); (D) shows the effect of RF7 on plasma recalcification time (PRT); (E) shows the effect of RF7 on fibrinogen clotting time (FCT); (F) shows the effect of RF7 on thrombin clotting time (TCT); Note: In the figure, ns is P > 0.05, is P < 0.05, is P < 0.01, is P < 0.001, is P < 0.0001.

[0039] Figure 8 Results graph of the in vitro antithrombotic activity of RF7. Among them, (A) shows the morphology of the blood clot after incubation with RF7; (B) shows the weight of the blood clot after incubation with RF7; (C) shows the morphology of the blood clot formed after incubating the sample with whole blood for 10 min; (D) shows the dry weight of the blood clot formed after incubating the sample with whole blood for 10 min. Note: In Figure A, ① is normal saline, ② is earthworm enzyme, ③ is 5 mg / mL RF7, ④ is 10 mg / mL RF7, ⑤ is 15 mg / mL RF7, ⑥ is 20 mg / mL RF7, ⑦ is 25 mg / mL RF7, ⑧ is 30 mg / mL RF7; in the figure is P < 0.05, is P < 0.01, is P < 0.001, is P < 0.0001.

[0040] Figure 9 Results graph of the anticoagulant effect of RF7 in animals. Among them, (A) shows the effect of different concentrations of RF7 (50, 75, 100 mg / kg) on the morphology of the rat neck blood vessels induced by ferric chloride; (B) shows the HE staining results of the rat neck blood vessels induced by different concentrations of RF7 (50, 75, 100 mg / kg); (C) shows the morphological appearance of the tail tissues of each group of mice in the carrageenan-induced tail thrombus model; (D) shows the staining results of the mouse tail tissues; (E) shows the wet weight of the rat neck blood vessels after induction with ferric chloride; (F) shows the black tail length of the mice; (G) shows the black tail rate of the mice; Note: In the figure is P < 0.05, is P <0.01, is P <0.001, is P <0.0001.

[0041] Figure 10 It is the result graph for the safety investigation of polypeptide RF7. Among them, (A) is the effect of polypeptide RF7 on the survival rate of HUVEC cells; (B) is the effect of RF7 on the morphology of red blood cells; (C) is the hemolysis rate of RF7 at different concentrations; (D) is the schematic diagram of the mouse tail bleeding experiment; (E) is the mouse tail bleeding time; (F) is the mouse tail blood loss; (G) is the HE staining result of common organs of rats by RF7 (scale bar is 50 μm); Note: ns in the figure is P >0.05, is P <0.001.

[0042] Figure 11 It is the blood drug concentration-time curve of RF7 in rats. Among them, (A) is the blood drug concentration-time curve at different time points in rats after oral administration of 200 mg / kg of RF7 (n = 4); (B) is the blood drug concentration-time curve at different time points in rats after tail vein injection of 100 mg / kg of RF7 (n = 4). Specific embodiments

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0044] In the embodiments of the present invention, the extraction and separation method of the anticoagulant activity of Eupolyphaga sinensis Walker is optimized, so as to detect ultra-trace anticoagulant peptides from complex crude peptide extracts. The NanoLC-Q-Orbitrap-MS / MS method is used to perform high-throughput analysis and identification of the peptide sequences of the screened active anticoagulant peptides. Through the intelligent data processing system and the search and comparison of the polypeptide database, the biological information and pharmaceutical components of the anticoagulant peptides are comprehensively analyzed. Finally, by constructing molecular level, cell and animal models, pharmacokinetics and pharmacodynamics and other studies are carried out on the anticoagulant peptides with obvious activity, and the safety and effectiveness of the drug are evaluated, laying a research foundation for the development and drug-likeness evaluation of new anticoagulant drugs.

[0045] Example 1

[0046] In this example, four methods of maceration method, enzymatic hydrolysis method, repeated freeze-thaw of enzymatic hydrolysate and repeated freeze-thaw of degreasing enzymatic hydrolysis are adopted to extract crude peptides.

[0047] (1)Macroporous resin adsorption method. Take the fine powder of Eupolyphaga sinensis, add eight times the amount of water, soak for 1 h, sonicate for 30 min, extract at 4 °C for 10 h. Centrifuge at 2560 g for 10 min, take the supernatant, add seven times the amount of water to the precipitate, sonicate for 30 min, extract at 4 °C for 8 h. Centrifuge at 2560 g for 10 min, take the supernatant. Combine the supernatants and freeze-dry, store at 4 °C for later use.

[0048] (2)Enzymolysis method. Take 2.0 g of the powder and dissolve it in 40 mL of hydrochloric acid solution with a pH of 1.5 (1:20, w / v), shake at 37 °C for half an hour, add 2% pepsin, and shake at 37 °C for 3 hours. Adjust the pH to 8.0 with 1.0 M NaOH, add 4% trypsin, and shake at 37 °C for 3 hours. Take out the crude extract and heat it in a boiling water bath for 15 minutes to inactivate trypsin. Wait for the solution to cool to about 40 °C, centrifuge at 2560 g for 10 minutes, and freeze-dry the supernatant.

[0049] (3)Repeated freeze-thaw of the enzymolysis solution. Take 2 g of the powder and dissolve it in 40 mL of hydrochloric acid solution with a pH of 1.5 (1:20, w / v), shake at 37 °C for half an hour, add 2% pepsin, and shake at 37 °C for 3 hours. Adjust the pH to 8.0 with 1.0 M NaOH, add 4% trypsin, and shake at 37 °C for 3 hours. Take out the crude extract and heat it in a boiling water bath for 15 minutes to inactivate trypsin. Wait for the solution to cool to about 40 °C, centrifuge at 2560 g for 10 minutes, take the supernatant, and repeatedly freeze-thaw it three times in a -20 °C refrigerator. Remove the precipitate precipitated during the freeze-thaw process, and freeze-dry the supernatant.

[0050] (4)Degreasing, enzymolysis, and repeated freeze-thaw. Take 2 g of the sieved powder of Eupolyphaga sinensis, add 10 times the amount of petroleum ether, shake overnight at 37 °C, centrifuge at 2560 g for 10 minutes, remove the supernatant, and air-dry the degreased powder in a fume hood overnight. Dissolve the degreased powder after overnight in 40 mL of hydrochloric acid solution with a pH of 1.5 (1:20, w / v), shake at 37 °C for half an hour, add 2% pepsin, and shake at 37 °C for 3 hours. Adjust the pH to 8.0 with 1.0 M NaOH, add 4% trypsin, and shake at 37 °C for 3 hours. Take out the crude extract and heat it in a boiling water bath for 15 minutes to inactivate trypsin. Wait for the solution to cool to about 40 °C, centrifuge at 2560 g for 10 minutes, take the supernatant, and repeatedly freeze-thaw it three times in a -20 °C refrigerator. Remove the precipitate precipitated during the freeze-thaw process, and freeze-dry the supernatant.

[0051] More than 60% of the components of Eupolyphaga sinensis are proteins, and the activities of the crude peptides obtained by different extraction methods are different. The appearance properties, water solubility, extraction rate and protein content of the crude peptides obtained by four extraction methods, namely maceration, enzymatic hydrolysis, freeze-thaw with enzyme and defatted freeze-thaw with enzyme, were compared. In terms of color traits, the extract obtained by maceration is yellowish-brown, the extract obtained by enzymatic hydrolysis is a mixture of yellowish-brown fragments and yellowish-white powder, the extract obtained by freeze-thaw with enzyme is dark brown, and the extract obtained by defatted freeze-thaw with enzyme is brownish-yellow. The water solubility of the extract shows to some extent the amount of water-soluble amino acids, and some of the amino acids in water-soluble amino acids play important roles in cardiovascular diseases. For example, lysine has excellent hydration, can form a hydration layer, can resist the adhesion of blood components, and specifically binds to plasminogen (Plg) and its activator tissue-type plasminogen activator (t-PA) to lyse early thrombus (fibrin clot). Arginine can affect the conformation of thrombin, thus affecting the anticoagulant effect; in addition, L-arginine can prolong APTT and PT, inhibit the formation rate of blood clots, and has anticoagulant activity. Therefore, the water solubility index has certain reference for anticoagulant activity. The extract obtained by freeze-thaw with enzyme has the best water solubility. At a concentration of 20 mg / mL, it is completely dissolved and there is no precipitation in the solution. Traditional Chinese medicine Eupolyphaga sinensis is often administered orally, enters the blood circulation after gastrointestinal digestion and absorption, and exerts its medicinal effects through the degradation of pepsin and trypsin. As shown in Table 1, enzymatic hydrolysis uses pepsin and trypsin to simulate the in vivo environment, with mild extraction conditions, high extraction efficiency reaching 52.94%, and little damage to proteins, and the protein content reaching 93.38%. Freeze-thaw with enzyme undergoes three freeze-thaw cycles on the basis of enzymatic hydrolysis. During the freeze-thaw process, some endogenous animal proteins are removed, further purifying the active ingredients, so the extraction rate and protein content decrease. Defatted freeze-thaw with enzyme uses petroleum ether to remove some fat-soluble components, and at the same time, a part of protein polypeptides will also be lost, and the extraction rate and protein content are further reduced. Maceration, as one of the most traditional extraction methods, has simple and mild operations, but has problems such as low extraction rate and long time consumption. The time consumed by maceration is more than 12 h, and the obtained protein content is not high (45.53%).

[0052] Table 1 Extraction rate and protein content of different extraction methods of Eupolyphaga sinensis (Note: IM: Maceration; EHM: Enzymatic hydrolysis; FC: Freeze-thaw; DT: Defatting)

[0053]

[0054] The activity of the above four extraction methods was measured respectively to screen the optimal extraction method. The activity measurement of the four extraction methods is the anticoagulant activity measurement, and the optimal extraction method is screened through the anticoagulant activity measurement. And the crude peptide extract obtained by the optimal extraction method was subjected to fibrinolysis activity measurement, in vivo anticoagulant activity measurement and bleeding risk investigation.

[0055] 1. Determination of anticoagulant activity

[0056] (1) Determination of APTT, PT, TT and FCT. Blood was collected from the abdominal aorta of SD rats, anticoagulated with 3.8% sodium citrate (blood:sodium citrate = (9:1), V / V), centrifuged at 1306 g for 8 min, and the supernatant was taken to obtain platelet-poor plasma (PPP). Coagulation time was measured using an SC40 semi-automatic coagulation analyzer and a diagnostic kit. Briefly, PPP (40 μL) and APTT reagent (50 μL) were mixed with sample solutions (10 μL) or normal saline (10 μL) at various concentrations, pre-incubated at 37 °C for 3 min, CaCl2 (50 μL) was added to initiate the coagulation pathway, and APTT was recorded. PPP (40 μL) was mixed with sample solutions (10 μL) or normal saline (10 μL) at various concentrations, pre-incubated at 37 °C for 3 min, PT reagent (100 μL) was added to initiate the coagulation pathway, and PT was recorded. PPP (80 μL) was mixed with sample solutions (20 μL) or normal saline (20 μL) at various concentrations, pre-incubated at 37 °C for 3 min, TT reagent (100 μL) was added to initiate the coagulation pathway, and TT was recorded. The extract of Eupolyphaga sinensis, thrombin and fibrinogen were dissolved in Tris-HCl buffer (35 mM Tris, 25 mM NaCl, pH 7.4). After pre-incubating 100 μL of the extract at different concentrations with 50 μL of fibrinogen (1 mg / mL) at 37 °C for 3 min, 50 μL of thrombin (10 U / mL) was added, and the time for fibrinogen to be converted into fibrin was measured on a semi-automatic coagulation analyzer. Measurements were made in parallel three times, with Tris-HCl buffer as the blank control.

[0057] (2) Determination of plasma recalcification time. PPP was prepared as described above. 50 μL of PPP and 50 μL of the crude peptide extract of Eupolyphaga sinensis or normal saline at different concentrations (30, 60 and 90 mg / mL) were added to the test cup, mixed well, incubated at 37 °C for 3 min, 50 μL of 25 mM CaCl2 solution was added, and the semi-automatic coagulation analyzer was immediately started to measure the coagulation time.

[0058] (3) Determination of antithrombin activity. The crude peptide was taken and prepared into sample solutions with concentrations of 30, 60 and 90 mg / mL using Tris-HCl buffer. 50 μL of the sample was taken, 50 μL of (human) fibrinogen (1 mg / mL) was added, mixed well by pipetting, and incubated at 37 °C for 5 min. Thrombin (10 U / mL) was added dropwise until coagulation occurred. When adding thrombin, it was added dropwise once every 1 min, 5 μL each time, and gently shaken while adding; the volume of the thrombin solution consumed was recorded.

[0059] (4)Determination of thrombin clotting time. Take 100 μL of crude peptide extracts at different concentrations, add 50 μL of thrombin (10 U / mL), pre-incubate at 37 °C for 30 min, then add 50 μL of fibrinogen (1 mg / mL), and measure the thrombin clotting time on a semi-automatic coagulometer. Measure in parallel three times, and use Tris-HCl buffer as the blank control.

[0060] 2. Determination of fibrinolytic activity

[0061] (1)Fibrinogen plate. Prepare 20 mL of 1% agarose solution with PBS (50 mM NaH2PO4, 0.15 M NaCl, pH 7.2), heat it to completely melt, wait until it cools to about 40 °C, add 1 mL of fibrinogen (5 mg / mL), stir evenly and immediately pour it into a glass petri dish with a diameter of 9 cm. After the agar completely solidifies, place the petri dish on a white paper printed with a 1.6 cm × 1.6 cm small square matrix, use a punch with a diameter of 3 mm to punch holes at the matrix points, and after punching, carefully suck the liquid in the holes with a 200 μL pipette. After adding 10 μL of the sample to be tested, urokinase and PBS buffer into the small holes respectively, place the fibrin plate in an incubator at 37 °C for 15 h, stain with Coomassie brilliant blue staining solution (0.25% Coomassie brilliant blue R-250, 5% glacial acetic acid, 4.5% methanol) for 15 minutes, and then decolorize with the eluent until a clear solvent circle can be seen.

[0062] (2)Fibrin plate. Prepare 20 mL of 1% agarose solution with PBS, heat it to completely melt, wait until it cools to about 40 °C, add 1 mL of fibrinogen (5 mg / mL), immediately add 20 μL of thrombin at 100 U / mL, stir evenly, pour it into the petri dish, start punching and adding samples after incubating at room temperature for half an hour, after incubating for 15 h, stain with Coomassie brilliant blue staining solution, and then decolorize with the eluent until a clear solvent circle can be seen.

[0063] (3)SDS-PAGE. Incubate fibrinogen (1 mg / mL) with the sample at 37 °C for 30 min, add protein loading buffer, boil in a water bath for 5 min, centrifuge at 9000 g for 2 min, take 10 μL of the supernatant for loading, and run the gel at a constant voltage of 80 V. Stain with 0.25% Coomassie brilliant blue and decolorize until obvious bands appear. The gel block photos are processed by Image J 2.0 software.

[0064] 3. Determination of in vivo anticoagulant activity

[0065] Twenty-four SD rats were randomly divided into a blank control group and low-, medium-, and high-dose Eupolyphaga sinensis Walker groups, with 6 rats in each group. The control group was given an equal volume of normal saline by gavage, and the low-, medium-, and high-dose Eupolyphaga sinensis Walker groups were given 200, 400, and 800 mg / kg by gavage, respectively. At 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 3.0 h after administration, 0.5 mL of blood was collected from the orbital sinus, anticoagulated with 3.8% sodium citrate (1:9), centrifuged at 1306 g for 8 min to obtain the supernatant, and its APTT, PT, and TT were measured.

[0066] 4. Investigation of bleeding risk

[0067] Twenty-four ICR mice were randomly divided into a control group and low-, medium-, and high-dose Eupolyphaga sinensis Walker groups, with 6 mice in each group. The control group was given the corresponding normal saline by gavage, and the low-, medium-, and high-dose Eupolyphaga sinensis Walker groups were given 200, 400, and 800 mg / kg by gavage, respectively. Half an hour after administration, the mice were anesthetized, the tail was cut off 3 mm from the tip of the mouse tail, and the tail was placed in physiological saline at 37°C, and the time when bleeding stopped was recorded. At the end of the experiment, the mouse wound was cleaned with iodophor to avoid infection.

[0068] The results of crude peptide extraction and activity determination are as follows:

[0069] 1. Results of active screening of crude peptides

[0070] The formation of thrombus is a complex pathological process, generally caused by multiple links or factors. Usually, APTT, PT, TT, and FCT are used as anticoagulation detection indicators to compare the anticoagulant activities of crude peptides obtained by different extraction methods (macroporous resin adsorption method, enzymatic hydrolysis method, enzyme thawing and freezing method, and enzyme thawing and freezing degreasing method). As shown in A in Figure 1 and B in Figure 1 , except that the crude peptides obtained by the enzymatic hydrolysis method (20 and 30 mg / mL) had a significant effect on APTT, there were no significant differences in the effects of different crude peptides extracted by other methods on APTT and PT at different concentrations (10, 20, 30 mg / mL). At higher concentrations (20 and 30 mg / mL), the crude peptides obtained by the four methods slightly prolonged TT (as shown in C in Figure 1 ). As shown in D in Figure 1 , the crude peptides obtained by the above 4 extraction methods all had a significant effect on FCT. Especially the enzyme thawing and freezing method could significantly prolong FCT, showing a significant difference compared with other methods and a concentration dependence. Considering the anticoagulant effect, yield, and protein content, the best extraction method was the enzymatic hydrolysis and repeated freezing and thawing method.

[0071] 2. Anticoagulant effect of crude peptides

[0072] The anticoagulant effect of crude peptides after repeated freeze-thaw was further investigated. Compared with the blank group, crude peptides with a concentration of 160 mg / mL or more prolonged APTT by 3 seconds, and high concentrations (90 mg / mL) prolonged APTT by more than 4 seconds ( Figure 2 A in the figure). There was also a tendency to prolong PT, with the highest concentration (90 mg / mL) extending it by 2 seconds ( Figure 2 The crude peptide had a more significant effect on TT. The effect of 20 mg / mL crude peptide on TT began to show significant differences. 90 mg / mL prolonged TT by 33.7 seconds ( Figure 2 C in the figure). The APTT, PT, and TT data indicate that the crude peptide mainly affects the common pathway of the coagulation cascade and has a significant inhibitory effect on the conversion of fibrinogen to fibrin, possibly by inhibiting thrombin or fibrinogen. When only fibrinogen, thrombin, and crude peptide are present in the system, the crude peptide prolongs FCT by 11.67 seconds at the lowest dose (10 mg / mL) and by 521.77 seconds at the high dose (90 mg / mL). Figure 2 D), the thrombin clotting time was prolonged by 436 seconds ( Figure 2 E in the figure), fibrinogen is hardly converted into fibrin by thrombin. The anticoagulant activity of crude peptide was determined by the classic thrombin titration method. There was an obvious threading phenomenon at the end point of the titration, and the liquid did not drip due to gravity after inversion. As the concentration of crude peptide increased, the amount of thrombin consumed gradually increased. In the end, compared with the blank group, 30 μL more thrombin was consumed ( Figure 2 F in the sample). Citric acid can chelate calcium ions in plasma, interrupting the coagulation cascade and thus blocking the formation of thrombus. The addition of calcium ions can restore the coagulation cascade and allow the blood to coagulate normally. When anticoagulants are present in the blood, the plasma recalcification time will be prolonged. When the crude peptide is pre-incubated with plasma and calcium ions are added to initiate the coagulation cascade, the fibrin formation time is significantly prolonged compared to the blank, and at a high dose (90 mg / mL) it is prolonged by 164.7 seconds ( Figure 2 G in the figure). Both the fibrinogen plate and the fibrin plate are semi-quantitative methods. Under appropriate conditions, the size of the dissolution zone is proportional to the concentration of the added sample. It can detect both plasmin and plasminogen activators. When the added sample has fibrinolytic activity, it can hydrolyze fibrin into fragments. The staining ability of Coomassie Brilliant Blue for fragments is not as high as that for intact fibrin. Therefore, under the dark blue background, the area where the plasmin is located appears light blue. Urokinase is a first-generation thrombolytic drug that hydrolyzes fibrin and fibrinogen by activating plasminogen. Under the blue background, it can be observed that the positive drug urokinase (labeled 4-6) has an obvious dissolution zone, while the crude peptides of different concentrations (labeled 7-15) cannot see the dissolution zone, even if the concentration has reached 90 mg / mL (labeled 13-15) ( Figure 2H and I in). It indicates that the crude peptide may not have fibrinolytic activity and is unable to hydrolyze fibrin (ogen) or cleave fibrin and fibrinogen by activating plasminogen.

[0073] 3. Anticoagulant activity of the crude peptide in vivo

[0074] The crude peptide enters the rats through the gastrointestinal tract, and the pharmacodynamic effect of the crude peptide in rats is investigated by measuring APTT, PT and TT at different time points. After the crude peptide enters the rats, it is first absorbed through the gastrointestinal tract and enters the blood. Due to the different absorption of anticoagulant active ingredients, there is no obvious tendency to prolong APTT at 0.5 h. At 1 h, the low-dose group (200 mg / kg) prolongs by 4.25 s, showing a significant difference. After the crude peptide undergoes enterohepatic circulation, the blood drug concentration further increases. At 2 h, the prolongation time of APTT shows a dose-dependent relationship. The low, medium and high dose groups (200, 400, 800 mg / kg) prolong by 1.87, 4.3, 8.73 s respectively ( Figure 3 A in). The crude peptide has almost no significant effect on PT. Only at 1 h, the low-dose group (200 mg / kg) prolongs by 2.43 s ( Figure 3 B in). The crude peptide mainly exerts anticoagulant activity by prolonging TT. During the period of 0.5 - 3 h, TT is continuously prolonged, and the prolongation times at the time points of 0.5, 2, and 3 h have significant differences. At 0.5 h, they prolong by 4.37, 8.45, 14.4 s respectively. At the 2 h time point, the high-dose group (800 mg / kg) prolongs by 5.6 s. At the 3 h time point, the three dose groups (200, 400, 800 mg / kg) prolong by 4.62, 5, 6.42 s respectively ( Figure 3 C in). The anticoagulant activity of the crude peptide in rats is consistent with the in vitro pharmacodynamic effect, mainly prolonging the TT time, and also having a certain effect on APTT and PT. It shows that the crude peptide mainly exerts anticoagulant activity by inhibiting coagulation factors (thrombin, fibrinogen, fibrin, etc.) in the common pathway.

[0075] 4. Investigation of the bleeding risk of the crude peptide

[0076] The effect of the crude extract of Eupolyphaga sinensis on the normal blood coagulation function of mice is investigated through a mouse bleeding experiment. The average bleeding time of the blank control group is 321.5 ± 116.4 s. The average bleeding times of the low (200 mg / kg), medium (400 mg / kg), and high dose (800 mg / kg) groups of the crude peptide extract of Eupolyphaga sinensis are 289.0 ± 62.1 s, 318.5 ± 90.9 s, and 293.8 ± 61.0 s respectively, showing no significant difference compared with the blank control group, indicating that the crude peptide extract has no effect on the normal hemostasis of mice after tail amputation ( Figure 3In D). Currently, the main problem of anticoagulant drugs is the high risk of bleeding. The mouse tail bleeding experiment shows that the bleeding time of mice given the crude peptide extract is almost the same as that of mice given normal saline, indicating that the crude peptide extract has a low bleeding risk. This further shows that the crude extract of Eupolyphaga sinensis has great research value. Future research needs to further explore the specific components that exert the drug effect in the crude extract and investigate its mechanism of action.

[0077] Example 2

[0078] In this example, the anticoagulant peptides in the crude peptides extracted in Example 1 were identified and screened to select the optimal active peptides in the crude peptide extract of Eupolyphaga sinensis.

[0079] 1. Identification

[0080] After desalting the crude peptides of Eupolyphaga sinensis by dialysis, the samples were analyzed using a Thermo Nano LC system coupled with an Orbitrap Fusion Lumos. 3 μL of the sample was taken, and the sample was separated by a chromatographic column, eluted for 120 min at a flow rate of 600 nL / min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was pure acetonitrile. Gradient elution was performed: 3% B - 8% B in 5 min, 5% B - 28% B from 5 - 85 min, 28% - 38% B from 85 - 102 min, 38% B - 100% B from 102 min - 110 min, and 100% B from 110 - 120 min. Mass spectrometry conditions: positive ion mode, the first - stage mass detector was Orbitrap, the resolution was 60K, the mass scanning range was m / z 350 - 2000, the maximum injection time was 50 ms; MIPS filtering was used, the filtering mode was peptide, the ejection time was 40 s, and the mass tolerance was ppm. For the second - stage scan, the quadrupole was used for isolation, the HCD mode was used for collision fragmentation, the collision energy was 30%, the Orbitrap was used to detect the second - stage fragments, the resolution was 15K, the maximum injection time was 22 ms, and the AGC control was 5.0×10 4 . The original data obtained from the mass spectrometry was imported into the Xcalibur 4.1.31 software to obtain the total ion current chromatogram of the sample. The Eupolyphaga sinensis database provided by the Key Laboratory of Natural Medicinal Chemistry, China Pharmaceutical University, was imported into the Proteome Discoverer 2.1 software. According to the set parameters, the original mass spectrometry data was matched with the data in the database, and the matching protein and polypeptide sequences were found in the database through the masses of the first - stage and second - stage fragments of the peptide segments, realizing the identification of the polypeptide sequences in the sample.

[0081] 2. Computer - assisted activity screening

[0082] The identified polypeptides were analyzed for bioactivity using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), with scores ranging from 0 to 1. The higher the score, the higher the likelihood that the peptide has bioactivity. The water solubility of the peptide is an important indicator for exploring functional peptides. Therefore, Innovagen (http: / / www.innovagen.com / proteomics-tools) was used to evaluate its water solubility. Peptides with scores greater than 0.5 were selected as potential active peptides, and further toxicity prediction and sequence comparison were carried out. The toxicity of the peptide was predicted using the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php), and the online database BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / ) was used to investigate whether it has been reported and included in the database. Peptides with good bioactivity, good water solubility, no toxicity, and not included in the database were further studied by molecular docking.

[0083] 3. Molecular Docking

[0084] Docking studies were carried out using the Auto Dock Vina program, which provides predictions of the binding free energy and binding constant of the docked ligand. The affinity of the best docking site of the ligand and protein target complex is determined by the E value (kcal / mol). The specific operations are as follows: First, prepare the ligand: The 2D and 3D structures of the polypeptide were drawn by ChemDraw 20.0, and the 3D structure was optimized. Energy minimization was performed using the MM2 force field and saved as a PDB file. Second, prepare the receptor: The X-ray crystal structures of coagulation factors (thrombin, PDB code: 2BVR; fibrinogen, PDB code: 2HPC; FXIIIa, PDB code: 5MHO; FXIIa, PDB code: 6L63; FXIa, PDB code: 6HHC) were downloaded from the RCSB Protein Data Bank (www.rcsb.org). The downloaded PDB files were imported into the PyMOL software, and the commands "remove solvent" and "remove organic" were entered in sequence to remove the water molecules and the included ligands existing in the receptor protein. The receptor protein was imported into the AutoDockTools (version 1.5.6) software, hydrogen was added, it was selected as the receptor, and saved as a PDBQT file. Then, the ligand was imported, hydrogen was added, it was selected as the ligand, and the PDBQT file was exported. Set the docking parameters, select the entire receptor as the docking Box, change the number of docking times to 10 times, and run AutoDock Vina. Analyze the docking results, select the one with the lowest binding energy, calculate the binding affinity, and display it in units of kcal / mol. Finally, the interaction between the polypeptide and the coagulation factor was demonstrated through the PyMOL software.

[0085] The identification and screening results of the new anticoagulant peptides are as follows:

[0086] 1. Mass spectrometry identification

[0087] The crude peptides extracted by thawing and melting the enzyme were detected by mass spectrometry. The obtained total ion current chromatogram showed that there were various proteins and polypeptides in the crude peptide extract of Eupolyphaga sinensis. After searching the database, 221 polypeptides were identified from the extract of Eupolyphaga sinensis. Since there are many proteins on the human surface, such as keratin existing in the outer layer of the skin, hair, and nails, which may contaminate the experimental samples, therefore, when performing database search, it is necessary to import the contaminant protein database and remove the polypeptides derived from the contaminant protein database. Therefore, a total of 213 polypeptides were finally obtained, with molecular weights ranging from 745 to 2984 Da and the number of amino acids in the polypeptides ranging from 6 to 28.

[0088] 2. Computer virtual activity screening

[0089] Table 2 Screened active peptides

[0090]

[0091] To screen the bioactive peptides in the extract of Eupolyphaga sinensis Walker, PeptideRank was applied to evaluate the biological activities of various polypeptides. Since the PeptideRank score represents the probability of a peptide having biological activity, peptides with a PeptideRank score above 0.5 were selected for further analysis. As shown in Table 2, there were 4 polypeptides with PeptideRank scores greater than 0.5, indicating that these peptides might have higher biological activities. The safety affecting the application of bioactive peptides is an important issue. It is expected that the potential allergenicity and toxicity of polypeptides can exclude polypeptides with potential risks. The results of the ToxinPred tool showed that none of the four polypeptides were toxic and could be used as potential bioactive peptides. Water solubility is also a key factor restricting the bioavailability of polypeptides. The results of Innovagen showed that polypeptides 3 and 2 had good water solubility. According to the prediction results of the ToxinPred software, polypeptide 2 had a higher hydrophilicity, reaching 0.76. When imported into the BIOPEP-UWM online database, it was not included. The sequence of polypeptide 2 was RSSDIRF, and its number of amino acids was less than 10. Research has shown that the activity of a peptide decreases with the increase in the length of the peptide chain. Therefore, polypeptide RSSDIRF, that is, RF7, was selected for subsequent research.

[0092] 3. Molecular Docking

[0093] The docking score is the approximate potential energy of the binding of a ligand to a macromolecule. The lower the score, the stronger the affinity between the ligand and the target protein. A docking score < –5.0 kcal / mol usually indicates good binding between an anticoagulant peptide and an enzyme (target), thereby inhibiting the enzymatic activity of the target protein. Thrombin is the core in the process of blood coagulation. It cleaves soluble fibrinogen into fibrin chains and forms an insoluble clot together with substances such as platelets and blood cells. Therefore, thrombin is an important target in antithrombotic drugs. Thrombin generally has four binding sites, namely the active site, two positively charged anion binding sites (exosite 1 and exosite 2), and a sodium ion binding site. In addition, it also contains an autocatalytic hydrolysis loop and a W60d loop. The docking score of the anticoagulant peptide RF7 with thrombin reached -7.6 kcal / mol, and formed 10 hydrogen bonds with the residues Trp-60A, Ser-214, Trp-215, Gly-216, Gly-219, Asp-189, Ala-190, Ser-195 of thrombin. The average hydrogen bond distance (Å) was 2.69 ( Figure 4 in A). It mainly interacted with the + sodium binding site of thrombin. +The binding site mainly fixes sodium ions through coordination bonds to regulate the activity of thrombin. According to the sodium ion binding situation, it can be divided into fast thrombin and slow thrombin. The sodium-free form (referred to as the "slow form") has anticoagulant activity. Asp-189 is negatively charged and plays a crucial role in the binding of thrombin to Na + while Ser-195 and Asp-189 jointly affect the optimal conformation of the binding substrate. The anticoagulant peptide FR7 is positively charged under physiological conditions, which is conducive to interacting with the Na + binding site. Na + binding is considered to allosterically regulate thrombin activation. The polypeptide RF7 forms hydrogen bond interactions with the main amino acids of the Na + binding site, inhibits the binding of Na + to thrombin, resulting in the sodium-free mode of thrombin, thereby exerting anticoagulant activity.

[0094] Fibrin formed after fibrinogen is hydrolyzed by thrombin is an important component of thrombus. Inhibiting fibrinogen plays an important role in inhibiting thrombus formation. Fibrinogen is composed of three chains, α, β, and γ, through 29 disulfide bonds. Fibrinogen is negatively charged at pH 7.40, which is also conducive to the interaction between RF7 and fibrinogen due to different charges. The affinity of the anticoagulant peptide RF7 for fibrinogen is -7.9 kcal / mol, and it forms 14 hydrogen bonds with the Asp-318, Asp-320, Asn-319, Arg-275, Tyr-278, Asn-308, Gly-309, Asp-291, Ser-306, Lys-302, and Lys-31 residues of fibrinogen, and the average hydrogen bond distance (Å) is 2.87 ( Figure 4 in B). The above results indicate that there is an interaction between RF7 and fibrinogen.

[0095] FXIa is located in the intrinsic pathway of the coagulation cascade, which can effectively inhibit the progression of coagulation and has a relatively low bleeding risk. Studies have shown that compared with wild-type mice, mice lacking the FXI gene are much less susceptible to arterial and venous thrombosis. More importantly, FXI-deficient mice grow healthily without showing bleeding symptoms. In addition, patients with hemophilia C rarely experience thrombus events due to the lack of FXI in their blood. Therefore, FXIa is an attractive target for antithrombotic therapy. Each subunit of FXIa contains four apple domains (N-terminal) and a protease domain (C-terminal). The catalytic triad of FXIa is located in the protease domain and consists of His-57, Asp-102, and Ser-195. Therefore, current research on FXIa inhibitors mainly focuses on the protease domain. The apple domains of FXIa are involved in binding to platelets, heparin, high molecular weight kininogen, and many proteins (such as thrombin, factor XIIa, and glycoprotein Ibα). The anticoagulant peptide RF7 has an affinity of -5.3 kcal / mol for fibrinogen (FXIa) and forms 9 hydrogen bonds with the amino acids Ser-500, Leu-501, Ser-503, Leu-589, Tyr-511, Val-612, and Asn-610 of FXIa, with an average hydrogen bond distance (Å) of 2.39 ( Figure 5 in A of Figure 5 ). The docking results with FXIa showed that RF7 did not act on the protease domain of FXIa but inhibited the activity of FXIa by interacting with the apple domain and changing the three-dimensional structure of FXIa.

[0096] As the first step of the intrinsic coagulation pathway, FXIIa is activated by negatively charged substances in the blood and further activates factor FXIa. The docking score of the anticoagulant peptide RF7 with FXIIa is 7.45 kcal / mol, and it forms 8 hydrogen bonds with His-384, Gly-428, Glu-430, Tyr-605, Asp-602, and Gln-581 of FXIIa, with an average hydrogen bond distance (Å) of 2.175 ( Figure 5in B). Gly-193 and Ser-19 are conserved residues of FXIIa, which form an oxyanion hole, a characteristic of serine proteases and essential for their proteolytic cleavage of substrates. RF7 does not interact with Gly-193 and Ser-19 and has low inhibitory activity against FXIIa. FXIIIa is a transglutaminase that crosslinks glutamine and lysine residues of proteins via isopeptide bonds and affects the size of the formed thrombus. In addition, FXIIIa-mediated fibrin crosslinking increases the rigidity and stability of the clot, making the clot less prone to dissolution. The docking score of the anticoagulant peptide RF7 with FXIIIa is -4.06 kcal / mol, and it forms eight hydrogen bonds with Glu-415, Lys-418, Pro-564, Lys-565, and Arg-333 of FXIIa, with an average hydrogen bond distance (Å) of 2.4 ( Figure 5 in C). The active center of FXIIIa consists of a catalytic triad composed of Cys-314, His-373, and Asp-396, as well as a catalytic dyad (His-342, Glu-401) located on the active side of FXIIIa. The docking results indicate that RF7 does not act on the active center of FXIIIa but exerts its effect by changing its three-dimensional conformation.

[0097] Example 3

[0098] The preparation method of the anticoagulant peptide RF7 provided by the embodiment of the present invention is as follows:

[0099]

[0100] The anticoagulant peptide RF7 is prepared by solid-phase synthesis. After the Fmoc-Phe-Wang-Resin resin is activated, Fmoc-Arg-OH is connected; the activation site is blocked to obtain Compound 1; the protecting group Fmoc in Compound 1 is removed, and Fmoc-Ile-OH is connected to obtain Compound 2; the protecting group Fmoc in Compound 2 is removed, and Fmoc-Asp-OH is connected to obtain Compound 3; the protecting group Fmoc in Compound 3 is removed, and Fmoc-Ser-OH is connected to obtain Compound 4; the protecting group Fmoc in Compound 4 is removed, and Fmoc-Ser-OH is connected to obtain Compound 5; the protecting group Fmoc in Compound 5 is removed, and Fmoc-Arg-OH is connected to obtain Compound 6; the protecting group Fmoc in Compound 6 is removed, and the resin is cleaved to obtain the target product RF7.

[0101] The details are as follows: First, swell the Fmoc-Phe-Wang-Resin with dichloromethane (DCM) for half an hour (solid-liquid ratio is 1:20), wash it three times with N,N-dimethylformamide (DMF), block the activated sites on the resin with a mixed solution of dichloromethane, methanol and diisopropylethylamine (80:15:5), and wash it five times with DMF. Deprotect with diisobutyl ketone (DBLK) for 15 minutes, wash it six times with DMF, and start coupling the first amino acid Fmoc-Arg-OH for half an hour. After passing the detection (the solution is bright yellow and the resin is transparent), repeat the DMF washing, DBLK deprotection, and link the second amino acid until all amino acids are linked (the molar ratio of the amino acid added to the resin is twice). After the amino acid coupling is completed, deprotect with DBLK for 15 minutes, remove the Fmoc protecting group of the last coupled amino acid, then wash it three times with DMF, DCM, and methanol respectively, cleave the resin with trifluoroacetic acid, filter, precipitate the polypeptide with ice ether, and freeze-dry. Purify the dried polypeptide powder with a protein purifier, collect the purified fractions, and finally obtain the polypeptide RF7.

[0102] The characterization method of the prepared polypeptide RF7 is as follows:

[0103] Use an AB4000 mass spectrometer (AB Sciex Company, United States) and an AVANCE NEO 600 nuclear magnetic resonance (Bruke Company, Germany) to verify whether the polypeptide is successfully synthesized, and use an Agilent 1260 high-performance liquid chromatograph (Agilent Technologies Company, United States) to investigate the purity of the synthesized polypeptide. The detection wavelength is 220 nm, buffer B is 0.1% trifluoroacetic acid aqueous solution, buffer A is 0.1% trifluoroacetic acid acetonitrile, and gradient separation is carried out at a flow rate of 1 mL / min through a chromatographic column (250 × 4.6 mm, Boston Green ODS-AQ). The gradient of liquid phase separation is: from 0 to 25 min, the linear gradient of buffer B is from 85% to 60%; from 25 to 25.1 min, the linear gradient of buffer B is from 60% to 0%; from 25.1 to 30 min, buffer B remains at 0%. Use a JASCO-810 circular dichroism spectropolarimeter (JASCO Corporation, Japan) to characterize the secondary structure of the polypeptide, use a Malven Nano ZS90 particle size potentiometer (Malvern Instruments Limited, England) to investigate the charge it carries, and use a TENSOR 27 Fourier transform infrared spectrometer (Bruke Company, Germany) to characterize its functional group structure.

[0104] The characterization results of the polypeptide RF7 are as follows:

[0105] The anticoagulant peptide RF7 (C 37 H 61 N 13 O 12 , MW = 879.97) was prepared by solid-phase synthesis method. The electrospray ionization mass spectrometry (ESI-MS) scan yielded two ion peaks at m / z = 440.8 [M+2H] 2+ and m / z = 880.5 [M+H] + . The NMR data of RF7 are as follows: 1H NMR (600 MHz, D2O) δ 7.42 (dd, J = 8.2, 6.8 Hz, 2H), 7.39 – 7.34(m, 1H), 7.34 – 7.31 (m, 2H), 4.72 (dd, J = 8.9, 5.3 Hz, 1H), 4.63 (t, J =5.9 Hz, 1H), 4.55 (t, J = 5.4 Hz, 1H), 4.20 – 4.14 (m, 2H), 4.01 – 3.89 (m,4H), 3.33 – 3.26 (m, 3H), 3.21 (t, J = 7.0 Hz, 2H), 3.09 (dd, J = 14.1, 8.9Hz, 1H), 2.97 (dd, J = 16.9, 6.0 Hz, 1H), 2.87 (dd, J = 16.9, 7.3 Hz, 1H),2.07 – 1.98 (m, 2H), 1.89 – 1.67 (m, 5H), 1.59 (dddd, J = 23.8, 16.9, 13.7,6.7 Hz, 2H), 1.47 (ddd, J = 13.6, 7.4, 3.5 Hz, 1H), 1.26 – 1.14 (m, 2H), 0.90(t, J = 7.4 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H). The mass spectrometry and NMR data indicate that RF7 has been successfully synthesized. The high performance liquid chromatography (HPLC) purity test result reached 96.18%. RF7 has UV-Vis absorption in the range of 200 - 300 nm, and the maximum absorption is at 210 nm ( Figure 6 A in Figure 6 ). The circular dichroism spectrum shows that RF7 has double negative peaks at 222 nm and 208 nm, and a positive peak at 190 nm ( Figure 6 B in -1 ), indicating that the structure of RF7 is mainly α-helix. In the infrared chromatogram peak, RF7 has peaks at 1669.53 cm -1 and 1532.45 cm -1Exhibited characteristic absorption of amide, 3287.74 cm -1 Exhibited characteristic absorption of carboxyl group ( Figure 6 C in). The website predicted that the isoelectric point of RF7 was 10.35. In the physiological environment, it was theoretically positively charged. The potential results showed that RF7 was positively charged, which was consistent with the theory ( Figure 6 D in).

[0106] Conducted pharmaceutical research on the polypeptide RF7, including in vitro anticoagulant activity of RF7, in vitro inhibition of thrombus formation, investigation of in vivo anticoagulant effect, safety investigation of RF7, and pharmacokinetic study:

[0107] 1. Anticoagulant activity

[0108] (1) In vitro anticoagulant activity of RF7

[0109] Referring to the APTT, PT, TT kits and Example 1, the effects of the polypeptide RF7 on APTT, PT, TT, FCT, TCT and PRT were determined.

[0110] (2) In vitro inhibition of thrombus formation

[0111] Collect 2 mL of blood from the orbital venous plexus of SD rats, incubate at room temperature for 2 h, wait for it to coagulate naturally, wash the blood clot with normal saline, divide it, add 55 mg of blood clot and 100 μL of sample to each EP tube, incubate at 37 °C for 12 h, wash the blood clot with normal saline, and weigh it. Add 200 μL of whole blood to the EP tubes containing 50 μL of samples with different concentrations, let it stand naturally at room temperature for 10 min, take out the formed thrombus blocks from the EP tubes, absorb the liquid with filter paper, and weigh the wet weight of the thrombus; to reduce errors, air-dry the thrombus blocks naturally and weigh the dry weight of the thrombus. Among them, the negative blank control was added with normal saline, and the positive control was selected as earthworm enzyme.

[0112] 2. Investigation of in vivo anticoagulant effect

[0113] (1) Rat carotid artery thrombosis model induced by ferric chloride

[0114] SD rats were randomly divided into six groups, namely the blank group (normal saline), the model group (normal saline), the positive drug group (5 mg / kg earthworm enzyme), and the low, medium, and high-dose groups (50, 75, 100 mg / kg RF7), with 6 rats in each group. The model was established by the method of inducing common carotid artery thrombosis with FeCl3. After tail vein administration, ketamine (100 mg / mL) was intraperitoneally injected at a dose of 1 mL / kg to anesthetize the rats. The left common carotid artery was isolated, and a filter paper strip (0.5 cm x 0.5 cm) soaked with 10% FeCl3 solution was looped around the common carotid artery. After 10 min, the filter paper was removed, and the artery was rinsed 3 times with normal saline. After 15 min, a 0.5-cm vascular segment of the common carotid artery thrombosis site wrapped by the filter paper strip was precisely cut off, placed on the filter paper to absorb the floating blood, and the wet weight of the blood vessel containing the thrombus was weighed. The hearts, livers, spleens, lungs, kidneys, and blood vessels of the rats were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (HE) to observe the degree of tissue damage.

[0115] (2)Carrageenan-induced mouse tail thrombosis model

[0116] ICR mice weighing 26 - 30 g were randomly divided into six groups, namely the blank group, the model group, the positive drug group, and the low, medium, and high-dose groups of RF7, labeled as groups A, B, C, D, E, and H. All six groups were administered via the tail vein. Among them, groups A and B were given normal saline, group C was given earthworm enzyme (5 mg / kg), and groups D, E, and H were given 50, 75, and 100 mg / kg of RF7 solution, respectively. After administration, 1% carrageenan was immediately given at a dose of 50 mg / kg via intraperitoneal injection to establish the model. The mice were weighed and observed on the second and third days respectively. On the third day, the mice were sacrificed to observe the length of the tail thrombus, and the tail was cut at 4 cm from the tip of the mouse tail, decalcified and embedded, and HE staining was performed to observe the pathological damage.

[0117] 3. Safety investigation of RF7

[0118] (1)Biocompatibility

[0119] 100 μL of HUVECs cells were added to a 96-well plate and cultured overnight at 37℃ in an environment of 5% CO2 (cell density was 4×10 3cells / well). To investigate the cytotoxicity of the polypeptide RF7 on HUVECs cells, the concentration range of RF7 was set at 0.0005, 0.005, 0.05, 0.1, 0.5, 1, 5, 10 mg / mL, and an equal volume of PBS solution was added to the control group. After the HUVECs cells were incubated for another 24 h after drug administration, 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well. Then, after the cells were incubated for another 2 h under the same conditions, the absorbance value of each well at 450 nm was measured using a SpectraMax M2e multimode microplate reader (Molecular Devices, United States). The percentage of cell viability was calculated using the following formula, and each concentration point was operated in parallel 5 times.

[0120] Cell viability (%) = OD sample / OD control × 100%

[0121] where OD sample and OD control represent the absorbance values of the sample group and the control group at 450 nm, respectively.

[0122] (2) Hemolysis rate investigation

[0123] 2 mL of blood was collected from the orbital venous plexus of rats, anticoagulated with sodium heparin, centrifuged at 200 g, the supernatant was removed, the lower layer solution was fixed to 10 mL, centrifuged at 400 g for 5 min, the supernatant was removed, and this was repeated 3 times until there was no obvious red color in the supernatant. 500 μL of the lower layer of red blood cells was taken and fixed to 25 mL to obtain a 2% red blood cell suspension. All samples were prepared with normal saline. 70 μL of the red blood cell suspension was incubated with the same volume of the sample at 37 °C for 4 h, centrifuged at 800 g for 5 min, 100 μL of the supernatant was taken, and the absorbance value at 540 nm was measured. Among them, pure water was used as the positive control and normal saline was used as the negative control. The hemolysis rate was measured according to the following formula.

[0124] (A1 - A0) / (A2 - A0) × 100%

[0125] where A1: absorbance value of the experimental group, A0: absorbance value of the positive control, A2: absorbance value of the negative control

[0126] (3) Bleeding risk experiment

[0127] Thirty ICR mice were randomly divided into a blank group, a heparin group, and a low-, medium-, and high-dose group of peptide RF7, with 6 mice in each group. The control group was given the corresponding saline through the tail vein, the heparin group was injected with 5 mg / kg sodium heparin through the tail vein, and the low-, medium-, and high-dose groups of peptide RF7 were given 50, 75, and 100 mg / kg RF7 solution through the tail vein, respectively. Five minutes after administration, the mice were anesthetized, cut off 3 mm from the tip of the tail, and placed in 37°C saline. The time when bleeding stopped was recorded. At the end of the experiment, the wound of the mouse was cleaned with iodine to avoid infection. The blood flowing out of the tail of the mouse was collected, centrifuged at 4000 g, the supernatant was removed, the red blood cells were resuspended with 1 mL of 1% Triton X-100 solution, and 200 μL of the above red blood cell solution was taken to measure its absorbance at 562 nm. Among them, the higher the absorbance value, the more red blood cells and the higher the amount of bleeding.

[0128] 4. Pharmacokinetic studies

[0129] SD rats were given 100 mg / kg RF7 solution through the tail vein and 200 mg / kg RF7 solution orally. After administration, blood was collected from the orbit at 2, 5, 10, 20, 30, 45, 60, 80, 100, and 120 min, respectively, and plasma was collected by centrifugation. After biological sample pretreatment, the RF7 concentration in rat plasma at different time points was determined by LC-MS / MS, and the peak area was substituted into the accompanying standard curve to calculate the blood drug concentration. The blood drug concentration-time curve of RF7 was plotted, and the blood drug concentration-time data were processed using DAS 2.2.7 software to calculate the pharmacokinetic parameters.

[0130] The results of pharmaceutical studies on peptide RF7 are as follows:

[0131] 1. Anticoagulant activity

[0132] (1) In vitro anticoagulant activity

[0133] The anticoagulant activity of RF7 was evaluated by APTT, PT, TT, FCT, TCT and PRT. RF7 had no significant effect on APTT at concentrations below 8 mg / mL. When it reached 10 mg / mL, it prolonged APTT by 3.73 seconds, and at 20 mg / mL, it prolonged APTT by 10.1 seconds ( Figure 7 A in the figure). Molecular docking results showed that RF7 had certain interactions with FXIa and FIXa, and it may prolong APTT by inhibiting these two coagulation factors. RF7 may prolong PT time by inhibiting FVIIa. At 20 mg / mL, RF7 prolonged PT by 5.7 seconds ( Figure 7B). In the molecular docking results, the docking scores of RF7 with thrombin and fibrinogen were the lowest, suggesting that RF7 has the strongest inhibitory effect on them. The TT results showed that at a concentration of 2 mg / mL, RF7 had a significant difference in the prolonged TT time, reaching 2.93 seconds, and at the highest concentration (20 mg / mL), it could prolong the TT time to 32.43 seconds ( Figure 7 C). Calcium ion is an essential ion in the coagulation cascade and is also coagulation factor IV. Lack of calcium ions will inhibit the coagulation cascade. PRT was used to investigate the effect on the intrinsic pathway of the coagulation cascade. RF7 could inhibit coagulation factors in the intrinsic coagulation pathway, such as FXIa, FIXa, etc., and prolong PRT. The experimental results showed that when the concentration of RF7 reached 20 mg / mL, PRT could be prolonged by 109.2 seconds ( Figure 7 D). APTT, PT, and TT are commonly used clinical indicators for detecting the coagulation system. From the measurement results, it can be seen that RF7 has a significant effect on TT, mainly affecting the coagulation factors in the common pathway, that is, affecting fibrin(ogen) and thrombin. RF7 was incubated with thrombin and fibrinogen respectively, and the fibrinogen clotting time (FCT) and thrombin clotting time (TCT) were measured. When the incubation concentration of RF7 with thrombin reached 10 mg / mL, FCT reached the maximum measurement time and fibrin could not be formed ( Figure 7 E). When the incubation concentration of RF7 with fibrinogen reached 8 mg / mL, TCT reached the maximum measurement time and fibrin could not be formed ( Figure 7 F). The results showed that RF7 has an inhibitory effect on both fibrinogen and thrombin, and its effect on fibrinogen may be stronger than that on thrombin. In summary, RF7 has obvious anticoagulant activity, mainly by inhibiting the activities of thrombin and fibrinogen, and then inhibiting the common pathway to play a role.

[0134] (2) Antithrombotic effect in vitro

[0135] Rat blood coagulates naturally without anticoagulant to obtain blood clots. Blood clots of the same weight were incubated with the sample to investigate whether RF7 could dissolve the blood clots. As Figure 8 shown in A, when RF7 was incubated with the blood clots, the volume of the blood clots gradually decreased with the increase of the RF7 concentration. The weight of the blood clots was measured. The blood clots in the blank group were not dissolved and the weight still reached 55.4 mg. With the increase of the RF7 concentration, when it reached 30 mg / mL, the blood clots only remained 14.23 mg ( Figure 8In B), it shows that RF7 plays a role in dissolving blood clots by hydrolyzing fibrin. In the investigation of the in vitro anticoagulation mechanism, RF7 can not only hydrolyze fibrinogen, but also inhibit thrombin and FXIa. After incubating RF7 with whole blood for a certain period of time, the in vitro antithrombotic effect of RF7 was investigated by measuring the weight of the blood clot after natural coagulation of the blood. The results are as Figure 8 shown in C. The blood clot formed in the blank group was the largest. As the concentration of RF7 increased, the formed blood clot gradually decreased. The wet weight of the blood clot was measured. The wet weight of the blood clot in the normal saline group was 96.83 ± 2.59 mg, and the weight of the blood clot in the positive drug control group, the earthworm enzyme group (5 mg / mL), was 69.93 ± 25.61 mg. Compared with the normal saline group, the formation of blood clots decreased after incubation of RF7 with whole blood, and as the concentration increased, the blood clot and its weight gradually became smaller. The weight of the blood clot in the highest concentration group (30 mg / mL) was only 30.2 ± 6.7 mg. Since the blood clot contains a high level of water, to reduce errors, the dry weight of the blood clots in each group was measured. The results are as Figure 8 shown in D. The weight of the blood clot in the normal saline group was 17.6 ± 2.59 mg, the weight of the blood clot in the earthworm enzyme group was 69.93 ± 25.61 mg, and the highest concentration group of RF7 (30 mg / mL) was 3.87 ± 1.33 mg. The above results indicate that RF7 can inhibit the formation of blood clots. To sum up, RF7 can dissolve the already formed blood clots by hydrolyzing fibrin, and can also inhibit the formation of blood clots by inhibiting coagulation factors such as FXIa and thrombin, and has significant antithrombotic activity in vitro.

[0136] 2. Investigation of the in vivo anticoagulation effect

[0137] (1) Ferric chloride model

[0138] To evaluate the antithrombotic effect of RF7 in animals, a rat acute carotid artery thrombosis model was established. Ferric chloride can damage endothelial cells, activate platelets and the coagulation cascade system, and form local arterial thrombosis. As Figure 9As shown in A of , RF7 can significantly reduce the wet weight of FeCl₃-induced carotid artery thrombosis and inhibit thrombosis formation. After the carotid arteries were fixed with paraformaldehyde, the blood vessels in the blank group were white as a whole, and no thrombosis was formed inside the blood vessels. The model group was dark brown, and the blood vessels were completely blocked by thrombus, indicating that the model was successful; in the positive drug group, a small amount of thrombus was formed inside the blood vessels, and the blood vessel walls showed yellow; the low, medium, and high-dose groups showed an obvious dose-dependence, and the thrombus inside the blood vessels gradually decreased. The HE staining results showed that the blood vessel walls in the blank group consisted of three layers: intima, media, and adventitia from inside to outside, with a complete tissue structure. A small number of endothelial cells could be seen on the intima, the elastic membrane in the media was wavy, with a clear structure, and blood vessels and nerve fibers could be seen in the adventitia. There was no obvious thinning or damage to the blood vessel walls, and no thrombosis was formed inside the lumen. In the model group, the walls were thinned, the structure was unclear, the wavy structure of the elastic membrane disappeared and became flat. Thrombosis could be seen inside the lumen, suspected to be a mixed thrombus, accounting for about 80% of the entire lumen. After administration, the thrombus content inside the lumen gradually decreased ( Figure 9 as shown in B of ). The blood vessels in the neck of the rats were weighed. The weight of the blood vessels in the blank group was 2.98 ± 0.45 mg. Thrombus was formed inside the blood vessels in the model group, and the weight of the removed blood vessels reached 6.38 ± 1.28 mg. After administration, it could significantly reduce the formation of thrombus inside the blood vessels. The efficacy of the high-dose group (100 mg / kg) was higher than that of the positive drug group, and it could significantly reduce the weight of the vascular ring ( Figure 9 as shown in E of ).

[0139] (2) Carrageenan in mice

[0140] Carrageenan is a sulfated polysaccharide extracted from seaweed. After entering the animal body, it can cause tissue inflammation, damage vascular endothelial cells, activate the coagulation system, and cause thrombosis formation. No thrombosis was formed in the tails of the mice in the blank control group, and the tail tissues were normal, and the blood vessels of the mice could be clearly seen. In the model group and the administration group of mice, thrombosis gradually formed from the tip of the tail, causing the tail tissues of the mice to turn black ( Figure 9 as shown in C of ). By sectioning the mice's tails at 4 cm from the tip and performing hematoxylin-eosin staining, the antithrombotic treatment effect of RF7 in the mouse tail thrombus model was further demonstrated, as shown in Figure 9As shown in D of [reference], no thrombosis was observed in the blood vessels of the blank group mice, while thrombosis was observed in the blood vessels of the tail tissues of the model group mice, which tended to be mixed thrombus, accounting for about 75% of the lumen. After administration, the thrombus in the tail blood vessels of the earthworm enzyme group (5 mg / kg) mice accounted for 30%-50% of the lumen, and a small amount of inflammatory cell infiltration was observed around some blood vessels. After administration of polypeptide RF7, at the medium dose (75 mg / kg), no thrombosis was observed in the blood vessels. The antithrombotic activity of RF7 in vivo was further evaluated by measuring the length of the black thrombus in the tails of mice. The normal group mice had no black tail length, and the black tail length of the model group reached 53.12 ± 13.10 mm. After administration of the positive drug earthworm enzyme and polypeptide RF7 respectively, the formation of thrombus in the tails of mice was inhibited to varying degrees, and the length of the thrombus in the tails of mice decreased significantly. The black tail length of the low-dose RF7 group (50 mg / kg) was 32.92 ± 14.03 mm, and the degree of thrombus inhibition was greater than that of the earthworm enzyme group (42.65 ± 8.94 mm). When the dose of polypeptide RF7 administered reached 100 mg / kg, the black tail length of the mice was only 13.93 ± 11.32 mm ( Figure 9 F of [reference]). Due to the different lengths of the tails of each mouse, in order to further reduce the error, the black tail rate of the mice was calculated. The black tail rate of the model group mice was 57.52 ± 13.79%, which decreased to 43.47 ± 8.40% after administration of earthworm enzyme. After administration of different concentrations of RF7 (50, 75, 100 mg / kg), the black tail rate gradually decreased, being 35.21 ± 14.82%, 33.38 ± 12.09%, and 17.31 ± 12.02% respectively ( Figure 9 G of [reference]). In summary, RF7 can slow down the formation of thrombus in the tails of carrageenan-induced mice and has good antithrombotic activity.

[0141] 3. Safety evaluation of RF7

[0142] The cytotoxicity of RF7 was investigated using HUVEC cells. The experimental results showed that the cell viability was close to 100% within the concentration range of 10 mg / mL ( Figure 10 A of [reference]), indicating that RF7 basically has no cytotoxicity. After co-culturing RF7 with 2% red blood cells for 4 h, no rupture of red blood cells was observed, and the results were consistent with those of the normal saline group ( Figure 10 B of [reference]). When the concentration of RF7 reached 20 mg / mL, the hemolysis rate was less than 1% ( Figure 10In C), it shows that RF7 does not cause hemolysis and has good biosafety. The bleeding risk is the biggest side effect of current anticoagulants. The bleeding risk of RF7 was investigated through the mouse tail bleeding experiment. RF7 at low, medium, and high concentrations (50, 75, 100 mg / kg) was administered via the tail vein. After 5 min, the mice were anesthetized, the blood vessels of the mouse tails were damaged, and they were placed in physiological saline and allowed to coagulate naturally. The bleeding time was recorded. The collected blood was centrifuged, the red blood cells were lysed, and the bleeding volume was investigated through the absorbance value and compared with the positive control heparin (5 mg / kg). Figure 10 In D). Saline was injected as a blank control, and its bleeding time was 160.13 s. The heparin group (5 mg / kg) had a serious bleeding risk, and the bleeding time reached 559.33 s, showing a significant difference from the blank group. The bleeding times of the low, medium, and high-dose groups of RF7 (50, 75, 100 mg / kg) were 161.83, 180.5, and 197.97 s respectively, showing no significant difference from the blank group. Figure 10 In E). The bleeding volumes of different groups were further shown through the levels of the absorbance value. The higher the absorbance value, the greater the bleeding volume and the higher the bleeding risk. The absorbance value of the blank group was 0.09, that of the heparin group (5 mg / kg) was 1.13, and the absorbance values of the low, medium, and high-dose groups of RF7 (50, 75, 100 mg / kg) were 0.21, 0.20, and 0.46 respectively, showing no significant difference from the blank group. Figure 10 In F). The results of the bleeding time and bleeding volume both indicate that the bleeding risk of RF7 is much lower than that of heparin, showing no significant difference from the blank group, with a low bleeding risk, and it can be used as a candidate drug for anticoagulants. After SD rats were intravenously administered RF7 at low, medium, and high concentrations (50, 75, 100 mg / kg), the rats were anesthetized and sacrificed by decapitation, and then important organs (heart, liver, spleen, lung, and kidney) were taken for HE staining. The results are as Figure 10 shown in G. The staining results of the low, medium, and high-dose groups (50, 75, 100 mg / kg) were consistent with those of the blank group, and there were no obvious acute or chronic pathological toxicities or inflammatory cells in the main organs. In summary, RF7 has no cytotoxicity, no hemolysis risk, low bleeding risk, no damage to the main organs of rats, and high safety.

[0143] 4. Pharmacokinetic study of RF7

[0144] Table 3 Pharmacokinetic parameters of RF7

[0145]

[0146] After SD rats were given RF7 solution by tail vein and oral gavage, 0.3 mL of blood was collected from the rats' orbits at 2, 5, 10, 20, 30, 45, 60, 80, 100, and 120 min, respectively. After biological sample pretreatment, LC-MS / MS was used to determine the RF7 concentration in rat plasma at different time points, and the peak area was substituted into the accompanying standard curve to calculate the blood drug concentration, and the blood drug concentration-time curve of RF7 was drawn ( Figure 11 The blood drug concentration-time data were processed by DAS 2.2.7 software, and the pharmacokinetic parameters of RF7 were analyzed by non-compartmental model fitting. The results are shown in Table 3. After oral absorption, RF7 reached C max (299.50 ± 114.18 ng / mL), AUC 0-∞ was (7564.65 ± 1497.56) ng·h / mL; after tail vein administration, C max (1171.00 ± 664.34 ng / mL), AUC 0-∞ The absolute bioavailability of RF7 was calculated to be 13.90%, which is not suitable for oral administration. The HPLC method was established to investigate the plasma stability and gastrointestinal stability of RF7. RF7 would break in plasma and then be gradually hydrolyzed into amino acids. The plasma stability of RF7 was low, and only 46.53% remained after incubation with plasma for 30 minutes.

[0147] Table 4 Stability of RF7 in artificial gastric juice and artificial intestinal juice

[0148]

[0149] Artificial gastric juice and artificial intestinal juice were used to simulate the human gastrointestinal tract to investigate the stability of RF7 in the gastrointestinal tract. The results are shown in Table 4. RF7 is relatively stable in gastric juice, still containing 78.42% after 3 hours of incubation, but it is unstable in artificial gastric juice, with only 1.3% left after 3 hours. RF7 is an acidic polypeptide, which is more stable in an acidic environment than in an alkaline environment. Secondly, RF7 is a linear polypeptide, which is much less stable than a cyclic peptide and is easily hydrolyzed into amino acids by various enzymes in plasma. Therefore, RF7 has a low half-life and low absolute bioavailability.

Claims

1. An anticoagulant peptide, characterized in that, Its structural formula is as follows: 。 2. A method for preparing the anticoagulant peptide according to claim 1, characterized in that, It includes the following steps: After the activation of Fmoc-Phe-Wang-Resin, Fmoc-Arg-OH is connected; the activated sites are blocked to obtain Compound 1; Remove the protecting group Fmoc in Compound 1 and connect Fmoc-Ile-OH to obtain Compound 2; Remove the protecting group Fmoc in Compound 2 and connect Fmoc-Asp-OH to obtain Compound 3; Remove the protecting group Fmoc in Compound 3 and connect Fmoc-Ser-OH to obtain Compound 4; Remove the protecting group Fmoc in Compound 4 and connect Fmoc-Ser-OH to obtain Compound 5; Remove the protecting group Fmoc in Compound 5 and connect Fmoc-Arg-OH to obtain Compound 6; Remove the protecting group Fmoc in Compound 6 and cleave the resin to obtain the target product RF7.

3. The preparation method of the anticoagulant peptide according to claim 2, characterized in that The reagent used to block the activation site is a mixed solution of dichloromethane, methanol and diisopropylethylamine.

4. The preparation method of the anticoagulant peptide according to claim 2, wherein After removing the protecting group Fmoc in Compound 6, wash the obtained product, cleave the resin, filter, and precipitate to obtain purified RF7.

5. The preparation method of the anticoagulant peptide according to claim 4, characterized in that, After removing the protecting group Fmoc in Compound 6, wash the product with N,N-dimethylformamide, dichloromethane and methanol respectively.

6. The preparation method of the anticoagulant peptide according to claim 4, wherein The reagent used to cleave the resin after removing the protecting group Fmoc in Compound 6 is trifluoroacetic acid.

7. The preparation method of the anticoagulant peptide according to claim 4, characterized in that, The reagent used for precipitation after removing the protecting group Fmoc in Compound 6 is ice-cold diethyl ether.

8. Use of the anticoagulant peptide according to claim 1 in the preparation of an anticoagulant drug.

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