Application of phosvitin and phosphopeptides thereof in preparation of in-vitro anticoagulant drugs

Abstract

The invention provides application of phosvitin and phosphopeptides thereof in preparation of in-vitro anticoagulant drugs, specifically, blood coagulation is caused by a series of enzymatic reactions, the chemical nature of the blood coagulation is that fibrinogen in a sol state is converted into fibrin in a gel state, and the main substance for catalyzing the reactions is thrombin. The phosvitin can inhibit the activity of thrombin in blood so as to prolong the blood coagulation time to achieve the anticoagulation effect. The application proves that phosvitin has the potential of being developed into an in-vitro anticoagulant.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the application of phosvitin in the preparation of in vitro anticoagulant drugs. Background Art

[0002] Hyperlipidemia, blood viscosity and atherosclerosis are the three main causes of CVD. Venous and arterial thrombosis affect the occurrence and development of CVD, including acute coronary syndrome, deep vein thrombosis and acute myocardial infarction. Antiplatelet aggregation and fibrinolytic drugs are important for the treatment of acute thrombotic events and are widely used to treat CVD. Among these drugs, anticoagulant chemical drugs, including rivaroxaban, warfarin and apixaban, are the most effective. In recent decades, in addition to mainstream drugs such as heparin, warfarin and aspirin, there has been an increasing number of clinical efforts to explore new molecular entities with anticoagulant activity from natural medicines.

[0003] Anticoagulants have been used as a first line of defense to inhibit the formation and expansion of blood clots. Heparin, the most commonly used anticoagulant, is an acidic mucopolysaccharide composed of alternating glucosamine, L-iduronide, N-acetylglucosamine, and D-glucuronic acid. It is currently one of the most important drugs for the prevention and treatment of thrombotic diseases. Traditional heparin is mainly extracted from the small intestinal mucosa and lung tissue of livestock. It is expensive and has a complex extraction process. It also carries a high safety risk of residual viruses and clinically carries the risk of bleeding and thrombocytopenia. The thrombocytopenia it induces is an immune-related adverse reaction that can lead to serious thrombotic complications.

[0004] Therefore, research and development of new antithrombotic drugs with reduced risk of side effects are of great significance for preventing thromboembolism.

[0005] Phosvitin (PSV), a phosphoprotein found in egg yolk, has been shown to possess antimicrobial and anti-inflammatory activities. PSV constitutes approximately 4% of the dry weight of egg yolk and is composed of 216 amino acid residues, 56% of which are serine, 80% of which are phosphorylated. As the most highly phosphorylated natural protein, PSV possesses excellent metal ion chelation capacity, which confers various biological activities such as antioxidant and antimicrobial properties. Its high phosphorylation level and strong electronegativity enable it to strongly interact with cationic or positively charged protein molecules. Studies have shown that PSV, as a phosphate-rich polyanion, also exhibits antithrombin activity. At 30 μg / mL, PSV increased the rate of heparin cofactor II (HCII) inhibition of thrombin by 1600-fold. Furthermore, PSV accelerates the formation of the HCII-thrombin complex, leading us to speculate that PSV plays a positive role in inhibiting blood coagulation. Summary of the Invention

[0006] This application aims to solve the above-mentioned technical problems by providing the application of phosvitin in the preparation of in vitro anticoagulant drugs, aiming to solve the problems of high cost and complex extraction process of existing in vitro anticoagulant drugs, as well as the wide source of raw materials, green and healthy, and no impact on blood safety.

[0007] The present application provides the use of phosvitin in the preparation of anticoagulant drugs.

[0008] The present application also provides a use of phosvitin phosphopeptide in the preparation of anticoagulant drugs.

[0009] Beneficial effects

[0010] The present application provides an application of phosvitin and phosphopeptide in the preparation of an in vitro anticoagulant drug, thereby avoiding the problem of difficult to cure and easy to relapse in the prior art treatment.

[0011] Although anticoagulant drugs for the treatment of cardiovascular diseases in existing technologies can control the disease to a certain extent, the following problems still exist: for example, traditional heparin is mainly extracted from the small intestinal mucosa and lung tissue of livestock, which is expensive and has a complex extraction process. There is a high safety risk of residual viruses and the risk of bleeding and thrombocytopenia in clinical practice. The thrombocytopenia it induces is an immune-related adverse reaction that can lead to serious thrombotic complications.

[0012] The phosvitin provided herein is a phosphate-rich polyanion with a high net negative charge, resulting in a high degree of phosphorylation and strong electronegativity. During the coagulation process, thrombin converts fibrinogen into insoluble fibrin, thereby causing blood coagulation. Phosvitin, a serine protease, can interact with phosvitin to inhibit its activity. Therefore, phosvitin possesses anticoagulant activity and can exert significant anticoagulant effects in vitro. It also has good clot-dissolving ability and may play a thrombolytic role in subsequent clinical studies, showing potential for the treatment of clinical pathological thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Effect of PSV on coagulation of fresh blood 5 minutes after addition, including (A) blank control - normal saline; (B) PSV - 100 μg / mL; (C) PSV - 500 μg / mL;

[0014] Figure 2 Effects of 5mg / mL PSV and phosphopeptides on plasma recalcification time (PRT);

[0015] Figure 3Effects of PSV on the three coagulation parameters APTT, PT, and TT. Figure (A) shows the effects of different concentrations of PSV on the three coagulation parameters; Figure (B) shows the effects of 5 mg / mL PSV and phosphopeptides on the three coagulation parameters.

[0016] Figure 4 Turbidity measurement reflects the blood coagulation process. Figure (A) shows the change in absorbance during blood coagulation, which reflects the degree of blood coagulation. Figure (B) shows the effect of adding 5 mg / mL PSV and phosphopeptide on blood coagulation time.

[0017] Figure 5 Fibrinolytic activity assay, where (A) is the area of ​​the fibrinolytic zone formed by different concentrations of urokinase standard on agarose-fibrin plates; (B) is the area of ​​the fibrinolytic zone formed by PSV and phosphopeptide on agarose-fibrin plates; (C) is the standard curve of the urokinase-fibrinolytic zone area;

[0018] Figure 6 Effects of PSV on the in vitro clot dissolution rate, where (A) shows the effects of different PSV concentrations on the clot dissolution rate; (B) shows the effects of 5 mg / mL PSV and phosphopeptides on the clot dissolution rate.

[0019] Figure 7 Determination of the ability of PSV to inhibit thrombin-catalyzed fibrinogen coagulation, where (A) is the inhibition rate of different concentrations of PSV on thrombin; (B) is the inhibition rate of 5 mg / mL PSV and phosphopeptide on thrombin. DETAILED DESCRIPTION

[0020] The present invention will be further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0021] One embodiment of the present application provides the use of phosvitin in the preparation of anticoagulant drugs.

[0022] In one embodiment, the anticoagulant drug is an extracorporeal anticoagulant drug.

[0023] In one embodiment, the concentration of the phosvitin is 0.5 mg / mL-5 mg / mL.

[0024] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0025] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier, wherein the carrier comprises one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant, and a preservative.

[0026] In one embodiment, the dosage form of the drug is tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, and solutions.

[0027] One embodiment of the present application provides a use of phosvitin phosphopeptide in the preparation of an anticoagulant drug.

[0028] In one embodiment, the anticoagulant drug is an extracorporeal anticoagulant drug.

[0029] In one embodiment, the concentration of phosvitin phosphopeptide is 0.5 mg / mL-5 mg / mL.

[0030] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0031] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier, wherein the carrier comprises one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant, and a preservative.

[0032] In one embodiment, the dosage form of the drug is tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, and solutions.

[0033] Extraction of phosvitin

[0034] Carefully break the shell of a fresh egg, separate the egg white with an egg yolk separator, roll the egg yolk on filter paper to remove the egg white and ovary, cut the egg yolk membrane with a toothpick, collect a total of 200 mL of egg yolk liquid, and place the egg yolk liquid in an ice water bath to keep it cold; add 1 L of water to the egg yolk liquid, adjust the pH to 5, and stir at 4°C for 6 h.

[0035] After stirring, the egg yolk solution was centrifuged at 7000 rpm for 1 h, the precipitate was collected, 800 mL of 0.05 mol NaCl solution was added, and the mixture was stirred for 4 h. The stirred suspension was centrifuged at 7000 rpm for 30 min, and the precipitate was collected.

[0036] Place the collected precipitate in a beaker and add a mixture of 600 mL of n-hexane and 200 mL of ethanol. Stir in a fume hood at 4°C for 6 hours. After stirring, filter the mixture to form a cake. After the cake-like precipitate is collected and air-dried in a fume hood, add 400 mL of 1.74 M NaCl solution and stir at 4°C for 4 hours.

[0037] The supernatant was collected after centrifugation at 7000 rpm and 15° C. for 40 min, filtered, dialyzed for 48 h and freeze-dried to obtain phosvitin.

[0038] Example 1 Phosvitin Anticoagulant Effect Test - Determination of Whole Blood Coagulation Time CT

[0039] To preliminarily determine whether phosvitin extracted from eggs has an anticoagulant effect, the present application measured the whole blood clotting time (CT). This refers to the time it takes for blood to completely coagulate after leaving the blood vessels. It reflects the overall activity of coagulation factors in the blood and the efficiency of the coagulation cascade. When isolated venous blood comes into contact with a standard glass test tube, coagulation factor XII is activated, initiating the endogenous coagulation system and generating fibrin, which causes blood to coagulate. The time required for this process is the whole blood clotting time (CT).

[0040] In the present embodiment, four test tubes with an inner diameter of 8 mm were taken and numbered. PSV was dissolved in physiological saline and filtered with a 0.22 μm membrane to remove bacteria. PSV solutions of different concentrations (100, 500 μg / mL) were prepared. Blood was collected from the mouse eyeball and dripped into a glass test tube with an anticoagulant added in advance. The blood volume from each mouse eyeball was about 0.5 to 1 mL. Blood collection was stopped after 15 drops of eyeball blood were dripped. The test tubes were placed in a water bath at 37°C for 3 minutes. After the blood was separated from the body for 3 minutes, the first test tube was gently tilted every 30 seconds until the blood stopped flowing. The blood coagulation time was observed and recorded. The experimental treatment was as follows: Figure 1 shown.

[0041] The test results are shown in Table 1. Compared with the blank group, the whole blood coagulation time of mice in the high-dose phosvitin group was significantly prolonged (P<0.01), indicating that the high-dose phosvitin group can significantly prolong the whole blood coagulation time of mice compared with the blank control group and has a good anticoagulant effect. Figure 1 It can be seen that after five minutes, the coagulation degree of mouse blood was: distilled water>100μg / mL>500μg / mL.

[0042] Table 1 Effect of phosvitin on whole blood clotting time in mice

[0043]

[0044] The above results preliminarily indicate that phosvitin can prolong blood clotting time. Based on the above analysis, it can be concluded that PSV can be used as an anticoagulant drug to increase blood clotting time.

[0045] Example 2 Determination of the Anticoagulant Titer of Phosvitin by Azure A Colorimetry

[0046] In order to further evaluate the anticoagulant effect of PSV, the present application embodiment selects the Azure A colorimetric method to measure its anticoagulant titer. Take heparin standard (100U / ml), mix it according to the ratio of heparin standard: distilled water = 1:99, and obtain 1U / ml heparin standard solution. Take an appropriate amount of Azure colorimetric solution, and mix it according to the ratio of Azure colorimetric solution: distilled water = 1:5, which is the Azure colorimetric working solution. Mix the heparin standard solution and Azure A colorimetric solution in proportion, use an ultraviolet spectrophotometer (UV-2600i) to detect the absorbance at 505nm, and draw a heparin titer standard curve. Measure 50μg / mL, 100μg / mL, and 500μg / mL of PSV sample solutions and Azure A colorimetric solution in proportion, and detect the absorbance at 505nm. Calculate the anticoagulant titer of PSV samples of different concentrations according to the following formula.

[0047]

[0048] Where: P is the number of units (U / mL) found on the standard curve based on the absorbance value of the sample to be tested; V is the total volume of the sample to be tested (mL); Vi is the volume of the sample to be tested used in the determination (mL); M is the mass of the sample (mg).

[0049] The results are shown in Table 2. The anticoagulant potency of 500 μg / mL PSV was 94.5±1.5 U / mg, which is approximately 75.6% of the potency of common heparin sodium on the market (125 U / mg).

[0050] Table 2 Anticoagulant titer of different concentrations of PSV

[0051]

[0052] According to the experimental conclusions in Table 2, the trend of the anticoagulant potency of phosvitin determined by the Azure A colorimetric method is consistent with the trend of the whole blood clotting time in Example 1, indicating that PSV has significant anticoagulant activity compared to normal saline.

[0053] Example 3 Preparation of phosvitin phosphopeptides treated with different enzymes

[0054] Bioactive peptides typically consist of 2 to 20 amino acid residues and can exert beneficial biological activities beyond the nutritional value of their parent proteins. Therefore, PSV was enzymatically hydrolyzed for subsequent anticoagulant efficacy measurement. The enzymatic hydrolysis process follows existing published methods (e.g., Himali Samaraweera, et al. 2014).

[0055] The ratio and enzymatic digestion time were adjusted for each enzyme, as shown in Table 3. PSV (10 mg / mL) was dissolved in distilled water and preheated at 100°C for 60 minutes to improve hydrolysis before use. Three enzymatic digestion treatments were performed on PSV. Before adding the protease, the pH of the PSV solution was adjusted with 1N HCl or 1N NaOH. The optimal temperature and pH conditions for each enzyme were: trypsin at 37°C, pH 8.0; pepsin at 37°C, pH 2.0; for co-treatment with pepsin and trypsin, the pH was first adjusted to 2.0 before adding pepsin at 37°C, followed by pH 8.0; and trypsin at 37°C. The enzyme / substrate ratio was 1:100 (w / w) for all enzymes. Enzymatic digestion was carried out in a shaking water bath at 37°C for 24 hours. Finally, the sample was placed in a boiling water bath for 10 minutes to arrest enzymatic degradation. The hydrolyzed products were lyophilized to obtain phosvitin phosphopeptides after different enzyme treatments.

[0056] Table 3 Conditions for enzymatic hydrolysis of PSV by different enzymes

[0057]

[0058] Example 4 Plasma recalcification time PRT determination

[0059] To preliminarily investigate the anticoagulant effect and mechanism of action of phosvitin, we measured plasma recalcification time (PRT). Prolonged recalcification time may indicate deficiencies in coagulation factors such as factors VIII, IX, and XI, as well as severe deficiencies in factors II, V, and X, or fibrinogen deficiency. Furthermore, elevated levels of anticoagulants in the blood can also lead to a prolonged recalcification time.

[0060] PSV and its phosphopeptide were dissolved in PBS (pH 7.4) buffer at a concentration of 5 mg / mL. Rabbit platelet-free plasma (PPP) (100 μL) was mixed with each test component in a 96-well plate and incubated in a 37°C water bath for 1 minute. Then, 100 μL of 0.025 M CaCl₂ was added, and a timer was started. The rabbit plasma and components were gently swirled using a glass capillary tube at approximately 15-second intervals, and the appearance of clots was observed. The time from the addition of CaCl₂ to the appearance of the first clot was recorded as the PRT.

[0061] The results are as follows Figure 2As shown, 5 mg / mL of PSV and its phosphopeptide significantly increased plasma recalcification time compared to the blank control group (p < 0.01), with the pepsin-hydrolyzed PSV showing the greatest effect. This suggests that PSV is related to its molecular weight after hydrolysis and its interaction with the thrombin site, and that PSV can act as an in vitro anticoagulant to prolong intrinsic clotting time. The prolongation of intrinsic clotting time is associated with decreased activity and quantity of coagulation factors VIII, IX, and XI, as well as the presence of anticoagulants in the blood. Therefore, phosvitin and phosphopeptides can achieve a certain anticoagulant effect by prolonging intrinsic clotting time.

[0062] Example 5 Analysis of the Anticoagulant Pathway of PSV and Phosphopeptides

[0063] To further investigate the anticoagulant activity and pathways of phosvitin, the in vitro anticoagulant activity of PSV samples was determined using APTT, PT, and TT kits. Fresh platelet-poor rabbit plasma was purchased from a reagent company. PBS buffer served as a negative control, and a heparin standard served as a positive control. For the APTT assay, 50 μL of APTT reagent was incubated with 50 μL of rabbit platelet-free plasma, with 50 μL of each sample at different concentrations. After a 5-minute incubation, 50 μL of 25 mM CaCl₂ preheated at 37°C was added, and clotting time was measured using an SK5001 semi-automatic coagulometer. For the PT assay, 50 μL of plasma and 50 μL of sample were incubated at 37°C for 3 minutes, followed by 50 μL of PT reagent, and clotting time was measured using an SK5001 semi-automatic coagulometer. In the TT experiment, 50 μL of PPP was first incubated at 37°C. Then, 50 μL of each sample was incubated with 50 μL of PPP at 37°C for 60 seconds. Coagulant steel beads were added, and 50 μL of TT reagent was added simultaneously with the timer. The timer was pressed and the results were automatically recorded using an SK5001 semi-automatic coagulometer. This experiment was repeated three times.

[0064] The prolongation of APTT indicates that the intrinsic or common pathway is inhibited; PT is used to characterize exogenous coagulation factors; TT reflects the coagulation state of fibrinogen conversion to fibrin, and TT together with fibrinogen is considered the third coagulation phase. Figure 3 As shown in Figure A, within the experimental concentration range, the APTT, TT, and PT of phosvitin were significantly different from those of the blank control PBS buffer solution, with a dose-dependent effect, and reached a peak at 5 mg / mL. Figure 3 The activated partial thromboplastin time (APTT) showed that at 5 mg / mL, PSV could significantly prolong the APTT time by 31.5 seconds, which was not significantly different from that of 1 mg / mL heparin sodium. The significant prolongation of APTT by PSV suggests that the protein may exert its anticoagulant effect by inhibiting the intrinsic pathway of coagulation factors VIII, IX, XI, and XII. Figure 3It can be seen from the prothrombin time (PT) in Figure 5 that PSV can significantly prolong the prothrombin time, and compared with the blank control, 5 mg / mL PSV can prolong the PT by 6.65S. This shows that PSV may exert an anticoagulant effect through the exogenous coagulation system, such as inhibiting the activity of coagulation factors I, II, V, VII, and X. Figure 3 As shown in the activated partial thromboplastin time (TT), which reflects the coagulation activity of the common coagulation system, PSV significantly prolongs TT, suggesting that PSV may act as an anticoagulant or cause fibrinogen abnormalities. This suggests that PSV can exert its anticoagulant activity by affecting intrinsic, extrinsic, and common coagulation pathways, with the strongest inhibitory effect on the intrinsic coagulation pathway. Furthermore, the anticoagulant activity of phosvitin at different doses is relatively mild, potentially reducing bleeding side effects.

[0065] Figure 3 Figure B reflects the three coagulation activities of phosvitin after treatment with different enzymes. A comprehensive comparison shows that phosvitin treated with pepsin has the best in vitro anticoagulation effect, which is consistent with the effect of plasma recalcification time in Example 5.

[0066] According to the above experimental results, PSV and phosphopeptides can inhibit the activation of coagulation factors in the coagulation pathway to varying degrees to exert anticoagulant effects, among which the anticoagulant effect of PSV is dose-dependent.

[0067] Example 6 Turbidity Determination of PSV and Phosphopeptides - Plasma Coagulation Process

[0068] To further investigate the inhibitory effects of PSV and phosphopeptides on plasma coagulation, turbidimetric measurements were performed to determine the initial and complete coagulation times of plasma after the addition of different samples. Turbidimetric measurements were performed in a 96-well plate at 37°C. 50 μL of plasma and 50 μL of sample were mixed in a 96-well plate and preheated at 37°C for 1 minute. Then, 50 μL of 25 mM warm CaCl₂ was added to the mixture, and the absorbance was measured every 15 seconds at 620 nm for 20 minutes using a multifunctional microplate reader (victorX3). Changes in absorbance were observed during this process, and an absorbance value of 0.026 was selected as the initial coagulation time of plasma. The time required for the plasma coagulation process was recorded when the absorbance value no longer changed significantly.

[0069] Depend on Figure 4In the A-phase assay, changes in absorbance reflect the coagulation process of plasma after the addition of different samples. Compared to the blank control group, where clot formation began at 110 seconds and complete coagulation occurred at 6000 seconds, with no change in absorbance, phosvitin and its phosphopeptides significantly inhibited the onset and complete coagulation of fibrin clots (p < 0.01). The high-dose PSV group exhibited the greatest inhibitory effect compared to the phosphopeptides, with clot formation beginning at 250 seconds and complete coagulation at 1100 seconds. Heparin sodium completely inhibited plasma coagulation within 1200 seconds. In this experiment, after the reaction began in a mixture containing plasma, sample, and CaCl2, the density of the plasma clot gradually increased over a 20-minute period. PSV, as an in vitro anticoagulant, prolonged clot formation, indicating that the protein affects the intrinsic coagulation cascade (factors II, V, VIII, IX, X, and XI).

[0070] Figure 4 Figure B reflects the coagulation time of plasma after adding different samples. The absorbance value of 0.026 was selected as the plasma coagulation start time, and when the absorbance value no longer changed significantly, it was taken as the plasma complete coagulation time. According to the experimental results, compared with the blank control, PSV and phosphopeptide can significantly inhibit the plasma coagulation process (p<0.01).

[0071] According to the above experimental results, PSV and phosphopeptides can also exert anticoagulant effects by inhibiting the formation of plasma clots.

[0072] Example 7 Evaluation of the fibrinolytic activity of PSV and phosphopeptides

[0073] Research on the antithrombotic pathways of common anticoagulants on the market typically focuses on five aspects: anticoagulation, antiplatelet aggregation, fibrinolysis, endothelial cell protection, and improvement of hemorheological properties. For example, heparin can enhance the activation of plasminogen to plasmin by enhancing the activation of u-PA and t-PA, increasing the inhibitory effect of PAI-1 during PCI, and inhibiting the activation of thrombin-activated fibrinolytic inhibitor (TAFI) by thrombin IIa, thereby increasing the degree of plasmin activation and thus thrombolysis. Therefore, in vitro fibrinolytic activity is measured using the agarose-fibrin plate method.

[0074] Figure 5 A reflects the area of ​​the fibrinolytic zone produced by different concentrations of urokinase standard, and the standard curve equation of the fibrinolytic zone size (x) and fibrinolytic activity (y) produced by different concentrations of urokinase is Y = 40.392X-15.562, R 2 =0.9919, good correlation ( Figure 5 Middle C). Figure 5Figure B reflects that neither PSV nor phosphopeptides exhibited a fibrinolytic zone on the fibrin plate, indicating that neither directly dissolves fibrin and possesses fibrinolytic activity. Therefore, the anticoagulant effects of PSV and phosphopeptides are unrelated to the fibrinolytic pathway.

[0075] Example 8 Evaluation of the Thrombolytic Activity of PSV and Phosphopeptides

[0076] In order to evaluate whether PSV can dissolve the formed thrombus and thus exert its anticoagulant activity, the thrombolytic activity was evaluated by a blood clot dissolution experiment. After one week of adaptive feeding, SPF male KM mice were fed blood through the eyeball and placed in a 1.5mL centrifuge tube, 1mL per tube. Place it at room temperature, wait for the blood to coagulate, remove the blood clot, absorb the water and weigh it to obtain the initial weight of the blood clot W1. After adding 1mL of normal saline, sodium heparin (1mg / mL), PSV and phosphopeptide (5mg / mL) solution to the centrifuge tube, it was placed in a constant temperature box at 37°C. After 5 hours, the blood clot was removed, the water was absorbed, and the weight of the blood clot after dissolution was obtained by weighing W2. The blood clot dissolution rate was calculated according to the following formula to evaluate its thrombolytic activity.

[0077]

[0078] The blood clot dissolution rate of different concentrations of PSV and phosphopeptide is as follows Figure 6 As shown. Figure 6 The results of Experiment A show that compared with PBS buffer solution, both heparin and PSV can improve the dissolution rate of blood clots to varying degrees, and are dose-dependent. Among them, the dissolution rate of PSV in the high-dose group was significantly increased by 42.7% (p<0.01) compared with PBS buffer solution. The mechanism of common thrombolytic drugs currently on the market depends on the fibrinolytic system of the blood, which gradually dissolves the thrombus by degrading the fibrin in the thrombus. According to the results of Example 7, PSV has no fibrinolytic activity. Analysis of the reason for its dissolution of blood clots may be that PSV has a strong affinity for divalent metal ions due to its large number of phosphate groups and a large amount of negative charge. Therefore, it is concluded that PSV can directly act on thrombus components, weaken its stability by chelating metal ions (such as calcium ions) in the thrombus, and promote dissolution. It is also possible to dissolve thrombi by changing the physical environment of the thrombus or indirectly enhancing the endogenous fibrinolytic system, rather than relying on the traditional plasmin system. At the same time, by Figure 6 The results in Figure B show that PSV at the same concentration has better thrombolytic ability than its phosphopeptide.

[0079] Example 9 Evaluation of the Thrombolytic Activity of PSV and Phosphopeptides

[0080] Based on the above experimental results, PSV has a significant anticoagulant effect and can inhibit coagulation cascade reactions such as intrinsic coagulation. Thrombin is the core enzyme of the coagulation cascade. Anticoagulants can exert their anticoagulant effect by directly or indirectly inhibiting thrombin activity, preventing fibrin formation and platelet activation. Therefore, to further explore the anticoagulant mechanism of PSV, we used absorbance to determine whether it has the ability to inhibit thrombin-catalyzed fibrin coagulation.

[0081] Different concentrations of PSV were used to verify the inhibitory effect of PSV on thrombin, and 1 mg / mL heparin sodium was used as a positive control. Figure 7 The results of Experiment A show that within the range of 0.5-5 mg / mL, the inhibitory effect of PSV on prothrombin activity is proportional to the dose, and as the PSV concentration increases, its inhibitory effect increases, reaching a peak at 5 mg / mL. Compared with heparin sodium at the same concentration, the inhibitory effect of PSV on thrombin is 63.1% of that of heparin sodium, indicating that PSV has a better inhibitory effect on thrombin. Figure 3 In Figure A, when the PSV concentration reached 5 mg / mL, the inhibitory effect of PSV reached 92.9%, which was not significantly different from the inhibitory effect of 1 mg / mL heparin sodium, which reached 95.5%. The inhibitory effect of PSV peptide on thrombin was verified using a concentration of 5 mg / mL PSV and peptides hydrolyzed by different enzymes, and 1 mg / mL heparin sodium was used as a positive control. Figure 7 The results of experiment B show that the inhibitory effect on thrombin is: PSV>pepsin>trypsin>stomach+trypsin, and its trend is consistent with the blood clot coagulation time and plasma recalcification time, further confirming that its mechanism of blood coagulation may be achieved by inhibiting thrombin activity. Similarly, PSV that has not been hydrolyzed by protease has the best effect.

[0082] These results demonstrate that phosvitin isolated from eggs can significantly prolong whole blood clotting time, plasma recalcification time (PRT), and delay the onset of plasma coagulation in vitro, thereby exerting an anticoagulant effect in vitro. Based on these intuitive experimental results, we further characterized its anticoagulant activity by measuring anticoagulant potency and three coagulation times using the Azure A colorimetric assay. To investigate its mechanism of action, PSV inhibits the activation of coagulation factors and dissolves existing thrombi by chelating metal ions in the blood. Furthermore, it inhibits thrombin activity in a dose-dependent manner, thereby prolonging blood clotting time.

[0083] In summary, these results suggest that PSV has great potential as a clinical antithrombotic drug for the treatment of CVD, or may provide a reference for future in-depth research on vascular health foods.

[0084] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of phosvitin in the preparation of anticoagulant drugs.

2. The use according to claim 1, characterized in that The anticoagulant drug is an in vitro anticoagulant drug.

3. The use according to claim 1, characterized in that The concentration of the phosvitin is 0.5 mg / mL-5 mg / mL.

4. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 1, characterized in that The drug further comprises a pharmaceutically acceptable carrier, which comprises one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant and a preservative.

6. The use according to claim 1, characterized in that The dosage forms of the medicine are tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders and solutions.

7. Use of a phosvitin phosphopeptide in the preparation of anticoagulant drugs.

8. The use according to claim 7, characterized in that The anticoagulant drug is an in vitro anticoagulant drug.

9. The use according to claim 7, characterized in that The concentration of phosvitin phosphopeptide is 0.5 mg / mL-5 mg / mL.

10. The use according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

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