Seaweed polysaccharide, preparation method and application thereof, and anticoagulant and / or antithrombotic drug for targeting endogenous coagulation pathway

Abstract

The invention relates to the technical field of medicines, and provides sea sodium polysaccharide, a preparation method and application thereof, and an anticoagulant and / or antithrombotic drug of a targeted endogenous coagulation pathway. The sea sodium polysaccharide provided by the invention is fucosylated chondroitin sulfate polysaccharide, the weight-average molecular weight is 90000-130000, the molar ratio of glucuronic acid to N-acetylgalactosamine to fucose is 1: (0.8-1.2): (0.5-0.8), and the mass percentage of sulfate group is 25-40%. The sea sodium polysaccharide provided by the invention can act on a terminal rate-limiting enzyme (iFXase) in an endogenous blood coagulation pathway in a targeting manner, is not obvious in effect on blood coagulation factors in other blood coagulation pathways, realizes the effects of anticoagulation and no bleeding, can be widely applied to acute-stage and recovery-stage treatment of cerebral arterial thrombosis, and has a wide application prospect. The problem of safety of anticoagulant and antithrombotic drugs used by clinical cerebral arterial thrombosis patients is solved, and a new field of research and development of targeted endogenous coagulation pathway rate-limiting enzyme anticoagulant drugs is developed.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to sodium thunbergii polysaccharide, a preparation method and application thereof, and an anticoagulant and / or antithrombotic drug targeting an endogenous coagulation pathway. Background Art

[0002] Anticoagulants and / or antithrombotic drugs are a class of drugs that inhibit blood coagulation by interfering with the body's coagulation process, thereby preventing thrombus formation or preventing the further development of an already formed thrombus. Timely use of anticoagulants and / or antithrombotic drugs in the acute and subacute stages of ischemic stroke is crucial to relieving symptoms. The coagulation pathways in the body are divided into intrinsic coagulation pathways, extrinsic coagulation pathways, and shared pathways. The intrinsic coagulation pathway causes pathological thrombi, the extrinsic coagulation pathway is related to inhibiting damage to the blood vessel wall, and the shared pathway affects both. Intrinsic factor coagulation factor X enzyme (Intrinsic tenase, FIXa-FVIIIa-PL-Ca 2+ The iFXase complex (iFXase) is the terminal rate-limiting enzyme of the intrinsic coagulation pathway, which is closely related to pathological thrombosis and is not necessary for hemostasis. Currently, the anticoagulant and / or antithrombotic drugs used in clinical practice directly act on coagulation factors IIa and / or Xa of the common pathway. Due to bleeding side effects, current international and domestic guidelines do not recommend the early use of direct IIa inhibitors or factor Xa inhibitors. Anticoagulant and / or antithrombotic drugs targeting the intrinsic coagulation pathway have always been the focus of research worldwide, but no related drugs are currently on the market.

[0003] The body wall of sea cucumber contains abundant polysaccharides, mainly including two categories: fucosan 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, anti-virus, anti-inflammatory, etc., especially good anticoagulant and anti-thrombotic activities. However, the natural fucosylated chondroitin sulfate currently extracted from the body wall of sea cucumber usually has a large bleeding side effect, that is, it cannot target the endogenous coagulation pathway. In the related art, the extracted natural polysaccharide is degraded to reduce the bleeding side effect by reducing the molecular weight of sea cucumber fucosylated chondroitin sulfate, but at the same time it will lead to a weakening of the anticoagulant effect.

[0004] In summary, there is an urgent need to provide an anticoagulant and / or antithrombotic drug that can target the intrinsic coagulation pathway, has a good anticoagulant effect, and has a small bleeding side effect. Summary of the invention

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

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A sodium thunbergii polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium thunbergii polysaccharide is 90,000 to 130,000, the monosaccharide composition comprises glucuronic acid, N-acetylgalactosamine and fucose, the molar ratio of the glucuronic acid, N-acetylgalactosamine and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sodium thunbergii polysaccharide is 25 to 40%.

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

[0009]

[0010] In Formula I: R 1 H, SO 3- or fucosyl, R 2 H or SO 3- , R 3 H or SO 3- , R 4 is H or fucosyl; the R 1 and R 4 at least one is fucosyl; represents a cation;

[0011] The R 1 and R 4 The structure of the fucosyl group in is shown in Formula I-1:

[0012]

[0013] In Formula I-1: R 5 H or SO 3 - , R 6 H or SO 3 - , R 7 H or SO 3 - .

[0014] Preferably, the sodium thunbergii polysaccharide 1In the HNMR spectrum, there is a terminal proton signal peak of the fucose fragment at a relative displacement of 5.70 to 4.90 ppm, a terminal proton signal peak of the glucuronic acid fragment at 4.80 to 4.45 ppm, a terminal proton signal peak of the N-acetylgalactosamine fragment at 4.90 to 4.50 ppm, an acetyl proton signal peak of the N-acetylgalactosamine fragment at 2.40 to 1.80 ppm; and a methyl proton signal peak of the fucose fragment at 1.60 to 1.10 ppm.

[0015] Said sodium hyaluronate polysaccharide 13 In the CNMR spectrum, there is a terminal carbon signal peak of glucuronic acid fragment at a relative displacement of 107.0-105.0ppm, a terminal carbon signal peak of N-acetylgalactosamine fragment at 103.0-101.0ppm, a terminal carbon signal peak of fucose fragment at 104.0-103.0ppm and 101.0-98.0ppm, a 2-position carbon signal peak of N-acetylgalactosamine fragment at 56.0-51.0ppm, an acetyl carbon signal peak of N-acetylgalactosamine fragment at 27.0-24.0ppm, and a methyl carbon signal peak of fucose fragment at 20.0-16.0ppm;

[0016] Said sodium hyaluronate 1 HNMR spectra and 13 In the CNMR spectrum test, heavy water was used as solvent and sodium 3-(trimethylsilyl)deuterated propionate was used as 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 the sodium ion in the sodium ion polysaccharide is 5 to 15%.

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

[0020] The present invention also provides a method for preparing the sodium thunbergii polysaccharide described in the above scheme, comprising the following steps:

[0021] (1) subjecting sea cucumber body wall powder to enzymolysis and acid hydrolysis in sequence to obtain an acid hydrolysis supernatant; the enzyme used in the enzymolysis is a protease;

[0022] (2) adjusting the pH value of the acid hydrolysis supernatant to 6.5 to 7.5 and then performing alcohol precipitation, and sequentially washing, centrifuging and freeze-drying the obtained alcohol precipitated solid to obtain a crude polysaccharide;

[0023] (3) Purifying the crude polysaccharide by column chromatography, collecting fractions with a weight average molecular weight of 90,000 to 130,000, and subjecting the obtained fractions to alcohol precipitation to obtain the sodium thunbergii polysaccharide.

[0024] The present invention also provides the use of the sodium thunbergii polysaccharide described in the above scheme or the sodium thunbergii polysaccharide prepared by the preparation method described in the above scheme in the preparation of anticoagulant and / or antithrombotic drugs or coagulation factor X enzyme targeted inhibitors.

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

[0027] The present invention provides a sodium sea polysaccharide, which is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium sea polysaccharide is 90,000 to 130,000, the monosaccharide composition includes glucuronic acid, N-acetylgalactose and fucose, the molar ratio of glucuronic acid, N-acetylgalactose and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate ester group in the sodium sea polysaccharide is 25 to 40%. The sodium sea polysaccharide provided by the present invention can target the terminal rate-limiting enzyme (iFXase) in the endogenous coagulation pathway, and has no obvious effect on coagulation factors in other coagulation pathways at a therapeutic dose, achieving the effect of anticoagulation and no bleeding, and can be widely used in the acute and recovery period treatment of ischemic stroke, reducing the risk of anticoagulation and bleeding in the treatment of thrombotic diseases, solving the safety problem of clinical ischemic stroke patients using anticoagulant and antithrombotic drugs, and opening up a new field of research and development of anticoagulant drugs targeting the rate-limiting enzyme of the endogenous coagulation pathway.

[0028] The present invention has conducted an evaluation study on the anticoagulant activity of sodium thiocyanate polysaccharide on mice, and the results show that sodium thiocyanate polysaccharide can significantly prolong the APTT coagulation time of mice and rats, and does not affect the PT and TT coagulation times. The above research results show that the sodium thiocyanate polysaccharide of the present invention targets the intrinsic coagulation pathway and does not affect the extrinsic coagulation pathway.

[0029] The present invention refers to the heparin bioassay method in Pharmacopoeia General Chapter 1208, and tests the anti-factor IIa, anti-factor Xa, ATⅢ-Ⅱa, and ATⅢ-Xa activities of sodium hyaluronate polysaccharide. The results show that at an experimental dose of 500 μg / mL, sodium hyaluronate polysaccharide has no activity on factor IIa, factor Xa, and ATⅢ-Ⅱa, and only has a weak effect on ATⅢ-Xa (inhibition rate of 36.7%); the ELISA method is used to test the activity of the rate-limiting enzyme of the endogenous coagulation pathway "intrinsic factor coagulation factor X enzyme (iFXase)", and the results show that the test drug can significantly inhibit the rate-limiting enzyme iFXase of the endogenous coagulation pathway, IC50 The value is 207.5ng / mL; the above experiment further clarifies from the mechanism that the sodium thiamethoxam polysaccharide of the present invention can selectively inhibit the activity of iFXase, the rate-limiting enzyme in the endogenous coagulation pathway, and target the endogenous coagulation pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the linear regression analysis result of sodium ion in Example 4;

[0031] Figure 2 is the linear regression analysis result of sulfate group in Example 5;

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

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

[0034] Figure 5 This is the HSQC test spectrum of sodium thunbergii polysaccharide in Example 6;

[0035] Figure 6 The effect of sodium hyaluronate polysaccharide on the coagulation function in mice in Example 7, wherein 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 thiocyanate polysaccharide on venous thrombosis in mice in Example 8, wherein A is a picture of the length of thrombosis in the mouse tail, and B is the percentage of the length of thrombosis in the mouse tail to the total length of the mouse tail;

[0037] Figure 8 This is the detection mechanism of the FVIII activity assay kit used in Example 10. DETAILED DESCRIPTION

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

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

[0040] In the present invention, the sodium hyaluronate polysaccharide is a fucosylated chondroitin sulfate polysaccharide, the skeleton of which is a copolymer of disaccharide units composed of acetylgalactosamine and glucuronic acid, and the disaccharide units are connected with fucose branches; the weight average molecular weight of the sodium hyaluronate polysaccharide is 90,000 to 130,000, preferably 90,000 to 120,000; the polydispersity index of the sodium hyaluronate polysaccharide is preferably ≤2, more preferably ≤1.32, specifically 1.27 to 1.32.

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

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

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

[0044]

[0045] In Formula I: R 1 H, SO 3- or fucosyl, R 2 H or SO 3- , R 3 H or SO 3- , R 4 is H or fucosyl; the R 1 and R 4 at least one is fucosyl; represents a cation;

[0046] The R 1 and R 4 The structure of the fucosyl group in is shown in Formula I-1:

[0047]

[0048] In Formula I-1: R 5 H or SO 3 - , R 6 H or SO 3 - , R 7 H or SO 3 - .

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

[0050] In the present invention, the sodium thunbergii polysaccharide 1 In the HNMR spectrum, there is a terminal proton signal peak of the fucose fragment at a relative displacement of 5.70 to 4.90 ppm, a terminal proton signal peak of the glucuronic acid fragment at 4.80 to 4.45 ppm, a terminal proton signal peak of the N-acetylgalactosamine fragment at 4.90 to 4.50 ppm, an acetyl proton signal peak of the N-acetylgalactosamine fragment at 2.40 to 1.80 ppm; and a methyl proton signal peak of the fucose fragment at 1.60 to 1.10 ppm.

[0051] Said sodium hyaluronate polysaccharide 13 In the CNMR spectrum, there is a terminal carbon signal peak of glucuronic acid fragment at a relative displacement of 107.0-105.0 ppm, a terminal carbon signal peak of N-acetylgalactosamine fragment at 103.0-101.0 ppm, terminal carbon signal peaks of fucose fragment at 104.0-103.0 ppm and 101.0-98.0 ppm, a 2-carbon signal peak of N-acetylgalactosamine fragment at 56.0-51.0 ppm, an acetyl carbon signal peak of N-acetylgalactosamine fragment at 27.0-24.0 ppm, and a methyl carbon signal peak of fucose fragment at 20.0-16.0 ppm.

[0052] In the present invention, the sodium thunbergii polysaccharide 1 HNMR spectra and 13 In the CNMR spectrum test, heavy water was used as the solvent, sodium 3-(trimethylsilyl)deuterated propionate was used as the internal standard, and the displacement of the methyl proton signal peak of sodium 3-(trimethylsilyl)deuterated propionate was set to 0.00 ppm.

[0053] In the present invention, the sea sodium polysaccharide is extracted from the body wall of sea cucumbers; the sea cucumbers include but are not limited to one or more of sea cucumbers of the jade foot, rough sea cucumber, sea cucumber, black sea cucumber and black milk sea cucumber, preferably sea cucumber of the jade foot.

[0054] The present invention also provides a method for preparing the sodium thunbergii polysaccharide described in the above scheme, comprising the following steps:

[0055] (1) subjecting sea cucumber body wall powder to enzymolysis and acid hydrolysis in sequence to obtain an acid hydrolysis supernatant; the enzyme used in the enzymolysis is a protease;

[0056] (2) adjusting the pH value of the acid hydrolysis supernatant to 6.5 to 7.5 and then performing alcohol precipitation, and sequentially washing, centrifuging and freeze-drying the obtained alcohol precipitated solid to obtain a crude polysaccharide;

[0057] (3) Purifying the crude polysaccharide by column chromatography, collecting fractions with a weight average molecular weight of 90,000 to 130,000, and subjecting the obtained fractions to alcohol precipitation to obtain the sodium thunbergii polysaccharide.

[0058] The present invention sequentially enzymatically hydrolyzes and acidolyzes the sea cucumber body wall powder to obtain an acidolysis supernatant. In the present invention, the method for preparing the sea cucumber body wall powder preferably comprises: soaking the sea cucumber body wall in acetone and then drying and grinding to obtain the sea cucumber body wall powder; the soaking temperature is preferably 3 to 5°C, more preferably 4°C, and the soaking time is preferably 12 to 48 hours, more preferably 24 hours; the present invention has no special requirements for the drying and grinding, and the conditions familiar to those skilled in the art can be used. The types of sea cucumbers are not described here.

[0059] In the present invention, the protease is preferably papain; the present invention preferably suspends the sea cucumber body wall powder in a sodium acetate buffer containing papain for enzymolysis; the weight ratio of the sea cucumber body wall powder and 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 temperature of the enzymolysis is preferably 50-70°C, more preferably 65°C, and the time is preferably 12-48h, more preferably 24h; after the enzymolysis is completed, the obtained feed liquid is centrifuged (referred to as the first centrifugation) to obtain a supernatant, and the supernatant is subsequently acid-hydrolyzed; the temperature of the first centrifugation is preferably 3-5°C, more preferably 4°C, the rotation speed is preferably 3000-5000rpm, more preferably 4000rpm, and the centrifugation time is preferably 20-40min, more preferably 30min.

[0060] In the present invention, the acid used for the 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 time of the acid hydrolysis is preferably 1-3 h, more preferably 2 h, the temperature of the acid hydrolysis is preferably room temperature, and the acid hydrolysis is preferably carried out under stirring conditions; after the acid hydrolysis is completed, the obtained feed liquid is preferably centrifuged (referred to as the second centrifugation) to obtain an acid hydrolysis supernatant; the temperature of the second centrifugation is preferably 3-5°C, more preferably 4°C, the rotation speed is preferably 3000-5000rpm, more preferably 4000rpm, and the centrifugation time is preferably 20-40min, more preferably 30min.

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

[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 a crude polysaccharide; the detergent used for the washing is preferably ethanol, and the volume fraction of the ethanol is preferably 95%; the temperature of the fifth centrifugation is preferably 3-5°C, more preferably 4°C, the rotation speed is preferably 3000-5000rpm, more preferably 4000rpm, and the centrifugation time is preferably 20-40min, more preferably 30min; the freeze-drying is specifically to dissolve the washed solid in distilled water and then freeze-dry it.

[0063] After obtaining the crude polysaccharide, the crude polysaccharide is purified by column chromatography, and fractions with a weight average molecular weight of 90,000 to 130,000 are collected. The obtained fractions are precipitated with alcohol to obtain the sodium thunbergii polysaccharide. In the present invention, the chromatography column used for the column chromatography purification is preferably a DEAE cellulose column, and the specification of the DEAE cellulose column is preferably 10cm×4cm; the present invention preferably first uses acetic acid-sodium acetate buffer to pre-balance the DEAE cellulose column, and then loads the crude polysaccharide onto the pre-balanced DEAE cellulose column; the elution process of the column chromatography purification includes a first stage and a second stage, and the eluent used in the first stage and the second stage is an acetic acid-sodium acetate buffer of 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 the present invention, during the elution process, preferably 500 mL of each fraction is collected, and each fraction is detected by molecular weight and molecular weight arrangement HPLC method, and the chromatographic peak fraction with a weight average molecular weight of 90,000 to 130,000 is collected.

[0064] In the present invention, the number of times the fraction is subjected to alcohol precipitation is preferably 1 time, the alcohol used for the alcohol precipitation is preferably ethanol, the volume fraction of the ethanol is preferably 95%, the volume ratio of the ethanol to the fraction is preferably 0.5-1.5:1, and more preferably 1:1; the temperature of the alcohol precipitation is preferably -5--15°C, and more preferably -10°C, and the time is preferably 20-30h, and more preferably 24h; after the alcohol precipitation is completed, centrifugation is performed (recorded as the sixth centrifugation) to collect the precipitate; the temperature of the sixth centrifugation is preferably 3-5°C, and more preferably 4°C, the rotation speed is preferably 3000-5000rpm, and more preferably 4000rpm, and the centrifugation time is preferably 20-40min, and more preferably 30min.

[0065] In the present invention, after the sixth centrifugation, the obtained precipitate is preferably concentrated and freeze-dried in sequence; the concentration is preferably ultrafiltration membrane concentration, and the retention molecular weight of the ultrafiltration membrane is preferably 10,000; the number of concentrations is preferably 4 times, specifically, the precipitate obtained by the sixth centrifugation is dissolved in distilled water, concentrated to 1 / 2 of the original volume with an ultrafiltration membrane, then distilled water is added to the original volume, and then concentrated to 1 / 2 with an ultrafiltration membrane, and so on, and the concentration is repeated 4 times to obtain a concentrated solution; after obtaining the concentrated solution, the present invention freeze-dries the concentrated solution to obtain a pure sodium thunbergii polysaccharide; the present invention has no special requirements for the freeze-drying conditions, and those familiar to those skilled in the art can be used.

[0066] In the present invention, the cations in the sodium ions of the sea sodium polysaccharide extracted according to the above method are sodium ions; in a specific embodiment of the present invention, the sodium ions of the sea sodium polysaccharide can be further subjected to cation exchange to obtain sodium ions of the sea sodium polysaccharide whose cations are other ions. The present invention has no special requirements for the method of cation exchange, and the method well known to those skilled in the art can be used.

[0067] The present invention also provides the use of the sodium thunbergii polysaccharide described in the above scheme or the sodium thunbergii polysaccharide prepared by the preparation method described in the above scheme in the preparation of anticoagulant and / or antithrombotic drugs or coagulation factor X enzyme targeted inhibitors. The present invention has no special requirements for the method of the application, and the method well known to those skilled in the art can be used.

[0068] The present invention also provides a targeted inhibitor of coagulation factor X enzyme, comprising the sodium thiocyanate polysaccharide described in the above scheme or the sodium thiocyanate polysaccharide prepared by the preparation method described in the above scheme. The present invention has no special requirements for other components in the targeted inhibitor of coagulation factor X enzyme, and those familiar to those skilled in the art can be used. In the present invention, the targeted inhibitor of coagulation factor X enzyme can target the terminal rate-limiting enzyme (coagulation factor X enzyme) in the endogenous coagulation pathway, and can be used in the treatment of cardiovascular and cerebrovascular diseases and thrombotic diseases or in drug development.

[0069] The present invention also provides an anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the sodium thiamethoxam polysaccharide described in the above scheme or the sodium thiamethoxam polysaccharide prepared by the preparation method described in the above scheme; the present invention has no special requirements for the pharmaceutically acceptable excipients, and those familiar to those skilled in the art can be used. The present invention has no special requirements for the dosage form of the drug, and those familiar to those skilled in the art can be used, specifically tablets, powders, capsules, granules or liquid preparations, etc.

[0070] The present invention also provides a method for treating vascular diseases, using the sodium thiamethoxam polysaccharide described in the above scheme, the coagulation factor X enzyme targeted inhibitor or the anticoagulant and / or antithrombotic drug described in the above scheme for treatment; the vascular diseases include thrombotic diseases or cardiovascular and cerebrovascular diseases; the dosage of the sodium thiamethoxam polysaccharide is preferably 10 to 30 mg / day / person; based on the sodium thiamethoxam polysaccharide, the dosage of the coagulation factor X enzyme targeted inhibitor or the anticoagulant and / or antithrombotic drug is preferably 10 to 30 mg / day / person; the thrombotic disease includes ischemic stroke, and can be specifically used for the acute phase and recovery treatment phase of ischemic stroke.

[0071] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] Example 1 Extraction of sodium thunbergii polysaccharide

[0073] After the body wall of the sea cucumber (Holothurialeucospilota (Brandt)) was carefully separated from other tissues, it was immediately immersed in acetone and stored at 4°C for 24 hours. The dried tissue (500g) was ground into powder, suspended in 10L of 0.1mol / L sodium acetate buffer (pH 6) containing 50g papain, incubated at 65°C for 24 hours, and then the incubation liquid was centrifuged at low temperature (4000rpm, 4°C centrifugation for 30min) to obtain the enzymatic supernatant. 6mol / L hydrochloric acid aqueous solution was added to the enzymatic supernatant to adjust the pH to 2.5±0.5, reacted for 2 hours under stirring, and centrifuged at low temperature (4000rpm, 4°C centrifugation for 30min) to obtain the acid hydrolysis supernatant. Add 40wt% sodium hydroxide aqueous solution to the acid hydrolysis supernatant to adjust the pH value to 7.0±0.5. Under stirring, add 95vol% ethanol in a volume of 1 times the supernatant to the reaction system. After maintaining at -10°C for 24h, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min) to collect the formed precipitate. The precipitate is dissolved in 500mL of distilled water, and 1L95vol% ethanol is added. After maintaining at -10°C for 24h, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min) to collect the formed precipitate. Add 500mL95vol% ethanol to the precipitate for washing, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min), collect the solid, dissolve in 500mL of distilled water, and freeze-dry to obtain 4g of crude sea sodium polysaccharide.

[0074] 4g of crude sodium polysaccharide was placed on a DEAE cellulose column (10cm×4cm) pre-equilibrated with 0.1mol / L HAc-NaAc buffer (pH 6), and washed with 5L of HAc-NaAc buffer (pH 6) containing 0.5mol / L NaCl and 2L of HAc-NaAc buffer (pH 6) containing 1mol / L NaCl, respectively, with a column flow rate of 50mL / min, and 500mL of each fraction was collected. Each fraction was detected by molecular weight and molecular weight distribution HPLC method, and the chromatographic peak fraction with a weight average molecular weight of 90,000 to 130,000 was collected, and 1 volume of 95vol% ethanol of the effluent was added, and after maintaining at -10°C for 24h, low-temperature centrifugation (4000rpm, 4°C centrifugation for 30min) was performed to collect the formed precipitate. The precipitate was dissolved in distilled water and concentrated to 1 / 2 of the original volume using an ultrafiltration membrane with a molecular weight cutoff of 10,000, water was added to the original volume, ultrafiltration was performed to 1 / 2 of the volume, water was added to the original volume, and then concentrated to 1 / 2 of the original volume, water was added to the original volume, and then concentrated to 1 / 2 of the original volume, and then water was added to the original volume, and then concentrated to 1 / 2 of the original volume, and the concentrate was collected and freeze-dried to obtain 2.5 g of pure sodium polysaccharide (the cation is sodium). According to the above operation, 5 batches of parallel samples were continuously prepared, namely 202401, 202402, 202403, 202404 and 202405 batches of sodium polysaccharide.

[0075] Example 2 Molecular weight and molecular weight arrangement test

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

[0077] Chromatographic conditions: mobile phase A: 0.1 mol / L sodium sulfate aqueous solution; flow rate: 0.5 mL / min; elution condition: 100% A; chromatographic column: TSK Gel G4000PWxl (30 cm×7.5 mm, 10 μm); column temperature: 35°C; differential detector temperature 35°C.

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

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

[0080] Determination method: The molecular weight distribution of different batches of sodium hyaluronate polysaccharide was determined according to the molecular exclusion chromatography (General Rule 0514 Chinese Pharmacopoeia Part IV). The results are shown in Table 1.

[0081] Table 1 Molecular weight distribution results

[0082]

[0083]

[0084] According to the data in Table 1, it can be seen that the weight average molecular weight of the sodium thunbergii polysaccharide of the present invention is 90,000 to 130,000, and the polydispersity coefficient DPI is ≤1.32.

[0085] Example 3 Analysis of Monosaccharide Composition of Sodium Hyaluronate Polysaccharide

[0086] 1. Determination of glucuronic acid content:

[0087] The content of glucuronic acid in sodium hyaluronate polysaccharide was tested by m-hydroxybiphenyl colorimetric method.

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

[0089] Preparation of standard curve: accurately measure 0.00, 0.10, 0.20, 0.30, 0.40 and 0.50 mL of reference solution and place them in stoppered test tubes respectively. Add water to each tube to make it 0.50 mL accurately. Add 3 mL of sodium tetraborate sulfuric acid test solution (take 2.4 g of sodium tetraborate and dissolve it in 500 mL of sulfuric acid) to each tube in an ice bath, shake well, heat it in a boiling water bath for 5 minutes, take it out and cool it in an ice bath, then accurately add 50 μL of m-hydroxybiphenyl test solution (take 75 mg of m-hydroxybiphenyl and dissolve it in 50 mL of 0.5% sodium hydroxide solution) to each tube and shake well. Use the test tube with reference solution as "0.00" as a blank control, measure the absorbance at a wavelength of 520 nm according to the spectrophotometric method (reference: Pharmacopoeia of the People's Republic of China), and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0090] Determination method: Take about 10 mg of the sample to be tested, weigh it accurately, place it in a 50 mL volumetric flask, add water to dissolve and dilute to the scale. Accurately measure 0.50 mL, and according to the method under the standard curve, starting from "accurately add 3 mL of sodium tetraborate sulfate test solution to each tube in an ice bath", determine the absorbance according to the law, read the content of glucuronic acid in the test solution from the standard curve, and calculate to obtain 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 of concentrated sulfuric acid to 1 part of water (V / V) to prepare a sulfuric acid test solution, which is cooled for use; prepare a cysteine-phenol test solution containing 1.0wt% cysteine ​​hydrochloride and 0.075wt% phenol; use distilled water to prepare a 40μg / mL fucose standard solution and a 50μg / mL sample solution.

[0093] Preparation of standard curve: Take 0.00, 0.05, 0.10, 0.15, 0.20, 0.25mL of standard solution in stoppered test tubes respectively, and make up to 0.25mL with distilled water. After cooling in an ice bath, add 1.25mL of sulfuric acid test solution. Shake well and immerse each tube in a boiling water bath at the same time. Strictly control the heating time. After 3 minutes, take out all together and transfer to an ice bath to cool. Add 0.25mL of cysteine-phenol test solution. After shaking well, keep it in an ice bath for 1 hour. Use the test tube with the standard solution as "0.00" as a blank control, measure the light absorption at 398nm and draw a standard curve.

[0094] Determination method: Pipette 0.25mL of sample solution, and operate the rest according to the method under the standard curve. Use the test tube with "0.00" as a blank control, measure the light absorption at 398nm, 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 0.1 g / mL PDABA test solution for use.

[0097] Standard curve preparation and sample measurement:

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

[0099] (2) Preparation of standard curve and determination of galactosamine in samples: Accurately pipette 0.00, 0.10, 0.20, 0.30, and 0.40 mL of the above standard solution into a stoppered test tube, and add distilled water to 0.40 mL. Add 0.3 mL of acetylacetone test solution, keep warm in a 25°C water bath for 2 hours, and then add 1 mL of PDABA test solution. After mixing, keep warm at 50°C for 15 minutes. After standing at room temperature for 30 minutes, measure the light absorption at 530 nm, and use the "0.00" tube as a blank control. Draw a standard curve based on concentration versus light absorption. Pipette 0.4 mL of the sample solution, mix it with 0.3 mL of acetylacetone test solution, and operate according to the method for preparing the standard curve. Use the "0.00" test tube as a blank control, measure the light absorption, and calculate the galactosamine content in the sample from the standard curve (note that the standard substance is converted from galactosamine hydrochloride to N-acetylgalactosamine).

[0100] 4. Test results:

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

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

[0103]

[0104]

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

[0106] Table 3 Molar content of monosaccharides in sodium hyaluronate polysaccharide (calculated based on 100 g sodium hyaluronate 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 sodium hyaluronate polysaccharide was calculated according to Table 3, and the results are shown in Table 4.

[0109] Table 4 Monosaccharide composition molar ratio of sodium hyaluronate 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, in the sodium thunbergii polysaccharide of the present invention, the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose is 1:0.9-1.1:0.6-0.7.

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

[0113] 1. Instrumental method

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

[0115] Table 5 Information of samples, instruments and chromatography system for determination of sodium ion content

[0116]

[0117] 2. Content determination

[0118] Preparation of reference solution: weigh 2.54159 g of sodium chloride into a 100 mL volumetric flask, dissolve it in water and make up to the scale to prepare a 10000 μg / mL stock solution; use a pipette to transfer 0.5 mL into a 50 mL volumetric flask, dilute it with water and make up to the scale to prepare a 100 μg / mL working solution; 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 7 25 mL volumetric flasks, respectively, dilute it with water and make up to the scale 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 sample solution: Weigh 10.00 mg of the test sample, place it in a 10 mL volumetric flask, dissolve and dilute it with purified water, mix well, and obtain the test sample stock solution; accurately transfer 1.0 mL of the test sample stock solution into a 10 mL volumetric flask, dilute and dilute it with water, and filter to obtain the test sample solution (concentration is 99.756 μg / mL).

[0120] Linearity experiment 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 respectively, and the results are shown in Table 6.

[0121] Table 6 Sodium ion linear test results

[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 substance to be tested (X, μg mL -1 ) as the horizontal axis, and the peak area (Y) as the vertical axis, and linear regression analysis was performed. The linear regression analysis results are shown in Figure 1 As shown, the regression equation of sodium ions is: Y=534.6389x+124.0996, and the correlation coefficient r=0.99988.

[0124] The above results show that the sodium ion has a good linear relationship in the range of 0.5001 to 20.01 (μg / mL), which meets the requirements of the validation scheme (linear range: at least in the range of 5% to 200% of the injection concentration). In this range, the linear regression coefficient R2 is 0.99997, which meets the requirements of the validation scheme (the regression coefficient (R 2 )≥0.9990).

[0125] 3. Content determination results

[0126] Take different batches of test samples, accurately weigh them, and prepare the test sample solutions according to the "Preparation of test sample solutions". Take an appropriate amount of Na ion reference substance, accurately weigh it, and prepare two reference solution in parallel according to the "Preparation of reference solution", record them as control-1 and control-2, and use the reference solution to perform system suitability test, where control-1 is injected 5 times and control-2 is injected 2 times. Accurately pipette 15μL of the mixed reference solution and the test sample solution, inject them into the ion chromatograph, record the chromatogram, and calculate the Na ion content according to the external standard method. The test results of the content determination system suitability solution are shown in Table 7, and the test sample determination results are shown in Table 8.

[0127] Table 7 Test results of the applicability solution for content determination

[0128]

[0129]

[0130] Table 8 Sodium ion content determination test results

[0131]

[0132] The above results show that the mass percentage of sodium ions in the sodium ions of ...

[0133] Example 5 Determination of Sulfate Content of Sodium Hydroxide Polysaccharide

[0134] The sulfate ester content (calculated as sulfate ions) can be determined by ion chromatography and colorimetry. There are differences in the sulfate ester content detected by different methods. The specific detection process is as follows:

[0135] Method I: Ion chromatography to detect polysaccharide sulfate content

[0136] 1. Instruments and methods

[0137] The sulfate content of sodium thiamethoxam polysaccharide was tested by ion chromatography. The instrument method is shown in Table 9.

[0138] Table 9 Sulfate ester content determination instrument and chromatography system information

[0139]

[0140] 2. Content determination

[0141] Preparation of reference solution: Accurately measure 0.02mL, 0.05mL, 0.1mL, 0.2mL, 0.5mL, 1mL, 1.5mL, 2mL, and 2.5mL of sulfate ion stock solution (100μg / mL) into 9 10mL volumetric flasks, dilute with water and make up to the scale to make 0.2μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, and 25μg / mL sulfate ion standard solutions.

[0142] Preparation of test solution: weigh about 30.00 mg of the test sample, accurately weigh it in a 20 mL bottle, add 15 mL of 2 mol / L hydrochloric acid, and heat at 90 ° C for 2 hours. Remove the hydrochloric acid under reduced pressure, dissolve it with mobile phase and dilute it to 50 mL, accurately transfer 1 mL to a 20 mL volumetric flask, dilute it with mobile phase and make up the volume, mix it, and filter it to get the test solution (concentration is 0.03 mg / mL).

[0143] Linearity test 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 respectively. The results are shown in Table 10.

[0145] Table 10 Sulfate ion linearity test

[0146] Test product 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 substance to be tested (X, μg mL -1 ) as the horizontal axis, the peak area (Y) as the vertical axis, and linear regression analysis was performed. The results are as follows Figure 2 The regression equation of sulfate ion is y=262.47x+82.247, and the correlation coefficient is r=0.9998.

[0148] The above results show that the sulfate ion has a good linear relationship in the range of 0.201 to 25.144 (μg / mL), which meets the requirements of the validation scheme (linear range: at least in the range of 5% to 200% of the injection concentration). In this range, the linear regression coefficient R 2 is 0.9998, which meets the requirements of the validation scheme (the regression coefficient (R 2 )≥0.9990).

[0149] 3. Content determination results

[0150] Take different batches of test samples, accurately weigh them, and prepare the test sample solutions according to the "Preparation of test sample solutions". Take an appropriate amount of sulfate ion reference substance, accurately weigh it, and prepare two reference solution in parallel according to the "Preparation of reference solution", record them as control-1 and control-2, and use the reference solution to test the system suitability, where control-1 is injected 5 times and control-2 is injected 2 times. Accurately draw 25μL of the mixed reference solution and the test solution, inject them into the ion chromatograph, record the chromatogram, and calculate the sulfate ion content according to the external standard method. The test results of the content determination system suitability solution are shown in Table 11, and the test sample determination results are shown in Table 12 below.

[0151] Table 11 Test results of content determination system applicability solution

[0152]

[0153] Table 12 Sulfate ester content determination test results

[0154]

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

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

[0157] Preparation of reference solution and test solution:

[0158] Take 10mL of 2mol / L hydrochloric acid solution, 2mL of 0.005mol / L barium chloride solution, and 8mL of 0.02mol / L sodium bicarbonate solution, mix them and add anhydrous ethanol to 100mL to prepare barium chloride buffer solution: dissolve 5mg of sodium rhodanoate in 20mL of water, then add 100mg of ascorbic acid, shake well, add ethanol to 100mL, and prepare sodium rhodanoate solution before use; accurately weigh 36.29mg (dried to constant weight at 105-110℃) of potassium sulfate, place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale to make a stock solution. Take 1mL of the stock solution in a 10mL volumetric flask, add water to the volume to obtain a standard sulfate solution (1mL is equivalent to 20μg sulfate). Accurately weigh 1mg of the sample and place it in a 2mL ampoule. Dissolve 1.0mg of the sample in 1.0mL of 2mol / L hydrochloric acid. Heat at 100℃ for 1 hour. After cooling, drain under reduced pressure at 65℃. Add a little water, and again remove the residual acid by vacuum pumping. The hydrolysis residue is dissolved in water at a ratio of 1 mg:4 mL to make the test solution for sulfate determination.

[0159] Preparation of standard curve: Accurately pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5 mL of sulfuric acid standard solution into each stoppered test tube, add water to 0.5 mL, add 2 mL of ethanol and 1 mL of barium chloride buffer solution in sequence. Shake well and then add 1.5 mL of sodium rose acid solution. Shake well and let stand for 10 minutes before measuring the light absorption at 510 nm. Use the test tube with "0.00" as a blank control. Draw a standard curve based on the concentration versus light absorption value.

[0160] Determination method: Take 0.1mL of the sample solution, add water to 0.5mL, and perform the remaining operations as in the preparation of the standard curve, and measure the light absorption. The total sulfate content can be calculated from the standard curve.

[0161] Test results:

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

[0163] Table 13 Determination results of sulfate content of sodium hyaluronate polysaccharide (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 show that the mass percentage of sulfate groups in the sodium thiamethoxam polysaccharide of the present invention is in the range of 32-40% as determined by colorimetry.

[0166] Example 6 Structure Identification

[0167] Weigh 20 mg of sodium thiamethoxam polysaccharide (batch 202401) and dissolve it in 1 mL of heavy water. Use 3-(trimethylsilyl) sodium deuterated propionate as the internal standard (δ H ,0ppm), and the sodium hyaluronate was analyzed by Bruker 600MHz NMR spectrometer. 1 HNMR, 13 CNMR and HSQC spectra were tested, and the results were as follows Figure 3 to Figure 5 shown.

[0168] The structural characteristics were analyzed by combining literature data and typical signal peaks. 1 In the HNMR spectrum, δ H The terminal proton signals of fucose modified with sulfate groups (or without sulfate groups) at different positions appeared at 5.70-4.90 ppm; combined with the HSQC spectrum, δ H The signal peaks at 4.80-4.45 ppm contain the terminal proton signals of glucuronic acid modified with sulfate groups (or without sulfate groups) at different positions; combined with the HSQC spectrum, δ H The signal peaks at 4.90-4.50 ppm contain the terminal proton signals of N-acetylgalactosamine with sulfate modification (or no sulfate modification) at different positions. HThe signal peak of the acetyl proton of N-acetylgalactosamine fragment appears at 2.40-1.80ppm; H The fucose fragment methyl proton signal peak appears at 1.60-1.10ppm. 13 In the CNMR spectrum, δ C The signal peak of the terminal carbon of the glucuronic acid fragment appears at 107.0-105.0ppm; C The terminal carbon signal peak of N-acetylgalactosamine fragment appears at 103.0-101.0ppm; C 104.0~103.0ppm and δ C The terminal carbon signal peak of the fucose fragment appears at 101.0-98.0ppm; C The carbon 2 position (N-CH 2 -) signal peak; δ C The signal peak of the acetyl carbon of N-acetylgalactosamine fragment appears at 27.0-24.0ppm; C The signal peak of methyl carbon of fucose fragment appears at 20.0-16.0 ppm.

[0169] According to the nuclear magnetic resonance spectrum information, the sea sodium polysaccharide of the present invention belongs to the fucosylated chondroitin sulfate component derived from sea cucumbers. The skeleton of this type of component is a copolymer of disaccharide units composed of acetylgalactosamine and glucuronic acid, and the structural difference is only the difference in the content and modification site of fucosyl and sulfate ester groups, as well as the difference in molecular weight distribution.

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

[0171]

[0172] Where: R 1 H, SO 3- or fucosyl, R 2 H or SO 3- , R 3 H or SO 3- , R 4 is H or fucosyl; the R 1 and R 4 at least one is fucosyl; represents a cation; the structure of the fucosyl group is as follows:

[0173]

[0174] Where: R 5 H or SO 3 - , R6 H or SO 3 - , R 7 H or SO 3 - .

[0175] In combination with Examples 2-5, it can be seen that the weight average molecular weight of the sea sodium polysaccharide of the present invention is 90,000 to 130,000 (PDI<1.32), wherein the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose is 1:0.9-1.1:0.6-0.7, the mass percentage of sodium ions is 9-11%, and the mass percentage of sulfate groups is 25-40%. The compound is a new type of fucosylated chondroitin sulfate polysaccharide, and the polysaccharide with the above-mentioned specific structure can target the terminal rate-limiting enzyme (iFXase, coagulation factor X enzyme) in the endogenous coagulation pathway, and has high activity, can achieve anti-coagulation and no bleeding effects, and has broad prospects in the research and development of anticoagulant drugs targeting the rate-limiting enzyme of the endogenous coagulation pathway.

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

[0177] 1. Experimental Materials

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

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

[0180] 2. Experimental Principle

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

[0182] 3. Experimental methods

[0183] 3.1 Animal grouping

[0184] Random grouping: After receiving the animals, they were fed adaptively for 5-7 days. During the acclimation period, the appearance and general condition of the animals were observed. Only animals that passed the inspection could enter this experiment. After the adaptation 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] The animals in the successfully grouped test substance (low, medium and high dose) groups were injected with the drug via the tail vein, and the blank control group was given the corresponding dose of normal saline. The dosage of each group is shown in Table 14.

[0187] Table 14 Drug administration of animals in each group

[0188] Group Dosage Dosage Number of C57BL / 6 mice Blank control group Tail vein injection Equal volume of saline 6 Low dose group Tail vein injection 2mg / kg body weight 6 Medium dose group Tail vein injection 4mg / kg body weight 6 High dose group 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 testing 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 1 hour after the tail vein injection, there was no significant change in the plasma PT and TT levels of the mice in the test group compared with the blank control group. ** The levels of HUVECs (P<0.01 vs. Control) increased significantly in a dose-dependent manner, indicating that sodium hyaluronate polysaccharide exerts its anticoagulant effect by inhibiting the endogenous coagulation pathway.

[0195] Example 8 Effect of Sodium Hydroxide Polysaccharide on Venous Thrombosis in Mice

[0196] 1. Experimental Materials

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

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

[0199] 2. Experimental Principle

[0200] A mouse model was developed in which tail thrombosis can be formed 24 hours after tail vein injection of carrageenan. The test substance was given to the mice 30 minutes in advance of tail vein injection to detect the effect of the test substance on thrombosis formation.

[0201] 3. Experimental methods

[0202] 3.1 Animal grouping

[0203] Random grouping: After the animals were received, they were fed adaptively for 5-7 days. During the acclimation period, the appearance and general condition of the animals were observed. Only animals that passed the inspection could enter this experiment. After the adaptation period, the Km mice were randomly weighed and randomly divided into 4 groups, namely the tail thrombosis model group, and different doses of DT-1 low, medium, and high dose groups.

[0204] 3.2 Establishment of tail thrombosis model

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

[0206] 3.3 Medication and modeling period:

[0207] DT-1 was injected into the tail vein of the Km mice in the test substance (low, medium and high dose) groups successfully divided 30 minutes before carrageenan induction, and the tail thrombosis index was detected 24 hours later. The dosage of each group is shown in Table 15.

[0208] Table 15 Drug administration of animals in each group

[0209] Group Dosage Dosage Number of Km mice Model Group No medication none 5 Low dose group Tail vein injection 0.2mg / kg body weight 5 Medium dose group Tail vein injection 0.4mg / kg body weight 5 High dose group 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 testing indicators

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

[0214] 4. Experimental data and results

[0215] The results are as follows Figure 7 As shown, Figure 7 A is a picture of the length of mouse tail thrombosis, and B is the percentage of the length of mouse tail thrombosis to the total length of the mouse tail; Figure 7 The results showed that the model group had obvious tail thrombus. Compared with the model group, the percentage of tail thrombus length in the DT-1 group was reduced in a dose-dependent manner ( ** P<0.01vs.Model, *** P<0.001vs.Model).

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

[0217] 1. Purpose of the test

[0218] The pharmacological effect of sodium thiocyanate is an anticoagulant drug. This example studies the activity of sodium thiocyanate on IIa, Xa, ATIII-IIa and ATIII-Xa. The activity of the above coagulation factors is tested with reference to the heparin bioassay.

[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 0.606 g of tris(hydroxymethyl)aminomethane, 1.23 g of sodium chloride, 0.28 g of disodium ethylenediaminetetraacetate, and 0.1 g of polyethylene glycol-60000, add 80 mL of water to dissolve the raw materials, adjust the pH value to 8.4 with hydrochloric acid, and dilute with water to 100 mL.

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

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

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

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

[0233] (4) Preparation of chromogenic substrate S-2765 solution: S-2765 was added with water to prepare a 0.003 mol / L stock solution, which was diluted 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 make 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 polysaccharide and add 10 mL of Tris buffer to obtain a 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 a 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 a 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 a 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 a 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 a working solution F with a concentration of 0.8 μg / mL.

[0236] (7) Preparation of reaction termination solution: weigh 1 g of citric acid and add 50 mL of ddHO. 2 O, prepared into 2% citric acid stop solution.

[0237] 4. Determination of polysaccharide activity

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

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

[0240] 4.3ATIII-IIa activity detection: 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 the blank well instead of polysaccharide working solution), incubate at 37°C for 10 min; add 40 μL of IIa solution and continue incubation at 37°C for 10 min; add 40 μL of 0.6 mmol / L S-2238 solution, incubate at 37°C for 15 min, add 40 μL of stop solution, and detect the absorbance at 405 nm.

[0241] 4.4ATIII-Xa activity detection: 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 the blank well instead of polysaccharide working solution), incubate at 37°C for 10 min; add 40 μL of Xa solution and continue incubation at 37°C for 10 min; add 40 μL of 1 mmol / L S-2765 solution, incubate at 37°C for 15 min, add 40 μL of stop solution, and detect the absorbance at 405 nm.

[0242] 5. Experimental results:

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

[0244] Table 18IIa 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 19Xa activity detection results

[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 20ATIII-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 21ATIII-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 to 21 show that after sodium hyaluronate polysaccharide acted on IIa and Xa, the OD values ​​of the different concentration drug-treated groups and the blank control group did not show a significant decrease (Tables 18 to 19), indicating that sodium hyaluronate polysaccharide did not show inhibitory activity on IIa and Xa at a dosage of 500 μg / mL; it did not show activity on ATIII-IIa and ATIII-Xa at a dosage of 100 μg / mL, and only showed weak inhibitory activity at 500 μg / mL (Tables 20 to 21), with inhibition rates of 11% and 36.7%, respectively.

[0253] Example 10 Test on the inhibition of iFXase (coagulation factor X enzyme) activity by sodium hyaluronate

[0254] 1. Purpose of the test

[0255] The pharmacological effect of sodium hyaluronate is mainly a non-heparin-like anticoagulant drug, and its mechanism of action is to selectively inhibit the terminal rate-limiting enzyme of the endogenous coagulation pathway, "endogenous coagulation factor X enzyme (Intrinsictenase, FIXa-FVIIIa-PL-Ca2+ complex, iFXase)". This example determines the IC of sodium thiamethoxam polysaccharide inhibiting iFXase 50 value.

[0256] Since FVIII standard products are easily available, the FVIII activity assay kit (FVIII: C) was used to determine the activity of this product in inhibiting iFXase.

[0257] 2. Determination 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 to release p-nitroaniline (pNA). pNA absorbs light at 405nm, and the amount of pNA is proportional to the absorbance at 405nm. The p-nitroaniline content is detected by an enzyme reader at 405nm to reflect the activity of the enzyme complex. The logarithm of the enzyme complex concentration is proportional to A405. The amount of sodium thiamethoxam that inhibits the activity of the enzyme complex formed by 1U FVIII is defined as 1 potency unit of this product. The detection mechanism of the FVIII activity assay kit used in the present invention is as follows Figure 8 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 kit solution:

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

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

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

[0270] Accurately weigh 25 mg of polysaccharide reference substance and dissolve it in 10 mL of ddHO. 2 Prepare 2.5 mg / mL polysaccharide mother solution 1 in 5% ddHO; take 100 μL of polysaccharide mother solution 1 and add 9900 μL of ddHO. 2 O, dilute to 25 μg / mL polysaccharide mother solution 2; respectively take polysaccharide mother solution 2, add ddHO 2 O 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, add 50mL of ddH 2 O, prepared into 2% citric acid stop solution.

[0272] 4.5 Polysaccharide activity assay

[0273] (1) Add ddH2O to the blank wells of the 96-well plate test wells. 2 O 20μL, add 20μL 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 / mL FVIII factor working solution to the above test wells in turn, and add 20 μL of R2 solution to each well, and incubate at 37°C for 15 min;

[0275] (3) Add 20 μL R1(FX) solution to each well and incubate at 37°C for 1 min;

[0276] (4) Add 20 μL of R3 (Xa factor chromogenic substrate SXa-11) solution to each well, incubate at 37°C for 15 min, add 30 μL of stop solution, and place in a microplate reader to measure the absorbance at 405 nm (A405).

[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 IC values ​​of polysaccharides for inhibiting iFXase activity were calculated using GraphPadPrism software. 50 The value is 207.5ng / mL, and the credible line is (185.9~233ng / mL).

[0279] Table 24 Polysaccharide inhibition of iFXase activity and IC 50 value

[0280]

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

[0282] The above is only a preferred embodiment 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sodium thunbergii polysaccharide, characterized in that The sodium thunbergii polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium thunbergii polysaccharide is 90,000 to 130,000, the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine and fucose, 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 groups in the sodium thunbergii polysaccharide is 25 to 40%.

2. The sodium thunbergii polysaccharide according to claim 1, characterized in that The structural unit of the sodium thunbergii polysaccharide is shown in Formula I: In formula I: R1 is H, SO3 - or fucosyl, R2 is H or SO3 - , R3 is H or SO3 - , R4 is H or fucosyl; at least one of R1 and R4 is fucosyl; X ⊕ represents a cation; The structure of the fucosyl in R1 and R4 is shown in Formula I-1: In formula I-1: R5 is H or SO3 - , R6 is H or SO3 - , R7 is H or SO3 - .

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

4. The sodium thunbergii polysaccharide according to claim 2, 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.

5. The sodium thunbergii polysaccharide according to claim 2, characterized in that The cation is a sodium ion; the mass percentage of the sodium ion in the sodium ion polysaccharide is 5-15%.

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

7. The method for preparing sodium thunbergii polysaccharide according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) subjecting sea cucumber body wall powder to enzymolysis and acid hydrolysis in sequence to obtain an acid hydrolysis supernatant; the enzyme used in the enzymolysis is a protease; (2) adjusting the pH value of the acid hydrolysis supernatant to 6.5 to 7.5 and then performing alcohol precipitation, and sequentially washing, centrifuging and freeze-drying the obtained alcohol precipitated solid to obtain a crude polysaccharide; (3) Purifying the crude polysaccharide by column chromatography, collecting fractions with a weight average molecular weight of 90,000 to 130,000, and subjecting the obtained fractions to alcohol precipitation to obtain the sodium thunbergii polysaccharide.

8. Use of the sodium thunbergii polysaccharide according to any one of claims 1 to 6 or the sodium thunbergii polysaccharide prepared by the preparation method according to claim 7 in the preparation of anticoagulant and / or antithrombotic drugs or targeted inhibitors of coagulation factor X enzyme.

9. A targeted inhibitor of coagulation factor X enzyme, characterized in that: It comprises the sodium thunbergii polysaccharide according to any one of claims 1 to 6 or the sodium thunbergii polysaccharide prepared by the preparation method according to claim 7.

10. An anticoagulant and / or antithrombotic drug targeting the intrinsic coagulation pathway, characterized in that: The invention comprises active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient is the sodium thunbergii polysaccharide according to any one of claims 1 to 6 or the sodium thunbergii polysaccharide prepared by the preparation method according to claim 7.

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