A marine sodium polysaccharide, a preparation method and application thereof, and an anticoagulant and / or antithrombotic drug targeting endogenous coagulation pathway

Abstract

The present application relates to the technical field of medicine, and provides a kind of haena polysaccharide and its preparation method and application and target endogenous coagulation pathway anticoagulant and / or antithrombotic drug.The haena polysaccharide provided by the present application is fucosylated chondroitin sulfate polysaccharide, the weight average molecular weight is 9-13 million, the molar ratio of glucuronic acid, N-acetylglucosamine and fucose is 1:0.8-1.2:0.5-0.8, and the mass percentage content of sulfate group is 25-40%.The haena polysaccharide provided by the present application can target endogenous coagulation pathway terminal rate-limiting enzyme (iFXase), and has no obvious effect on coagulation factors in other coagulation pathways, realizes the effect of anticoagulation and non-hemorrhage, and can be widely applied in acute and recovery period treatment of ischemic stroke, solves the safety problem of using anticoagulant and antithrombotic drug for clinical ischemic stroke patients, and opens up a new field of research and development of target endogenous coagulation pathway rate-limiting enzyme anticoagulant drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a sodium hyaluronate polysaccharide, its preparation method and application, as well as an anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway. Background Technology

[0002] Anticoagulants and / or antithrombotic drugs are a class of medications that prevent thrombus formation or inhibit the further development of existing thrombi by interfering with the body's coagulation process. Timely use of anticoagulants and / or antithrombotic drugs in the acute and subacute phases of ischemic stroke is crucial for symptom relief. The body's coagulation pathways are divided into intrinsic, extrinsic, and common pathways. The intrinsic coagulation pathway causes pathological thrombosis, the extrinsic coagulation pathway is related to inhibiting vascular wall damage, and the common pathway affects both. Intrinsic factors include coagulation factor X enzyme (FIXa-FVIIIa-PL-Ca). 2+ Factor IFXase (IFX) is the final rate-limiting enzyme in the intrinsic coagulation pathway, which is closely related to pathological thrombosis but is not essential for hemostasis. Currently used anticoagulants and / or antithrombotic drugs act directly on coagulation factors IIa and / or Xa in the common pathway. Due to bleeding side effects, current international and domestic guidelines do not recommend the early use of direct IIa inhibitors or Xa inhibitors. Targeting anticoagulants and / or antithrombotic drugs that act on the intrinsic coagulation pathway has been a key focus of research worldwide; however, no such drugs are currently on the market.

[0003] Sea cucumber body walls are rich in polysaccharides, mainly including two categories: fucoidan sulfate and fucosylated chondroitin sulfate. Among them, sea cucumber fucosylated chondroitin sulfate exhibits rich biological activities due to its unique structure, such as lowering blood lipids, anti-tumor, antiviral, and anti-inflammatory effects, especially good anticoagulant and antithrombotic activities. However, currently available natural fucosylated chondroitin sulfate extracted from sea cucumber body walls often has significant bleeding side effects, meaning it cannot target the intrinsic coagulation pathway. Related technologies degrade the extracted natural polysaccharides to reduce the molecular weight of sea cucumber fucosylated chondroitin sulfate and thus reduce bleeding side effects, but this simultaneously weakens the anticoagulant effect.

[0004] In summary, there is an urgent need for an anticoagulant and / or antithrombotic drug that can target the intrinsic coagulation pathway, has good anticoagulant effect, and has minimal bleeding side effects. Summary of the Invention

[0005] In view of this, the present invention provides a sea cucumber polysaccharide, its preparation method and application, as well as an anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway. The sea cucumber polysaccharide provided by the present invention is a natural fucoidylated chondroitin sulfate polysaccharide extracted directly from the body wall of sea cucumbers. It can target the terminal rate-limiting enzyme iFXase in the intrinsic coagulation pathway, exhibiting good anticoagulant effect and minimal bleeding side effects, thus achieving the effect of anticoagulation and no bleeding.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] A type of sea sodium polysaccharide, wherein the sea sodium polysaccharide is a fucoidylated chondroitin sulfate-like polysaccharide; the sea sodium polysaccharide has a weight-average molecular weight of 90,000 to 130,000, and its monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose, wherein the molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose is 1:0.8 to 1.2:0.5 to 0.8; and the mass percentage of sulfate ester groups in the sea sodium polysaccharide is 25% to 40%.

[0008] Preferably, the structural unit of the sea sodium polysaccharide is shown in Formula I:

[0009]

[0010] In Equation I: R1 represents H and SO 3- Or fucose, where R2 is H or SO 3- R3 is H or SO 3- R4 is H or fucose; at least one of R1 and R4 is fucose. Indicates a cation;

[0011] The structures of the fucosylates in R1 and R4 are shown in Formula I-1:

[0012]

[0013] In Equation I-1: R5 is H or SO3 - R6 is H or SO3 - R7 is H or SO3 - .

[0014] Preferably, the sea sodium polysaccharide 1 In the HNMR spectrum, a proton signal peak at the end of the fucose fragment is present at a relative shift of 5.70–4.90 ppm, a proton signal peak at the end of the glucuronic acid fragment is present at 4.80–4.45 ppm, a proton signal peak at the end of the N-acetylgalactoside fragment is present at 4.90–4.50 ppm, an acetyl proton signal peak of the N-acetylgalactoside fragment is present at 2.40–1.80 ppm, and a methyl proton signal peak of the fucose fragment is present at 1.60–1.10 ppm.

[0015] The sea sodium polysaccharide 13 In the CNMR spectrum, a terminal carbon signal peak of the glucuronic acid fragment is present at a relative shift of 107.0–105.0 ppm, a terminal carbon signal peak of the N-acetylgalactosyl fragment is present at 103.0–101.0 ppm, a terminal carbon signal peak of the fucose fragment is present at 104.0–103.0 ppm and 101.0–98.0 ppm, a carbon signal peak of the N-acetylgalactosyl fragment at position 2 is present at 56.0–51.0 ppm, an acetyl carbon signal peak of the N-acetylgalactosyl fragment is present at 27.0–24.0 ppm, and a methyl carbon signal peak of the fucose fragment is present at 20.0–16.0 ppm.

[0016] The sodium polysaccharide 1 HNMR spectra and 13 Heavy water was used as the solvent in the CNMR spectrum analysis, and sodium 3-(trimethylsilyl)deuterated propionate was used as the internal standard.

[0017] Preferably, the cation is a hydrogen ion, an alkali metal ion, an alkaline earth metal ion, an ammonium ion, or an iron ion.

[0018] Preferably, the cation is a sodium ion; the mass percentage of sodium ions in the sea sodium polysaccharide is 5-15%.

[0019] Preferably, the polydispersity index of the sea sodium polysaccharide is ≤2.

[0020] This invention also provides a method for preparing the sodium polysaccharide described in the above-mentioned scheme, comprising the following steps:

[0021] (1) The sea cucumber body wall powder was subjected to enzymatic hydrolysis and acid hydrolysis in sequence to obtain acid hydrolysis supernatant; the enzyme used in the enzymatic hydrolysis was protease;

[0022] (2) After adjusting the pH of the acid hydrolysis supernatant to 6.5-7.5, alcohol precipitation was performed. The resulting alcohol-precipitated solid was washed, centrifuged and freeze-dried in sequence to obtain crude polysaccharide.

[0023] (3) The crude polysaccharide was purified by column chromatography, and the fraction with a weight average molecular weight of 90,000 to 130,000 was collected. The obtained fraction was precipitated with alcohol to obtain the sea sodium polysaccharide.

[0024] The present invention also provides the application of the sodium hyaluronate described in the above-described scheme or the sodium hyaluronate prepared by the preparation method described in the above-described scheme in the preparation of anticoagulant and / or antithrombotic drugs or coagulation factor X enzyme targeting inhibitors.

[0025] The present invention also provides a coagulation factor X enzyme targeting inhibitor, comprising the sodium hyaluronate described in the above scheme or the sodium hyaluronate prepared by the preparation method described in the above scheme.

[0026] The present invention also provides an anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway, comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is the sodium hyaluronate described in the above-described scheme or the sodium hyaluronate prepared by the preparation method described in the above-described scheme.

[0027] This invention provides a sea sodium polysaccharide, which is a fucoidylated chondroitin sulfate-like polysaccharide. The sea sodium polysaccharide has a weight-average molecular weight of 90,000 to 130,000, and its monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose, with a molar ratio of 1:0.8–1.2:0.5–0.8. The mass percentage of sulfate groups in the sea sodium polysaccharide is 25–40%. The sea sodium polysaccharide provided by this invention can target the terminal rate-limiting enzyme (iFXase) in the intrinsic coagulation pathway. At therapeutic doses, it has little effect on coagulation factors in other coagulation pathways, achieving an anticoagulant and non-bleeding effect. It can be widely used in the acute and recovery phases of ischemic stroke, reducing the risk of bleeding associated with anticoagulation in thrombotic disease treatment. It solves the safety problem of using anticoagulant and antithrombotic drugs in clinical ischemic stroke patients and opens up a new field for the development of anticoagulant drugs targeting the rate-limiting enzyme of the intrinsic coagulation pathway.

[0028] This invention evaluated the anticoagulant activity of sodium hyaluronate in mice. The results showed that sodium hyaluronate significantly prolonged the APTT clotting time in mice and rats, without affecting PT and TT clotting times. These results indicate that the sodium hyaluronate of this invention targets the intrinsic coagulation pathway without affecting the extrinsic coagulation pathway.

[0029] This invention, referring to the heparin bioassay method in Pharmacopoeia General Chapter 1208, tested the anti-factor IIa, anti-factor Xa, ATIII-IIa, and ATIII-Xa activities of sea sodium polysaccharide. The results showed that at the experimental dose of 500 μg / mL, sea sodium polysaccharide showed no activity against factor IIa, factor Xa, and ATIII-IIa, and only a weak effect on ATIII-Xa (inhibition rate 36.7%). The activity of the rate-limiting enzyme of the intrinsic coagulation pathway, "intrinsic factor coagulation factor X enzyme (iFXase)," was tested using ELISA. The results showed that the test drug could significantly inhibit the rate-limiting enzyme iFXase of the intrinsic coagulation pathway, IC50. 50 The value was 207.5 ng / mL; the above experiments further clarified from a mechanistic perspective that the sodium polysaccharide of the present invention can selectively inhibit the activity of iFXase, the rate-limiting enzyme of the intrinsic coagulation pathway, and target the intrinsic coagulation pathway. Attached Figure Description

[0030] Figure 1 The results of linear regression analysis of sodium ions in Example 4;

[0031] Figure 2 The results of linear regression analysis of the sulfate groups in Example 5;

[0032] Figure 3 The sodium polysaccharide in Example 6 1 HNMR test spectrum;

[0033] Figure 4 The sodium polysaccharide in Example 6 13 CNMR test spectrum;

[0034] Figure 5 The HSQC test spectrum of sodium hyaluronate polysaccharide in Example 6;

[0035] Figure 6 The effect of sodium hyaluronate on coagulation function in mice in Example 7 is shown in Figure 7, where A is the TT test result, B is the APTT test result, and C is the PT test result.

[0036] Figure 7 The effect of sodium hyaluronate on venous thrombosis in mice in Example 8 is shown in Figure A, where A is the length of the thrombus formed in the mouse tail and B is the percentage of the length of the thrombus formed in the mouse tail to the total length of the mouse tail.

[0037] Figure 8 The detection mechanism of the FVIII activity assay kit used in Example 10 is described. Detailed Implementation

[0038] In this invention, sea sodium polysaccharide specifically refers to the natural polysaccharide extracted from the body wall of sea cucumber.

[0039] This invention provides a sea sodium polysaccharide, which is a fucoidylated chondroitin sulfate-like polysaccharide; the sea sodium polysaccharide has a weight-average molecular weight of 90,000 to 130,000, and its monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose, wherein the molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate ester groups in the sea sodium polysaccharide is 25% to 40%.

[0040] In this invention, the sea sodium polysaccharide is a fucoidylated chondroitin sulfate polysaccharide with a backbone that is a copolymer of disaccharide units composed of acetaminophen and glucuronic acid, and the disaccharide units are connected with fucose branches; the weight-average molecular weight of the sea sodium polysaccharide is 90,000 to 130,000, preferably 90,000 to 120,000; the polydispersity index of the sea sodium polysaccharide is preferably ≤2, more preferably ≤1.32, specifically 1.27 to 1.32.

[0041] In this invention, the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose in the sodium polysaccharide is 1:0.8-1.2:0.5-0.8, preferably 1:0.9-1.1:0.6-0.7.

[0042] In this invention, the mass percentage of sulfate ester groups in the sodium polysaccharide is 25-40%, preferably 26-35% (ion chromatography) or 32-40% (colorimetric method).

[0043] In this invention, the structural unit of the sea sodium polysaccharide is shown in Formula I:

[0044]

[0045] In Equation I: R1 represents H and SO 3- Or fucose, where R2 is H or SO 3- R3 is H or SO 3- R4 is H or fucose; at least one of R1 and R4 is fucose. Indicates a cation;

[0046] The structures of the fucosylates in R1 and R4 are shown in Formula I-1:

[0047]

[0048] In Equation I-1: R5 is H or SO3 - R6 is H or SO3 - R7 is H or SO3 - .

[0049] In this invention, the cation in Formula I is preferably hydrogen ion, alkali metal ion, alkaline earth metal ion, ammonium ion, or iron ion, wherein the alkali metal ion is preferably sodium ion or potassium ion, and the alkaline earth metal ion is preferably calcium ion; in a specific embodiment of this invention, the cation in Formula I is preferably sodium ion; when the cation is sodium ion, the mass percentage of sodium ion in the sea sodium polysaccharide is preferably 5-15%, preferably 8-11%.

[0050] In this invention, the sodium polysaccharide... 1 In the HNMR spectrum, a proton signal peak at the end of the fucose fragment is present at a relative shift of 5.70–4.90 ppm, a proton signal peak at the end of the glucuronic acid fragment is present at 4.80–4.45 ppm, a proton signal peak at the end of the N-acetylgalactoside fragment is present at 4.90–4.50 ppm, an acetyl proton signal peak of the N-acetylgalactoside fragment is present at 2.40–1.80 ppm, and a methyl proton signal peak of the fucose fragment is present at 1.60–1.10 ppm.

[0051] The sea sodium polysaccharide 13 In the CNMR spectrum, a terminal carbon signal peak of the glucuronic acid fragment is present at a relative shift of 107.0–105.0 ppm, a terminal carbon signal peak of the N-acetylgalactosyl fragment is present at 103.0–101.0 ppm, a terminal carbon signal peak of the fucose fragment is present at 104.0–103.0 ppm and 101.0–98.0 ppm, a carbon signal peak at position 2 of the N-acetylgalactosyl fragment is present at 56.0–51.0 ppm, an acetyl carbon signal peak of the N-acetylgalactosyl fragment is present at 27.0–24.0 ppm, and a methyl carbon signal peak of the fucose fragment is present at 20.0–16.0 ppm.

[0052] In this invention, the sea sodium polysaccharide 1 HNMR spectra and 13 In the CNMR spectrum analysis, heavy water was used as the solvent, sodium 3-(trimethylsilyl)deuterated propionate was used as the internal standard, and the shift of the methyl proton signal peak of sodium 3-(trimethylsilyl)deuterated propionate was 0.00 ppm.

[0053] In this invention, the sodium polysaccharide is extracted from the body wall of sea cucumber; the sea cucumber includes, but is not limited to, one or more of the following: sea cucumber with white feet, sea cucumber with rough skin, sea cucumber with spiny spine, sea cucumber with black skin, and sea cucumber with black milk, preferably sea cucumber with white feet.

[0054] This invention also provides a method for preparing the sodium polysaccharide described in the above-mentioned scheme, comprising the following steps:

[0055] (1) The sea cucumber body wall powder was subjected to enzymatic hydrolysis and acid hydrolysis in sequence to obtain acid hydrolysis supernatant; the enzyme used in the enzymatic hydrolysis was protease;

[0056] (2) After adjusting the pH of the acid hydrolysis supernatant to 6.5-7.5, alcohol precipitation was performed. The resulting alcohol-precipitated solid was washed, centrifuged and freeze-dried in sequence to obtain crude polysaccharide.

[0057] (3) The crude polysaccharide was purified by column chromatography, and the fraction with a weight average molecular weight of 90,000 to 130,000 was collected. The obtained fraction was precipitated with alcohol to obtain the sea sodium polysaccharide.

[0058] This invention involves sequentially enzymatically and acidically hydrolyzing sea cucumber body wall powder to obtain an acid hydrolysis supernatant. In this invention, the preferred method for preparing the sea cucumber body wall powder includes: soaking the sea cucumber body wall in acetone, followed by drying and grinding to obtain the sea cucumber body wall powder; the soaking temperature is preferably 3–5°C, more preferably 4°C, and the soaking time is preferably 12–48 hours, more preferably 24 hours; this invention does not have special requirements for the drying and grinding processes, and conditions well known to those skilled in the art can be used. The types of sea cucumbers mentioned are not described here.

[0059] In this invention, the protease is preferably papain; the sea cucumber body wall powder is preferably suspended in a sodium acetate buffer solution containing papain for enzymatic hydrolysis; the weight ratio of the sea cucumber body wall powder to papain is preferably 5-15:1, more preferably 10:1; the concentration of the sodium acetate buffer solution is preferably 0.1 mol / L, and the pH value is preferably 6; the enzymatic hydrolysis temperature is preferably 50-70℃, more preferably 65℃, and the time is preferably 12-48h, more preferably 24h; after the enzymatic hydrolysis is completed, the obtained liquid is centrifuged (referred to as the first centrifugation) to obtain the supernatant, and the supernatant is subjected to subsequent acid hydrolysis; the temperature of the first centrifugation is preferably 3-5℃, more preferably 4℃, the rotation speed is preferably 3000-5000 rpm, more preferably 4000 rpm, and the centrifugation time is preferably 20-40 min, more preferably 30 min.

[0060] In this invention, the acid used for acid hydrolysis is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 6 mol / L; the pH value of the acid hydrolysis is preferably 2-3, specifically 2.5; the acid hydrolysis time is preferably 1-3 h, more preferably 2 h; the acid hydrolysis temperature is preferably room temperature; and the acid hydrolysis is preferably carried out under stirring conditions. After the acid hydrolysis is completed, the resulting liquid is preferably centrifuged (referred to as the second centrifugation) to obtain the acid hydrolysis supernatant; the temperature of the second centrifugation is preferably 3-5℃, more preferably 4℃; the rotation speed is preferably 3000-5000 rpm, more preferably 4000 rpm; and the centrifugation time is preferably 20-40 min, more preferably 30 min.

[0061] After obtaining the acid hydrolysis supernatant, the pH value of the acid hydrolysis supernatant is adjusted to 6.5-7.5 and then subjected to alcohol precipitation to obtain an alcohol-precipitated solid. In this invention, the reagent used to adjust the pH value of the acid hydrolysis supernatant is preferably an aqueous sodium hydroxide solution, with a preferred mass concentration of 30-50%, more preferably 40%; the alcohol used for the alcohol precipitation is preferably ethanol, with a preferred volume fraction of 90-95%, more preferably 95%; the alcohol precipitation temperature is preferably -5 to -15°C, more preferably -10°C; the number of alcohol precipitations is preferably 2-3 times, and the time for each precipitation is preferably 20-30 hours, more preferably 24 hours; specifically, ethanol is first added to the acid hydrolysis supernatant for the first alcohol precipitation, followed by centrifugation (referred to as the third centrifugation) to collect the precipitate, and the precipitate is then... After the substance is dissolved in distilled water, ethanol is added for a second alcohol precipitation. The precipitate is collected by centrifugation (referred to as the fourth centrifugation), which is the alcohol-precipitated solid. The volume ratio of ethanol used in the first alcohol precipitation to the volume of the acid hydrolysis supernatant is preferably 1:0.5 to 1.5, more preferably 1:1. The volume ratio of ethanol used in the second alcohol precipitation to the volume of distilled water used to dissolve the precipitate is preferably 1 to 3:1, more preferably 2:1. The temperature of the third and fourth centrifugations is preferably 3 to 5°C, more preferably 4°C, the rotation speed is preferably 3000 to 5000 rpm, more preferably 4000 rpm, and the centrifugation time is preferably 20 to 40 min, more preferably 30 min.

[0062] After obtaining the alcohol-precipitated solid, the present invention sequentially washes, centrifuges (referred to as the fifth centrifugation), and freeze-dries the obtained alcohol-precipitated solid to obtain crude polysaccharide; the washing agent is preferably ethanol, and the volume fraction of ethanol is preferably 95%; the temperature of the fifth centrifugation is preferably 3-5℃, more preferably 4℃, the rotation speed is preferably 3000-5000 rpm, more preferably 4000 rpm, and the centrifugation time is preferably 20-40 min, more preferably 30 min; the freeze-drying specifically involves dissolving the washed solid in distilled water and then freeze-drying it.

[0063] After obtaining the crude polysaccharide, the crude polysaccharide was purified by column chromatography, and the fraction with a weight-average molecular weight of 90,000 to 130,000 was collected. The obtained fraction was then subjected to alcohol precipitation to obtain the sea sodium polysaccharide. In this invention, the column chromatography used for purification is preferably a DEAE cellulose column, and the DEAE cellulose column is preferably 10cm × 4cm. Preferably, the DEAE cellulose column is pre-equilibrated with an acetate-sodium acetate buffer before the crude polysaccharide is loaded onto the pre-equilibrated DEAE cellulose column. The elution process of the column chromatography purification includes a first stage and a second stage, where the eluent used in both stages is an acetate-sodium acetate buffer containing sodium chloride. The concentration of sodium chloride in the eluent used in the first stage is 0.4–0.6 mol / L, preferably 0.5 mol / L, and the concentration of sodium chloride in the eluent used in the second stage is 0.8–1.2 mol / L, preferably 1 mol / L. In a specific embodiment of this invention, during the elution process, preferably 500 mL of each fraction is collected, and each fraction is detected using HPLC methods based on molecular weight and molecular weight distribution, collecting chromatographic peaks with a weight-average molecular weight of 90,000–130,000.

[0064] In this invention, the fraction is preferably precipitated with alcohol once, the alcohol used for precipitation is preferably ethanol, the volume fraction of ethanol is preferably 95%, and the volume ratio of ethanol to fraction is preferably 0.5 to 1.5:1, more preferably 1:1; the temperature for precipitation is preferably -5 to -15°C, more preferably -10°C, and the time is preferably 20 to 30 hours, more preferably 24 hours; after precipitation, centrifugation (referred to as the sixth centrifugation) is performed to collect the precipitate; the temperature for the sixth centrifugation is preferably 3 to 5°C, more preferably 4°C, the rotation speed is preferably 3000 to 5000 rpm, more preferably 4000 rpm, and the centrifugation time is preferably 20 to 40 minutes, more preferably 30 minutes.

[0065] In this invention, after the sixth centrifugation, the obtained precipitate is preferably concentrated and freeze-dried sequentially; the concentration is preferably ultrafiltration membrane concentration, and the molecular weight cutoff of the ultrafiltration membrane is preferably 10,000; the concentration is preferably repeated 4 times, specifically, the precipitate obtained from the sixth centrifugation is dissolved in distilled water, concentrated to 1 / 2 of the original volume using an ultrafiltration membrane, then distilled water is added back to the original volume, and then concentrated to 1 / 2 again using an ultrafiltration membrane, and so on, repeating the concentration 4 times to obtain a concentrated solution; after obtaining the concentrated solution, the present invention freeze-dries the concentrated solution to obtain pure sodium polysaccharide; the present invention does not have special requirements for the freeze-drying conditions, and those well known to those skilled in the art can be used.

[0066] In this invention, the cation in the sea sodium polysaccharide extracted by the above method is sodium ion; in a specific embodiment of this invention, the sea sodium polysaccharide can be further subjected to cation exchange to obtain sea sodium polysaccharide with other ions as cations. This invention does not have special requirements for the cation exchange method, and any method well known to those skilled in the art can be used.

[0067] This invention also provides the application of the sodium hyaluronate described in the above-described scheme or the sodium hyaluronate prepared by the above-described preparation method in the preparation of anticoagulant and / or antithrombotic drugs or coagulation factor X enzyme-targeting inhibitors. This invention does not impose special requirements on the methods used for the application; methods well-known to those skilled in the art can be employed.

[0068] This invention also provides a coagulation factor X enzyme targeting inhibitor, comprising the sodium hyaluronate described in the above-described scheme or the sodium hyaluronate prepared by the preparation method described in the above-described scheme. This invention does not have special requirements for other components in the coagulation factor X enzyme targeting inhibitor; those well known to those skilled in the art can be used. In this invention, the coagulation factor X enzyme targeting inhibitor can target the terminal rate-limiting enzyme (coagulation factor X enzyme) in the intrinsic coagulation pathway and can be used in the treatment of cardiovascular and cerebrovascular diseases, thrombotic diseases, or in drug development.

[0069] This invention also provides an anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway, comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient is the sodium hyaluronate described in the above-described scheme or the sodium hyaluronate prepared by the preparation method described in the above-described scheme. This invention does not have special requirements for the pharmaceutically acceptable excipients; any excipient well-known to those skilled in the art can be used. This invention also does not have special requirements for the dosage form of the drug; any form well-known to those skilled in the art can be used, specifically tablets, powders, capsules, granules, or liquid formulations, etc.

[0070] This invention also provides a method for treating vascular diseases, using the sodium hyaluronate described in the above-described scheme, the coagulation factor X enzyme-targeting inhibitor described in the above-described scheme, or anticoagulant and / or antithrombotic drugs for treatment; the vascular diseases include thrombotic diseases or cardiovascular and cerebrovascular diseases; the preferred dosage of the sodium hyaluronate is 10-30 mg / day / person; based on the sodium hyaluronate, the preferred dosage of the coagulation factor X enzyme-targeting inhibitor or anticoagulant and / or antithrombotic drugs is 10-30 mg / day / person; the thrombotic diseases include ischemic stroke, specifically applicable to the acute phase and recovery phase of ischemic stroke.

[0071] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0072] Example 1: Extraction of sodium polysaccharide

[0073] After careful separation of the body wall from other tissues, the sea cucumber *Holothurialeucospilota* (Brandt) was immediately immersed in acetone and stored at 4°C for 24 hours. The dried tissue (500g) was ground into powder and suspended in 10L of 0.1mol / L sodium acetate buffer (pH 6) containing 50g papain. The mixture was incubated at 65°C for 24 hours, followed by low-temperature centrifugation (4000rpm, 4°C, 30min) to obtain the enzymatic supernatant. The pH was adjusted to 2.5±0.5 by adding 6mol / L hydrochloric acid solution to the supernatant, and the reaction was carried out with stirring for 2 hours. The mixture was then centrifuged at low temperature (4000rpm, 4°C, 30min) to obtain the acid hydrolysis supernatant. The pH of the acid hydrolysis supernatant was adjusted to 7.0 ± 0.5 by adding 40 wt% sodium hydroxide aqueous solution. While stirring, an equal volume of 95 vol% ethanol was added to the reaction system. After maintaining the temperature at -10℃ for 24 h, the mixture was centrifuged at low temperature (4000 rpm, 4℃ for 30 min) to collect the precipitate. The precipitate was dissolved in 500 mL of distilled water, and 1 L of 95 vol% ethanol was added. After maintaining the temperature at -10℃ for 24 h, the mixture was centrifuged at low temperature (4000 rpm, 4℃ for 30 min) to collect the precipitate. The precipitate was washed with 500 mL of 95 vol% ethanol, centrifuged at low temperature (4000 rpm, 4℃ for 30 min), and the solid was collected. The solid was dissolved in 500 mL of distilled water and freeze-dried to obtain 4 g of crude sodium polysaccharide.

[0074] Four g of crude sodium polysaccharide was placed on a DEAE cellulose column (10 cm × 4 cm) pre-equilibrated with 0.1 mol / L HAc-NaAc buffer (pH 6). The column was washed with 5 L of HAc-NaAc buffer (pH 6) containing 0.5 mol / L NaCl and 2 L of HAc-NaAc buffer (pH 6) containing 1 mol / L NaCl, respectively, at a flow rate of 50 mL / min. 500 mL of each fraction was collected. Molecular weight and molecular weight distribution were analyzed by HPLC. Fractions with a weight-average molecular weight of 90,000–130,000 were collected. One volume of 95 vol% ethanol was added to the eluent, and the mixture was incubated at -10°C for 24 h. The resulting precipitate was collected by low-temperature centrifugation (4000 rpm, 4°C for 30 min). The precipitate was dissolved in distilled water and concentrated to half its original volume using an ultrafiltration membrane with a molecular weight cutoff of 10,000. Water was added back to the original volume, and the mixture was ultrafiltered again to half its original volume. Water was added back to the original volume, and the mixture was concentrated to half its original volume again. This process was repeated, and the concentrated solution was collected and freeze-dried to obtain 2.5 g of pure sodium lysate (sodium cation). Following the same procedure, five parallel batches of sodium lysate were prepared consecutively: batches 202401, 202402, 202403, 202404, and 202405.

[0075] Example 2: Molecular weight and molecular weight distribution test

[0076] The molecular weight and molecular weight distribution of the sodium polysaccharide obtained in Example 1 were determined by HPLC.

[0077] Chromatographic conditions: Mobile phase A: 0.1 mol / L sodium sulfate aqueous solution; Flow rate: 0.5 mL / min; Elution conditions: 100% A; Column: TSK Gel G4000PWxl (30 cm × 7.5 mm, 10 μm); Column temperature: 35 ℃; Differential detector temperature: 35 ℃.

[0078] Reference solutions: Take dextran reference standards with molecular weights of 9750 Da, 13050 Da, 36800 Da, 64650 Da, 135350 Da and 300600 Da respectively, dissolve and dilute them in the mobile phase to prepare solutions with a concentration of 10 mg / mL.

[0079] Test solution: Weigh appropriate amounts of sodium polysaccharide from different batches 202401, 202402, 202403, 202404 and 202405, dissolve and dilute them with the mobile phase to prepare a 10 mg / mL test solution.

[0080] Determination method: The molecular weight distribution of different batches of sodium polysaccharide was determined by size exclusion chromatography (General Chapter 0514, Chinese Pharmacopoeia, Part IV). The results are shown in Table 1.

[0081] Table 1 Results of Molecular Weight Distribution Determination

[0082]

[0083]

[0084] As can be seen from the data in Table 1, the weight-average molecular weight of the sodium polysaccharide of the present invention is 90,000 to 130,000, and the polydispersity index (DPI) is ≤1.32.

[0085] Example 3: Monosaccharide Composition Analysis of Sodium Hydrocarbon Polysaccharide

[0086] 1. Determination of glucuronic acid content:

[0087] The content of glucuronic acid in sea sodium polysaccharide was determined by the m-hydroxybiphenyl colorimetric method.

[0088] Preparation of the reference solution: Accurately weigh 30 mg of glucuronic acid dried to constant weight at 80 °C, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with water, and shake well. Accurately measure 5 mL of the solution, place it in a 50 mL volumetric flask, dilute to the mark with water, and shake well to obtain the reference solution (each 1 mL contains 60 mg of glucuronic acid).

[0089] Preparation of the standard curve: Accurately measure 0.00, 0.10, 0.20, 0.30, 0.40, and 0.50 mL of the reference solution and place them in stoppered test tubes. Accurately add water to each tube to a final volume of 0.50 mL. In an ice bath, accurately add 3 mL of sodium tetraborate solution (dissolve 2.4 g of sodium tetraborate in 500 mL of sulfuric acid) to each tube, shake well, and heat in a boiling water bath for 5 minutes. After cooling in an ice bath, accurately add 50 μL of m-hydroxybiphenyl solution (dissolve 75 mg of m-hydroxybiphenyl in 50 mL of 0.5% sodium hydroxide solution) to each tube and shake well. Use the tube with the reference solution as the "0.00" mark as a blank control. Measure the absorbance at 520 nm using spectrophotometry (refer to the Pharmacopoeia of the People's Republic of China). Plot the standard curve with absorbance on the ordinate and concentration on the abscissa.

[0090] Determination method: Accurately weigh approximately 10 mg of the sample to be tested and place it in a 50 mL volumetric flask. Dissolve and dilute to the mark with water. Accurately measure 0.50 mL of the solution and, following the method under the standard curve section, starting from "accurately add 3 mL of sodium tetraborate solution to each tube in an ice bath," determine the absorbance according to the method. Read the content of glucuronic acid in the test solution from the standard curve and calculate the content of glucuronic acid in the sample to be tested.

[0091] 2. Determination of fucose content:

[0092] Preparation of reference solution and test solution: Slowly add 6 parts concentrated sulfuric acid to 1 part water (V / V) to prepare sulfuric acid test solution and cool it for later use; prepare cysteine-phenol test solution containing 1.0 wt% cysteine ​​hydrochloride and 0.075 wt% phenol; prepare 40 μg / mL fucose standard solution and 50 μg / mL sample solution with distilled water.

[0093] Preparation of standard curve: Take 0.00, 0.05, 0.10, 0.15, 0.20, and 0.25 mL of standard solution into stoppered test tubes, and bring the volume to 0.25 mL with distilled water. After cooling in an ice bath, add 1.25 mL of sulfuric acid solution. Shake well and simultaneously immerse each tube in a boiling water bath. Strictly control the heating time. After 3 minutes, remove all tubes and cool them in an ice bath. Add 0.25 mL of cysteine-phenol solution. Shake well and retain in an ice bath for 1 hour. Use the test tube with the standard solution as "0.00" as a blank control. Measure the absorbance at 398 nm and plot the standard curve.

[0094] Assay method: Pipette 0.25 mL of sample solution, and proceed as described under the standard curve section. Use a "0.00" test tube as a blank control, measure the light absorption at 398 nm, and calculate the fucose content according to the standard curve.

[0095] 3. Determination of acetylgalactosamine content:

[0096] Preparation of reference solution and test solution: Prepare 0.25 mol / L sodium tetraborate test solution; prepare 3.5% (V / V) acetylacetone test solution with sodium tetraborate solution; dissolve 0.16 g of dimethylaminobenzaldehyde (PDABA) in 1.5 mL of 12 mol / L isopropanol, and dilute to prepare PDABA test solution with a concentration of 0.1 g / mL for use.

[0097] Preparation of standard curve and sample determination:

[0098] (1) Standard galactose and sample pretreatment: Accurately weigh 1.0 mg of galactose hydrochloride or an appropriate amount of sample (equivalent to 0.2 mg of galactose) into ampoules, add 6 mol / L hydrochloric acid at a ratio of 1 mg: 1 mL, seal the ampoules, heat at 100 °C for 3 hours, cool, and then deacidify and evaporate to dryness at 70 °C. After drying, add a small amount of distilled water and repeat the deacidification process twice. Dissolve the deacidified galactose in distilled water at a ratio of 1 mg: 10 mL to prepare the standard solution for the following determinations; dissolve the deacidified sample in distilled water at a ratio of 1 mg: 4 mL to obtain the sample solution.

[0099] (2) Preparation of standard curve and determination of galactosamine in sample: Accurately pipette 0.00, 0.10, 0.20, 0.30, and 0.40 mL of the above standard solution into stoppered test tubes, and add distilled water to a final volume of 0.40 mL. Add 0.3 mL of acetylacetone reagent and incubate in a 25°C water bath for 2 hours, followed by the addition of 1 mL of PDABA reagent. After mixing, incubate at 50°C for 15 minutes. After standing at room temperature for 30 minutes, measure the absorbance at 530 nm, using the "0.00" tube as a blank control. Plot a standard curve based on concentration versus absorbance. Separately, pipette 0.4 mL of the sample solution and mix it with 0.3 mL of acetylacetone reagent, then proceed according to the standard curve preparation method. Use the "0.00" tube as a blank control and measure the absorbance. Calculate the galactosamine content in the sample from the standard curve (note that the standard should be converted from galactosamine hydrochloride to N-acetylgalactosamine).

[0100] 4. Test Results:

[0101] The results of the monosaccharide composition test for each batch of sea sodium polysaccharide samples are shown in Table 2.

[0102] Table 2. Monosaccharide composition results of sea sodium polysaccharide (mass fraction)

[0103]

[0104]

[0105] Based on 100g of sea sodium polysaccharide, the molar content of monosaccharides was calculated according to Table 2, and the results are shown in Table 3.

[0106] Table 3. Molar content of monosaccharides in sea sodium polysaccharide (calculated per 100g of sea sodium polysaccharide)

[0107] batch number 202401 202402 202403 202404 202405 mean Glucuronic acid (mol) 0.115 0.112 0.111 0.107 0.102 0.109 N-acetylgalactosamine (mol) 0.119 0.119 0.106 0.103 0.109 0.111 Fucose (mol) 0.078 0.077 0.073 0.0745304055 0.063 0.073

[0108] The molar ratio of monosaccharides in sea sodium polysaccharide was calculated based on Table 3, and the results are shown in Table 4.

[0109] Table 4. Molar ratio of monosaccharide composition of sea sodium polysaccharide

[0110] batch number 202401 202402 202403 202404 202405 mean Glucuronic acid (mol) 1 1 1 1 1 1 N-acetylgalactosamine (mol) 1.04 1.06 0.96 0.96 1.07 1.02 Fucose (mol) 0.67 0.69 0.66 0.7 0.62 0.67

[0111] According to the results in Table 4, the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose in the sodium polysaccharide of the present invention is 1:0.9-1.1:0.6-0.7.

[0112] Example 4: Determination of Na ion content in sea sodium polysaccharide

[0113] 1. Instrumentation and Methods

[0114] The sodium ion content in sea sodium polysaccharide was tested by ion chromatography. The instrumentation method is shown in Table 5.

[0115] Table 5 Information on Samples, Instruments, and Chromatographic Systems Used for Sodium Ion Content Determination

[0116]

[0117] 2. Content determination

[0118] Preparation of reference solution: Weigh 2.54159 g of sodium chloride into a 100 mL volumetric flask, dissolve in water and dilute to the mark to prepare a stock solution of 10000 μg / mL; transfer 0.5 mL of the stock solution into a 50 mL volumetric flask, dilute with water and dilute to the mark to prepare a working solution of 100 μg / mL; accurately measure 0.125 mL, 0.25 mL, 0.5 mL, 1.25 mL, 2.5 mL, 3.75 mL, and 5 mL of the working solution into seven 25 mL volumetric flasks, dilute with water and dilute to the mark to prepare sodium ion standard solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL.

[0119] Preparation of test solution: Weigh 10.00 mg of test sample and place it in a 10 mL volumetric flask. Dissolve and dilute with purified water and make up to volume. Mix well to obtain the test sample stock solution. Accurately transfer 1.0 mL of the test sample stock solution into a 10 mL volumetric flask, dilute with water and make up to volume. Filter to obtain the test solution (concentration of 99.756 μg / mL).

[0120] Linearity test results: A series of sodium ion standard solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL were injected and analyzed. The results are shown in Table 6.

[0121] Table 6. Results of sodium ion linearity test

[0122] sample Concentration (μg / mL) Peak area 5％ 0.5001 389.2919 10％ 1.000 564.9002 25％ 2.001 1304.71739 50％ 5.001 2761.19062 100％ 10.00 5513.44555 150％ 15.00 8139.68196 200％ 20.01 10806.76113

[0123] The mass concentration of the analyte (X, μg·mL) -1 Using the x-axis as the x-axis and the peak area (Y) as the y-axis, a linear regression analysis was performed. The results of the linear regression analysis are as follows: Figure 1 As shown, the regression equation for sodium ions is: Y = 534.6389x + 124.0996, with a correlation coefficient r = 0.99988.

[0124] The above results indicate that sodium ions exhibit good linearity in the range of 0.5001–20.01 μg / mL, meeting the requirements of the validation protocol (linear range: at least 5%–200% of the injected concentration). Within this range, the linear regression coefficient R02 The value is 0.99997, which meets the requirements of the validation scheme (regression coefficient (R²)). 2 ≥0.9990).

[0125] 3. Content determination results

[0126] Accurately weigh different batches of test samples and prepare the test sample solution according to the "Preparation of Test Sample Solution" section. Accurately weigh an appropriate amount of Na ion reference standard and prepare two parallel reference solutions, designated as Reference-1 and Reference-2, according to the "Preparation of Reference Solution" section. Use these reference solutions for system suitability testing, with Reference-1 injected 5 times repeatedly and Reference-2 injected 2 times repeatedly. Accurately pipette 15 μL each of the mixed reference solution and test sample solution and inject them into the ion chromatograph. Record the chromatograms and calculate the Na ion content using the external standard method. The results of the system suitability test for the content determination system are shown in Table 7 below, and the test sample determination results are shown in Table 8 below.

[0127] Table 7 Results of the solution test for content determination.

[0128]

[0129]

[0130] Table 8. Results of Sodium Ion Content Determination

[0131]

[0132] The above results indicate that the mass percentage of sodium ions in the sodium polysaccharide of this invention is in the range of 9-11%.

[0133] Example 5: Determination of the sulfate group content of sea sodium polysaccharide

[0134] The determination of sulfate ester group content (calculated based on sulfate ions) can be performed using ion chromatography and colorimetry. Differences exist in the detection of sulfate ester group content under different methods. The specific detection process is as follows:

[0135] Method I: Determination of polysaccharide sulfate group content by ion chromatography

[0136] 1. Instruments, Equipment, and Methods

[0137] The sulfate ester group content of sea sodium polysaccharide was tested by ion chromatography. The instrumentation method is shown in Table 9.

[0138] Table 9. Information on Instruments and Chromatographic Systems for the Determination of Sulfate Group Content

[0139]

[0140] 2. Content determination

[0141] Preparation of reference solutions: Accurately measure 0.02 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of sulfate ion stock solution (100 μg / mL) into nine 10 mL volumetric flasks, dilute with water and bring to the mark to prepare sulfate ion standard solutions with concentrations of 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL.

[0142] Preparation of test solution: Weigh approximately 30.00 mg of the test sample into a 20 mL bottle, add 15 mL of 2 mol / L hydrochloric acid, and heat at 90 °C under sealed conditions for 2 h. Remove the hydrochloric acid under reduced pressure, dissolve and dilute with the mobile phase to a final volume of 50 mL, accurately transfer 1 mL to a 20 mL volumetric flask, dilute and dilute with the mobile phase to a final volume, mix well, and filter to obtain the test solution (concentration 0.03 mg / mL).

[0143] Linear experimental results

[0144] A series of sulfate ion standard solutions with concentrations of 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL were injected and analyzed. The results are shown in Table 10.

[0145] Table 10. Linearity test of sulfate ions

[0146] Test sample Concentration (μg / mL) Peak area 2％ 0.2 102.3833 5％ 0.5 183.08611 10％ 1 385.69977 20％ 2 628.64865 50％ 5 1406.25683 100％ 10 2731.11205 150％ 15 3970.69577 200％ 20 5323.54837 250％ 25 6665.06959

[0147] The mass concentration of the analyte (X, μg·mL) -1 Using (x) as the x-axis and peak area (Y) as the y-axis, a linear regression analysis was performed, and the results are as follows: Figure 2 As shown, the regression equation for sulfate ions is y = 262.47x + 82.247, with a correlation coefficient r = 0.9998.

[0148] The above results indicate that sulfate ions exhibit good linearity in the range of 0.201–25.144 μg / mL, meeting the requirements of the validation protocol (linear range: at least 5%–200% of the injected concentration). Within this range, the linear regression coefficient R0 is [value missing]. 2 The value is 0.9998, which meets the requirements of the validation scheme (regression coefficient (R²)). 2 ≥0.9990).

[0149] 3. Content determination results

[0150] Accurately weigh different batches of test samples and prepare the test solution according to the "Preparation of Test Solution" section. Accurately weigh an appropriate amount of sulfate ion reference standard and prepare two parallel reference solutions, designated as Reference-1 and Reference-2, according to the "Preparation of Reference Solution" section. Use these reference solutions for system suitability testing, with Reference-1 injected 5 times and Reference-2 injected 2 times. Accurately pipette 25 μL each of the mixed reference solution and test solution and inject them into the ion chromatograph. Record the chromatograms and calculate the sulfate ion content using the external standard method. The results of the system suitability test for the content determination system are shown in Table 11, and the test sample determination results are shown in Table 12.

[0151] Table 11 Results of the test for the applicability of the content determination solution.

[0152]

[0153] Table 12 Results of Sulfate Group Content Determination

[0154]

[0155] The above results indicate that the mass percentage of sulfate groups in the sodium polysaccharide of the present invention is in the range of 26% to 35%.

[0156] Method II: Determination of sulfate content using the Terho method

[0157] Preparation of reference solution and test solution:

[0158] Prepare a barium chloride buffer solution by mixing 10 mL of 2 mol / L hydrochloric acid solution, 2 mL of 0.005 mol / L barium chloride solution, and 8 mL of 0.02 mol / L sodium bicarbonate solution, then adding anhydrous ethanol to a final volume of 100 mL. Prepare a sodium rosemate solution by dissolving 5 mg of sodium rosemate in 20 mL of water, then adding 100 mg of ascorbic acid, shaking well, and adding ethanol to a final volume of 100 mL. Accurately weigh 36.29 mg (dried to constant weight at 105–110 °C) of potassium sulfate, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with water to prepare a stock solution. Take 1 mL of the stock solution and place it in a 10 mL volumetric flask, then add water to a final volume to obtain a standard sulfate-based solution (1 mL is equivalent to 20 μg of sulfate). Accurately weigh 1 mg of the sample and place it in a 2 mL ampoule. Dissolve 1.0 mg of the sample in 1.0 mL of 2 mol / L hydrochloric acid. Heat at 100 °C for 1 hour. After cooling, dry under reduced pressure at 65 °C. Add a little water, then remove the residual acid by vacuuming again. Dissolve the hydrolyzed residue in water at a ratio of 1 mg: 4 mL to prepare the test solution for the determination of sulfate groups.

[0159] Preparation of standard curve: Accurately pipette 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of sulfate-based standard solution into each stoppered test tube, add water to 0.5 mL, then add 2 mL of ethanol and 1 mL of barium chloride buffer solution sequentially. Shake well, then add 1.5 mL of sodium rosette solution. Shake well again, let stand for 10 minutes, and measure the absorbance at 510 nm. Use the "0.00" test tube as a blank control. Plot a standard curve based on concentration versus absorbance.

[0160] Determination method: Take 0.1 mL of the sample solution, add water to make up to 0.5 mL, and perform the remaining operations as in the preparation of the standard curve. Measure the light absorption. Calculate the total sulfate content from the standard curve.

[0161] Test results:

[0162] The results of the determination of sulfate ester group content of each batch of sea sodium polysaccharide samples are shown in Table 13.

[0163] Table 13 Results of Determination of Sulfated Polysaccharide Content (mass fraction)

[0164] batch number 202401 202402 202403 202404 202405 sulfate content 33.34％ 34.52％ 36.74 38.23 39.05％

[0165] The above results indicate that, determined by colorimetric method, the mass percentage of sulfate groups in the sodium polysaccharide of this invention is in the range of 32-40%.

[0166] Example 6 Structural Identification

[0167] Weigh 20 mg of sea sodium polysaccharide (batch 202401) and dissolve it in 1 mL of heavy water, using sodium 3-(trimethylsilyl)deuterated propionate as an internal standard (δ). H The polysaccharide content of seaweed (0 ppm) was analyzed using a Bruker 600MHz nuclear magnetic resonance spectrometer. 1 HNMR, 13 CNMR and HSQC spectra were tested, and the results are as follows: Figures 3-5 As shown.

[0168] The structural characteristics were analyzed by combining literature data and typical signal peaks. 1 In the HNMR spectrum, δ H Fucoidose terminal proton signals were observed at different sites (5.70–4.90 ppm) with sulfate ester group modification (or no sulfate ester group modification); combined with HSQC spectra, δ H The signal peaks appearing at 4.80–4.45 ppm contain signals from glucuronic acid terminus protons at different sites with sulfate-modified (or unmodified) groups; combined with HSQC spectra, δ H The signal peaks appearing at 4.90–4.50 ppm contain N-acetylgalactose terminal proton signals with different sulfate-modified (or unmodified) sites. Simultaneously, δ HA signal peak for the N-acetylgalactosamine fragment acetyl proton appeared in the range of 2.40–1.80 ppm; δ H A methyl proton signal peak for the fucose fragment appeared at 1.60–1.10 ppm. 13 In CNMR spectra, δ C A signal peak at the terminal carbon of the glucuronic acid fragment appears in the range of 107.0–105.0 ppm; δ C A signal peak at the terminal carbon of the N-acetylgalactosamine fragment appeared at 103.0–101.0 ppm; δ C 104.0–103.0 ppm and δ C A fucose fragment terminal carbon signal peak appeared at 101.0–98.0 ppm; δ C A signal peak (N-CH2-) at the 2-carbon position of the N-acetylgalactosamine fragment appears at 56.0–51.0 ppm; δ C A signal peak at the acetyl carbon of the N-acetylgalactosyl fragment appeared at 27.0–24.0 ppm; δ C A methyl carbon signal peak of the fucose fragment appeared at 20.0–16.0 ppm.

[0169] Nuclear magnetic resonance spectroscopy reveals that the sea cucumber polysaccharide of this invention belongs to the fucoidan sulfate-derived chondroitin derivative class. The backbone of this class of components is a copolymer of disaccharide units composed of acetaminophen and glucuronic acid; the structural differences lie only in the content and modification sites of fucoidan and sulfate groups, as well as the molecular weight distribution.

[0170] Accordingly, the structural formula of the sea sodium polysaccharide prepared in Example 1 is as follows:

[0171]

[0172] Where: R1 represents H and SO 3- Or fucose, where R2 is H or SO 3- R3 is H or SO 3- R4 is H or fucose; at least one of R1 and R4 is fucose. It represents a cation; the structure of the fucose is as follows:

[0173]

[0174] Where: R5 is H or SO3 - R6 is H or SO3 - R7 is H or SO3 - .

[0175] As shown in Examples 2-5, the sea sodium polysaccharide of the present invention has a weight-average molecular weight of 90,000 to 130,000 (PDI < 1.32), wherein the molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose is 1:0.9 to 1.1:0.6 to 0.7, the mass percentage of sodium ions is 9 to 11%, and the mass percentage of sulfate ester groups is 25 to 40%. This compound is a novel fucoidylated chondroitin sulfate polysaccharide. Furthermore, the polysaccharide with the above-mentioned specific structure can target the terminal rate-limiting enzyme (iFXase, coagulation factor X enzyme) in the intrinsic coagulation pathway with high activity, achieving anticoagulation and non-bleeding effects. It has broad prospects in the development of anticoagulant drugs targeting the rate-limiting enzyme in the intrinsic coagulation pathway.

[0176] Example 7: In vivo anticoagulation and antithrombotic experiment in mice

[0177] 1. Experimental Materials

[0178] Experimental animals: 24 C57BL / 6 mice of uniform weight.

[0179] Test substance: Sodium polysaccharide prepared in Example 1 (batch number: 202401).

[0180] 2. Experimental Principle

[0181] Different doses of sodium hyaluronate (DT-1) were injected into mice, and mouse plasma was collected 1 hour later. Coagulation function indicators such as PT, APTT, and TT were detected to examine the effect of the test substance on the coagulation function of mice.

[0182] 3. Experimental Methods

[0183] 3.1 Animal grouping

[0184] Randomization: After receiving the animals, they were acclimatized for 5-7 days. During this period, their appearance and general condition were observed. Only animals that passed the inspection were allowed to enter the experiment. After the acclimatization period, the animals were weighed and randomly divided into a blank control group and a test substance group (low, medium, and high doses).

[0185] 3.2 Dosing period

[0186] Animals in the successfully grouped test substance (low, medium, and high dose) groups were administered the drug via tail vein injection, while the blank control group received the corresponding dose of physiological saline. The dosage for each group is shown in Table 14.

[0187] Table 14. Drug administration details for each group of animals.

[0188] Group Administration method Dosage Number of C57BL / 6 mice Blank control group Tail vein injection Equal volumes of physiological saline 6 Low-dose group of test substance Tail vein injection 2mg / kg body weight 6 medium dose group of test substance Tail vein injection 4mg / kg body weight 6 High-dose group of test substance Tail vein injection 8mg / kg body weight 6

[0189] 3.3 Observation period

[0190] General vital signs were observed during the experiment.

[0191] 3.4 Main detection indicators

[0192] Plasma PT, APTT, and TT levels.

[0193] 4. Experimental Data and Results

[0194] The results are as follows Figure 6 As shown, Figure 6 The results showed that one hour after tail vein injection, there were no significant changes in plasma PT and TT levels in the test group mice compared to the blank control group mice, while APTT levels in the treated group were significantly higher. ** The levels (P<0.01 vs. Control) were significantly increased in a dose-dependent manner, indicating that sodium hyaluronate exerts its anticoagulant effect by inhibiting the intrinsic coagulation pathway.

[0195] Example 8: Effects of sodium hyaluronate on venous thrombosis in mice

[0196] 1. Experimental Materials

[0197] Experimental animals: 24 Km mice of uniform weight.

[0198] Test substance: Sodium polysaccharide prepared in Example 1 (batch number: 202401).

[0199] 2. Experimental Principle

[0200] A mouse model of tail thrombosis can be formed by injecting carrageenan into the tail vein 24 hours later. The effect of the test substance on thrombosis can be detected by injecting the test substance into the tail vein 30 minutes in advance.

[0201] 3. Experimental Methods

[0202] 3.1 Animal grouping

[0203] Randomization: After receiving the animals, they were acclimatized for 5-7 days. During the acclimatization period, the animals' appearance and general condition were observed. Only animals that passed the inspection were allowed to enter this experiment. After the acclimatization period, the Km mice were randomly weighed and randomly divided into 4 groups: the tail thrombosis model group, and low, medium, and high dose groups of different doses of sodium thrombosaccharide (DT-1).

[0204] 3.2 Establishment of a tail thrombosis model

[0205] A mouse tail thrombosis model was established by tail vein injection of carrageenan. Healthy male Km mice (22±1g) were induced for 24 hours by tail vein injection of carrageenan (1mg / kg).

[0206] 3.3 Drug administration and modeling period:

[0207] DT-1 was administered via tail vein injection to Km mice in the successfully grouped test substance (low, medium, and high dose) groups 30 minutes before carrageenan induction. Tail thrombosis markers were detected 24 hours later. The dosage for each group is shown in Table 15.

[0208] Table 15. Drug administration details for each group of animals.

[0209] Group Administration method Dosage Number of Km mice Model group No medication none 5 Low-dose group of test substance Tail vein injection 0.2mg / kg body weight 5 medium dose group of test substance Tail vein injection 0.4mg / kg body weight 5 High-dose group of test substance Tail vein injection 0.8mg / kg body weight 5

[0210] 3.4 Observation period

[0211] General vital signs were observed during the experiment.

[0212] 3.5 Main detection indicators

[0213] Observe the length of the thrombus formation in the mouse tail and its percentage of the total tail length.

[0214] 4. Experimental Data and Results

[0215] The results are as follows Figure 7 As shown, Figure 7 Image A shows the length of the thrombus formation in the mouse tail, and image B shows the percentage of the total tail length to the length of the thrombus formation in the mouse tail. Figure 7 The results showed that the model group had significant tail thrombosis, and compared with the model group, the percentage of tail thrombus length in the DT-1 (saturated sodium thrombus) group was dose-dependently reduced. ** P<0.01 vs. Model, *** P < 0.001 vs. Model.

[0216] Example 9: Sodium polysaccharide inhibits the activity of coagulation factors IIa and Xa.

[0217] 1. Purpose of the experiment

[0218] The pharmacological action of sodium saccharide is as an anticoagulant. This example studies the activity of sodium saccharide against coagulation factors IIa, Xa, ATIII-IIa, and ATIII-Xa. The activity of the above coagulation factors was tested according to the heparin bioassay method.

[0219] 2. Instruments and Materials

[0220] The instruments and reagents required for the experiment are shown in Table 16, and the reagents and kits required for the experiment are shown in Table 17.

[0221] Table 16. Instruments required for the experiment

[0222]

[0223] Table 17 Reagents and Kits Required for the Experiment

[0224]

[0225] Among them, S-2238 and S-2765 are chromogenic substrates.

[0226] 3. Experimental Methods

[0227] 3.1 Preparation of TRIS buffer (pH = 8.4)

[0228] Take 0.606g of tris(hydroxymethyl)aminomethane, 1.23g of sodium chloride, 0.28g of disodium ethylenediaminetetraacetate, and 0.1g of polyethylene glycol-6000. Add 80mL of water to dissolve the raw materials, adjust the pH value to 8.4 with hydrochloric acid, and dilute with water to 100mL.

[0229] 3.2 Preparation of standard solutions and chromogenic substrate solutions

[0230] (1) Preparation of IIa solution: On the day of the experiment, add 1 mL of TRIS buffer to the IIa standard to prepare a 680 IU / mL IIa standard stock solution. Take 5 μL of the above stock solution, dilute it to 1 mL with TRIS buffer, mix well, and obtain a 3.4 IU / mL IIa solution.

[0231] (2) Preparation of Xa solution: On the day of the test, add TRIS buffer to Xa standard to prepare a 7.66 nkat / mL Xa standard solution.

[0232] (3) Preparation of S-2238 solution for chromogenic substrate: S-2238 was added to water to prepare a stock solution of 0.003 mol / L, and diluted with water to 0.6 mmol / L before use.

[0233] (4) Preparation of S-2765 solution for chromogenic substrate: Add water to S-2765 to prepare a stock solution of 0.003 mol / L, and dilute with water to 1 mmol / L before use.

[0234] (5) Preparation of antithrombin (ATIII) solution: Add Tris buffer (pH 8.4) to the ATIII standard to dissolve and dilute it to prepare a solution containing 0.15 IU / mL of antithrombin per 1 mL.

[0235] (6) Preparation of polysaccharide working solution: Take 25 mg of sodium hyaluronate and add 10 mL of Tris buffer to obtain working solution A with a concentration of 2.5 mg / mL; take 200 μL of working solution A and add 800 μL of Tris buffer to obtain working solution B with a concentration of 500 μg / mL; take 200 μL of working solution B and add 800 μL of Tris buffer to obtain working solution C with a concentration of 100 μg / mL; take 200 μL of working solution C and add 800 μL of Tris buffer to obtain working solution D with a concentration of 20 μg / mL; take 200 μL of working solution D and add 800 μL of Tris buffer to obtain working solution E with a concentration of 4 μg / mL; take 200 μL of working solution E and add 800 μL of Tris buffer to obtain working solution F with a concentration of 0.8 μg / mL.

[0236] (7) Preparation of reaction termination solution: Weigh 1g of citric acid and add 50mL of ddH2O to prepare a 2% citric acid termination solution.

[0237] 4. Polysaccharide activity assay

[0238] 4.1 IIa activity test: Add 20 μL of Tris buffer and 20 μL of polysaccharide working solution of different concentrations to each well (add 20 μL of Tris buffer to the blank well instead of polysaccharide working solution), add 40 μL of IIa solution, and incubate at 37℃ for 15 min; add 40 μL of 0.6 mmol / L S-2238 solution, incubate in a water bath at 37℃ for 15 min, add 40 μL of stop solution, and detect the absorbance at 405 nm.

[0239] 4.2 Xa activity test: Add 20 μL of Tris buffer, 20 μL of polysaccharide working solution of different concentrations to each well (add 20 μL of Tris buffer to the blank well instead of polysaccharide working solution), 40 μL of Xa solution, and incubate at 37℃ for 15 min; add 40 μL of 0.6 mmol / L S-2765 solution, incubate in a water bath at 37℃ for 15 min, add 40 μL of stop solution, and detect the absorbance at 405 nm.

[0240] 4.3 ATIII-IIa activity assay: Add 20 μL of ATIII solution and 20 μL of polysaccharide working solution of different concentrations to each well (add 20 μL of Tris buffer to blank wells instead of polysaccharide working solution), and incubate at 37℃ for 10 min; add 40 μL of IIa solution and continue incubation at 37℃ for 10 min; add 40 μL of 0.6 mmol / L S-2238 solution, incubate at 37℃ for 15 min, add 40 μL of stop solution, and detect the absorbance at 405 nm.

[0241] 4.4 ATIII-Xa activity assay: Add 20 μL of ATIII solution and 20 μL of polysaccharide working solution of different concentrations to each well (add 20 μL of Tris buffer to blank wells instead of polysaccharide working solution), and incubate at 37℃ for 10 min; add 40 μL of Xa solution and continue incubation at 37℃ for 10 min; add 40 μL of 1 mmol / L S-2765 solution, incubate at 37℃ for 15 min, add 40 μL of stop solution, and detect the absorbance at 405 nm.

[0242] 5. Experimental Results:

[0243] The results of the activity assays for IIa, Xa, ATIII-IIa, and ATIII-Xa are shown in Tables 18 to 21.

[0244] Table 18 IIa Activity Detection Results

[0245] Polysaccharide concentration (μg / mL) blank 0.8 4 20 100 500 OD405 0.776 0.78 0.772 0.767 0.773 0.758

[0246] Table 19. Results of Xa activity assay

[0247] Polysaccharide concentration (μg / mL) blank 0.8 4 20 100 500 OD405 1.81 1.84 1.879 1.862 1.796 1.870

[0248] Table 20 ATIII-IIa Activity Detection Results

[0249] Polysaccharide concentration (μg / mL) blank 0.8 4 20 100 500 OD405 0.707 0.695 0.705 0.681 0.649 0.629

[0250] Table 21 ATIII-Xa Activity Detection Results

[0251] Polysaccharide concentration (μg / mL) blank 0.8 4 20 100 500 OD405 0.806 0.793 0.769 0.753 0.744 0.51

[0252] The results in Tables 18-21 show that after sodium thalassemia was applied to IIa and Xa, the OD values ​​of the different concentration groups and the blank control group did not decrease significantly (Tables 18-19), indicating that sodium thalassemia did not show inhibitory activity against IIa and Xa at a dose of 500 μg / mL; it did not show activity against ATIII-IIa and ATIII-Xa at 100 μg / mL, and only showed weak inhibitory activity at 500 μg / mL (Tables 20-21), with inhibition rates of 11% and 36.7%, respectively.

[0253] Example 10: Assay of sodium polysaccharide inhibiting iFXase (coagulation factor X enzyme) activity

[0254] 1. Purpose of the experiment

[0255] The pharmacological effects of sodium hyaluronate are mainly as a non-heparin-like anticoagulant, with the mechanism of action being the selective inhibition of the terminal rate-limiting enzyme in the intrinsic coagulation pathway, "intrinsic factor X (FIXa-FVIIIa-PL-Ca)".2+ This embodiment measures the IC50 of sodium hyaluronate in inhibiting iFXase. 50 value.

[0256] Since FVIII is readily available as a standard, the FVIII activity assay kit (FVIII:C) was used to determine the activity of this product in inhibiting iFXase.

[0257] 2. Measurement Principle

[0258] In the presence of phospholipids (PLP) and calcium ions, VIII is activated by thrombin to form VIIIa. VIIIa, IXa phospholipids, and calcium ions form an enzyme complex (iFXase), thereby activating factor X. The generated factor Xa hydrolyzes the chromogenic substrate SXa-11, releasing p-nitroaniline (pNA). pNA has light absorption at 405 nm, and the amount of pNA is directly proportional to the absorbance at 405 nm. The activity of the enzyme complex is reflected by detecting the content of p-nitroaniline at 405 nm using a microplate reader. The logarithm of the enzyme complex concentration is directly proportional to A405. The amount of sodium polysaccharide that inhibits the enzyme complex activity of 1 U FVIII formation is defined as one potency unit of this product. The detection mechanism of the FVIII activity assay kit used in this invention is as follows: Figure 8 As shown.

[0259] 3. Instruments and Materials

[0260] The instruments and reagents required for the experiment are shown in Table 22, and the reagents and kits required for the experiment are shown in Table 23.

[0261] Table 22 Instruments required for the experiment

[0262]

[0263] Table 23 Reagents and kits required for the experiment

[0264]

[0265] 4. Experimental Methods

[0266] 4.1 Preparation of reagent solution:

[0267] Add 2.5 mL of ultrapure water to R1 (FX), R2 (FIXa, FIIa, phospholipid, Tris-HCl, CaCl), and R3 (SXa-11) respectively to obtain solutions R1, R2, and R3. R4 (Tris-BSA Buffer) is used to dilute the FVIII standard solution.

[0268] 4.2 Preparation of FVIII working solution: Take FVIII (4.9 IU / vial), dissolve it with 1.225 mL of R4 reagent to prepare a 4 IU / mL FVIII solution; take 1000 μL of the 4 IU / mL FVIII solution, add 333 μL of R4 to prepare a 3 IU / mL FVIII working solution.

[0269] 4.3 Preparation of polysaccharide mother liquor and working solution

[0270] Accurately weigh 25 mg of polysaccharide reference standard and dissolve it in 10 mL of ddH2O to prepare a 2.5 mg / mL polysaccharide stock solution 1; take 100 μL of polysaccharide stock solution 1 and add 9900 μL of ddH2O to dilute it to a 25 μg / mL polysaccharide stock solution 2; take 100 μL of polysaccharide stock solution 2 and add ddH2O to prepare 500 ng / mL polysaccharide working solution 1, 300 ng / mL polysaccharide working solution 2, 250 ng / mL polysaccharide working solution 3, 180 ng / mL polysaccharide working solution 4, 150 ng / mL polysaccharide working solution 5, and 125 ng / mL polysaccharide working solution 6.

[0271] 4.4 Preparation of reaction termination solution: Weigh 1g of citric acid and add 50mL of ddH2O to prepare a 2% citric acid termination solution.

[0272] 4.5 Polysaccharide Activity Assay

[0273] (1) Add 20 μL of ddH2O to the blank wells of the 96-well plate test wells, and add 20 μL of polysaccharide working solution of different concentrations to the polysaccharide test wells of the 96-well plate in sequence;

[0274] (2) Add 20 μL of 3 IU / mLFVIII factor working solution to each of the above test wells in sequence, and add 20 μL of L2 solution to each well. After adding, incubate at 37°C for 15 min.

[0275] (3) Add 20 μL of LR1 (FX) solution to each well and incubate at 37°C for 1 min;

[0276] (4) Add 20 μL of L3 (Xa factor chromogenic substrate SXa-11) solution to each well, incubate at 37°C for 15 minutes, add 30 μL of stop solution, and measure the absorbance at 405 nm (A405) using a microplate reader.

[0277] 5. Experimental Results:

[0278] The activity test results of polysaccharides at different unit concentrations are shown in Table 24. Based on the experimental data, the IC50 value of polysaccharides inhibiting iFXase activity was calculated using GraphPadPrism software. 50The value was 207.5 ng / mL, with a confidence level of 185.9–233 ng / mL.

[0279] Table 24 Polysaccharide Inhibition of iFXase Activity and IC50 50 value

[0280]

[0281] The above results further clarify from a mechanistic perspective that the sodium hyaluronate polysaccharide of the present invention can selectively inhibit the activity of iFXase, the rate-limiting enzyme in the intrinsic coagulation pathway, and target the intrinsic coagulation pathway.

[0282] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A sea sodium polysaccharide, characterized in that, The sodium thalassemia polysaccharide is a fucoidan-based chondroitin sulfate polysaccharide; the weight-average molecular weight of the sodium thalassemia polysaccharide is 90,000 to 130,000, and the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose, with a molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose of 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate ester groups in the sodium thalassemia polysaccharide is 25% to 40%. The structural unit of the sea sodium polysaccharide is shown in Formula I: Formula I; In formula I: R1 represents H and SO3 - Or fucose, where R2 is H or SO3 - R3 is H or SO3 - R4 is H or fucose; at least one of R1 and R4 is fucose; X ⊕ Indicates a cation; The structures of the fucosylates in R1 and R4 are shown in Formula I-1: Equation I-1; In Equation I-1: R5 is H or SO3 - R6 is H or SO3 - R7 is H or SO3 - .

2. The sodium polysaccharide according to claim 1, characterized in that, The sea sodium polysaccharide 1 In the HNMR spectrum, there is a fucose fragment end matrix signal peak at a relative shift of 5.70–4.90 ppm, a glucuronic acid fragment end matrix signal peak at 4.80–4.45 ppm, an N-acetylgalactoside fragment end matrix signal peak at 4.90–4.50 ppm, and an N-acetylgalactoside fragment acetyl matrix signal peak at 2.40–1.80 ppm. A methyl proton signal peak for the fucose fragment is present at 1.60–1.10 ppm; The sea sodium polysaccharide 13 In the CNMR spectrum, a terminal carbon signal peak of the glucuronic acid fragment is present at a relative shift of 107.0–105.0 ppm, a terminal carbon signal peak of the N-acetylgalactosyl fragment is present at 103.0–101.0 ppm, a terminal carbon signal peak of the fucose fragment is present at 104.0–103.0 ppm and 101.0–98.0 ppm, a carbon signal peak of the N-acetylgalactosyl fragment at position 2 is present at 56.0–51.0 ppm, an acetyl carbon signal peak of the N-acetylgalactosyl fragment is present at 27.0–24.0 ppm, and a methyl carbon signal peak of the fucose fragment is present at 20.0–16.0 ppm. The sea sodium polysaccharide 1 HNMR spectra and 13 In the CNMR spectrum analysis, heavy water was used as the solvent and sodium 3-(trimethylsilyl)deuterated propionate was used as the internal standard.

3. The sodium polysaccharide according to claim 1, characterized in that, The cation is a hydrogen ion, an alkali metal ion, an alkaline earth metal ion, an ammonium ion, or an iron ion.

4. The sodium polysaccharide according to claim 1, characterized in that, The cation is sodium ion; the mass percentage of sodium ion in the sea sodium polysaccharide is 5-15%.

5. The sodium polysaccharide according to claim 1, characterized in that, The polydispersity index of the sea sodium polysaccharide is ≤2.

6. The method for preparing the sodium polysaccharide according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) The sea cucumber body wall powder was subjected to enzymatic hydrolysis and acid hydrolysis in sequence to obtain acid hydrolysis supernatant; the enzyme used in the enzymatic hydrolysis was protease; (2) After adjusting the pH of the acid hydrolysis supernatant to 6.5-7.5, alcohol precipitation was performed. The resulting alcohol precipitated solid was washed, centrifuged and freeze-dried in sequence to obtain crude polysaccharide. (3) The crude polysaccharide was purified by column chromatography, and the fraction with a weight average molecular weight of 90,000 to 130,000 was collected. The obtained fraction was precipitated with alcohol to obtain the sodium polysaccharide.

7. The use of the sodium hyaluronate according to any one of claims 1 to 5 or the sodium hyaluronate prepared by the preparation method according to claim 6 in the preparation of anticoagulant and / or antithrombotic drugs or coagulation factor X enzyme-targeting inhibitors.

8. A coagulation factor X enzyme-targeting inhibitor, characterized in that, Includes the sodium hyaluronate as described in any one of claims 1 to 5 or the sodium hyaluronate prepared by the preparation method described in claim 6.

9. An anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is the sodium hyaluronate as described in any one of claims 1 to 5 or the sodium hyaluronate prepared by the preparation method described in claim 6.

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