WO2014183467A1

WO2014183467A1 - Use of black sea cucumber glycosaminoglycan in preparing medicine for prevention and treatment of thromboembolic disease

Info

Publication number: WO2014183467A1
Application number: PCT/CN2014/000471
Authority: WO
Inventors: 王志国; 刘全海; 董玉琼
Original assignee: 上海开润生物医药有限公司; 哈尔滨红豆杉生物制药有限公司
Priority date: 2013-05-13
Filing date: 2014-05-07
Publication date: 2014-11-20
Also published as: CN104147039A

Abstract

The present invention discloses the use of black sea cucumber glycosaminoglycan in preparing medicine for the prevention and treatment of thromboembolic disease. animal testing has shown that for a weight average molecular weight exceeding 20,000Da of black sea cucumber depolymerized sea cucumber glycosaminoglycan or of one or more segments of natural molecular segments of black sea cucumber sea cucumber glucosaminoglycan, the anti-coagulant activity thereof shows dose-dependency; in contrast to heparin and low molecular weight heparin, the dose-dependency thereof increases the incremental easing of blood coagulation, and, with an identical dose size, as weight average molecular weight increases, the onset time is delayed while the duration of efficacy is increased. For certain dosages, natural molecular segment sea cucumber glycosaminoglycan can have a duration of efficacy of up to 16 hours. The present invention has a higher level of safety when compared with heparin-type drugs and vitamin K antagonist-type drugs. In clinical use, the present invention has a wide treatment window for thromboembolic disease, has a high level of safety, and has good research and development value.

Description

Application of black sea cucumber glycosaminoglycan in preparing medicine for preventing and treating thromboembolic diseases

The invention relates to the medical use of the sea cucumber glycosaminoglycan of black sea cucumber, in particular to the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 20,000 Da or the sea cucumber glycosaminoglycan of the natural molecular segment in the preparation of a medicament for thromboembolic diseases Applications, thromboembolic diseases include atherothrombotic thrombosis, venous thromboembolic disease, hypercoagulable state, and prevention of thrombosis after surgery or treatment of thrombosis after surgery.

Background technique

The blood viscosity of the middle-aged and elderly people gradually increases, and the possibility of platelet aggregation with thrombosis (such as coronary artery and cerebral artery) increases. Thromboembolic disease has become a common threat to the health of humans, especially middle-aged and elderly people. disease. Thrombosis is the leading cause of arterial disease such as myocardial infarction and stroke, as well as venous thromboembolic disease and death. Anti-thrombosis drugs can be divided into anticoagulant drugs, anti-platelet drugs and direct thrombolytic drugs according to their mechanism of action, and can be used clinically to prevent and treat thrombosis. Among them, anticoagulant drugs prevent thrombosis or recurrence by affecting blood coagulation factors. Anticoagulant drugs have no dissolution effect on the formed thrombus, but can prevent thrombus expansion and new thrombus formation, and promote the early autolysis of thrombus. There are many kinds of anticoagulants available, but most of them are western medicine anticoagulants and their side effects are large. It is necessary to repeatedly detect blood coagulation during use to avoid bleeding. In addition, the administration method is complicated, and more importantly, the anti-drug There are safety hazards in clotting drugs. For example, the use of a wide range of anticoagulant heparin, low molecular weight heparin, and valerin requires the detection of blood coagulation conditions. Excessive or different constitutional use of various types of bleeding may cause serious safety hazards.

Therefore, in view of the aging of the population, the increase in the incidence of cardiovascular diseases, and the widespread use of existing anticoagulant drugs for the prevention and treatment of thromboembolic diseases and the severity of their safety risks. Screening and isolation from traditional Chinese medicines More effective and safe prevention and treatment of thromboembolic diseases is an inevitable trend in the prevention and treatment of thromboembolic diseases.

Sea cucumber is a sea cucumber of the echinoderms. There are more than 1,100 sea cucumbers in the world, and there are more than 140 species in China, most of which are inedible. China's edible sea cucumbers account for half of the total. Sea cucumbers are the same as ginseng, bird's nest and shark's fin. They are one of the eight treasures in the world. Sea cucumber is not only a precious food, but also a valuable medicine. According to the "Compendium of Compendium of Materia Medica", it is recorded: sea cucumber, sweet and salty, kidney, essence, urination, aphrodisiac, its warming, foot enemies, hence the name sea cucumber. Modern research shows that sea cucumber has prevention and treatment of arterial thrombosis, prevention and treatment of arteriosclerosis, and enhancement Immunity and anti-tumor effects.

Black sea cucumber (scientific name: Holothuria atra) is an animal of the sea cucumber family. It is distributed in India-Western Pacific, Taiwan, and China's Hainan Island, Xisha Islands, etc., and generally inhabits coral reefs and calm, seaweed and organic sandy bottoms. In the intertidal zone of the South China Sea and the sandy bottom of the coral reefs, the white linear mucus flowing out of the mouth can be used to treat traumatic hemorrhage and pain relief. After the internal organs, the whole can be used as a tonic for women's milk, but Its body wall is thin and its quality is far less than that of ginseng. It is the most edible sea cucumber. Many studies in recent years have shown that sea cucumber glycosaminoglycan is an acid mucopolysaccharide contained in the wall of sea cucumber. It is unique to sea cucumber. Sea cucumber glycosaminoglycans have significant anti-platelet aggregation and anticoagulant effects. Preferably, the sea cucumber of the present invention is selected from the group consisting of black sea cucumber, jade foot sea cucumber, sea cucumber, two-color table ginseng, plum ginseng, white-spotted anal ginseng, and preferably black sea cucumber and jade foot sea cucumber. Among them, black sea cucumber is produced in China's South China Sea, Beihai and other places. It is widely distributed, rich in resources and suitable in price. Moreover, sea cucumber has rich glycosaminoglycan content and is an ideal raw material.

Sea cucumber glycosaminoglycan has significant anticoagulant, anti-platelet aggregation, blood viscosity reduction, fibrinolysis, regulation of blood lipids and other biological activities, and can be used for the treatment of thromboembolic diseases. Experimental studies have found that the activity of different molecular weight segments of sea cucumber glycosaminoglycans Further experiments have found that the glycosaminoglycan activity mentioned in black sea cucumber is higher than the glycosaminoglycan activity reported so far, but the anticoagulant activity of black sea cucumber glycosaminoglycan and glycosaminoglycan is not currently studied. See the literature report, especially for the natural molecular segment samples of black sea cucumber and the anticoagulant activity of subcutaneous injection of degradation products have not been reported. Summary of the invention

The object of the present invention is to provide an application of black sea cucumber glycosaminoglycan in the preparation of a medicament for preventing and treating thromboembolic diseases, so as to overcome the above defects existing in the prior art and satisfy the needs of clinical application.

Animal experiments have shown that one or more of the depolymerized sea cucumber glycosaminoglycans or the sea cucumber glycosaminoglycans with a weight average molecular weight of more than 20,000 Da have a dose-dependent anticoagulant activity, with heparin and low molecular weight. Compared with heparin, it increases the coagulation with increasing dose, and at the same dose, the weight-dependent molecular weight increases with the onset of time delay and the duration of drug effect increases. The sea cucumber glycosaminoglycan of the natural molecular segment can last for up to 16 hours at a certain dose.

Therefore, the one or more of the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 20,000 Da or the sea cucumber glycosaminoglycan of the natural molecular segment can be used for the preparation of a medicament for preventing atherothrombotic disease, It can be used for the preparation of drugs for treating atherothrombotic diseases, can be used for the preparation of venous thromboembolic diseases, can be used for the preparation of drugs for treating venous thromboembolic diseases, and can be used for the preparation of drugs for preventing and treating blood hypercoagulable state, and can be used for Prevention of thrombosis after surgery or preparation of thrombotic drugs after treatment.

The "depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 20,000 Da or the sea cucumber sugar amine of a natural molecular segment" "Glycan" refers to a sea cucumber glycosaminoglycan having a weight average molecular weight of more than 20,000 Da, which has a weight average molecular weight or a natural molecular weight, or a depolymerized sea cucumber glycoside having a weight average molecular weight of more than 20,000 Da. a multi-stage mixture of any one weight average molecular weight or a natural molecular segment of sea cucumber glycosaminoglycan;

The weight average molecular weight is defined as follows: Weight-average Molecular Weight: The molecular weight of all synthetic polymer compounds and the molecular weight of most natural polymer compounds are not uniform, they are homologues of different molecular weights. mixture. The statistical average molecular weight of the polymer in the polymer with different molecular weights is the weight average molecular weight.

The weight average molecular weight was tested by high performance liquid chromatography.

Preferably, the weight average molecular weight of the depolymerized sea cucumber glycosaminoglycan is: 20,000 Da to 22,300 Da,

29,250Da~32,500Da, 39,050Da~42,100Da, 45,800Da~50,000Da, 54,506Da~57,450Da, 57,500Da~65,400Da, 65,500Da~75,400Da, 75,500Da~86,000Da, 86,050Da~96,000Da, 96,050Da Any of ~102,000Da;

Particularly preferably, the weight average molecular weight of the depolymerized sea cucumber glycosaminoglycan is:

20,000Da~22,300Da, 29,250Da~32,500Da, 39,050Da~42,100Da, 45,800Da~50,000Da, 54,600Da~57,00Da, 57,600Da~65,000Da, 65,600Da~75,300Da, 75,600Da~85,500Da, 86,200 Any of Da~95, 500Da, 96, 100Da~101,500Da;

The above-mentioned sea cucumber glycosaminoglycans which depolymerize sea cucumber glycosaminoglycan or natural molecular segment are all depolymerized sea cucumber glycosaminoglycans of black sea cucumber or black sea cucumber glycosaminoglycans of natural molecular segments;

The medicament comprising a therapeutically effective amount of the depolymerized sea cucumber glycosaminoglycan and a pharmaceutically acceptable carrier selected from the group consisting of mannitol, lactose, dextran, glucose, glycine, hydrolyzed gelatin More than one of povidone or sodium chloride, preferably mannitol;

The drug is an injection for intravenous or subcutaneous injection or a lyophilized powder injection;

The sea cucumber glycosaminoglycan having a molecular weight of more than 20,000 Da or the sea cucumber glycosaminoglycan of the natural molecular segment is administered by subcutaneous injection in a dose of 1 mg/kg to 100 mg/kg, preferably 2 mg/kg to 80 mg/kg _; The amount of intravenous administration is 0.1 mg/kg to 40 mg/kg, preferably 0.2 mg/kg to 30 mg/kg;

In the medicament, the depolymerized sea cucumber glycosaminoglycan has a purity of 90 to 99.99%, preferably 92% or more, more preferably 94% or more for achieving a more desirable effect; the sea cucumber glycosaminoglycan of the natural molecular segment The purity of the sugar is 90 to 99.99%, preferably 92% or more, and more preferably 95% or more in order to achieve a more desirable effect;

The purity is weight purity. The polydispersity of the depolymerized sea cucumber glycosaminoglycan is 1~2, preferably 1~1.6, more preferably 11.4;

The polydispersity refers to an index commonly used in the art to measure the molecular weight distribution of a polymer for characterizing the width of the molecular weight distribution of the polymer. Polydispersity, in this document or other literature, is also referred to as polydispersity index, polydispersity or distribution width index, which is the ratio of weight average molecular weight (Mw) to number average molecular weight (Mn), ie, Mw / Mn. This ratio varies with the width of the molecular weight distribution. In the case of monodispersion, M _W / M _n is equal to 1, and as the molecular weight distribution becomes wider, the Mw / Mn value becomes larger.

The depolymerized sea cucumber glycosaminoglycan can be prepared by using a commercial product such as sea cucumber glycosaminoglycan and depolymerized sea cucumber glycosaminoglycan produced by Harbin Sequoia Biopharmaceutical Co., Ltd., or by the following method:

(1) adding the enzyme to the ground sea cucumber, performing enzymatic hydrolysis and precipitation, collecting the crude sea cucumber glycosaminoglycan, purifying and decolorizing the crude sea cucumber glycosaminoglycan, and collecting the sea cucumber glycosaminoglycan;

The sea cucumber is selected from the group consisting of black sea cucumber;

The enzyme is a hydrolyzed protease and a composite trypsin. The hydrolyzed protease can be a commercial product, such as Alcalase of Novozymes (Shenyang) Biotechnology Co., Ltd., and the compound pancreatin can be used as a commercial product, such as Wuxi Xuemei enzyme preparation. Xuemei brand compound pancreatin of Science and Technology Co., Ltd., the amount of hydrolyzed protease is 2% of the weight of sea cucumber, and the amount of compound pancreatin is 2~3% of the weight of sea cucumber;

(2) adding the concentration of 5% acetic acid and 3% hydrogen peroxide to the product of step (1) for degradation, and collecting the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 20,000 Da;

The preparation method of the medicine is a conventional method in the field of preparation, such as the method described in the Handbook of Traditional Chinese Medicine Preparation, and the injection or lyophilized powder injection is obtained;

The depolymerized sea cucumber glycosaminoglycan drug of the present invention can be applied to a patient in need of treatment by subcutaneous or intravenous injection, and the dosage is administered by the physician according to the patient's specific conditions (such as age, weight, sex, time of illness, body). Status, etc.) OK. In general, the dosage of subcutaneous administration is 0.1 to 50 mg/kg, preferably 0.2 to 45 mg/kg, and the dosage for intravenous administration is 0.01 to 30 mg/kg, preferably 0.05 to the depolymerized sea cucumber glycosaminoglycan. 20 mg/kg.

(3) The desired molecular weight fraction is obtained by using a gel column of the sea cucumber glycosaminoglycan and the depolymerized sea cucumber glycosaminoglycan of the desired molecular weight range.

The gel adsorption column, such as a Sephadex-GlOO gel adsorption column, a Sephadex-G50 gel adsorption column or Sephadex-G200, the Sephadex-G100 gel adsorption column can be a GE gel of the United States GE company. column.

Sea cucumber glycosaminoglycan is an acidic mucopolysaccharide contained in the wall of sea cucumber, which is unique to sea cucumber. The invention Now, sea cucumber glycosaminoglycan has significant anti-coagulation, anti-platelet aggregation, blood viscosity reduction, fibrinolysis, regulation of blood lipids and other biological activities, and can be used for the treatment of thrombotic diseases.

The sea cucumber glycosaminoglycans are further depolymerized into depolymerized sea cucumber glycosaminoglycans of different molecular weight fractions, which exhibit different anticoagulant activities, and their anticoagulant activity is gradually increased with increasing dose, relative to heparin drugs and Vitamin K antagonists are safer. It is clinically used for the treatment of thromboembolic diseases with wide window, high safety and good research and development value.

DRAWINGS

Fig.1 Purity diagram of black sea cucumber-sea sea cucumber glycosaminoglycan and depolymerized sea cucumber glycosaminoglycan:

Figure 1-1 Purity diagram of sea cucumber glycosaminoglycan in the natural segment of black sea cucumber;

Figure 1-2 The weight average molecular weight of 21,254 Da

Figure 1-3 The weight average molecular weight of 31,887Da

Figure 1-4 The weight average molecular weight of 40,682 Da purity map

Figure 1-5 The weight average molecular weight of 48,769 Da

Figure 1-6 is a purity diagram of a weight average molecular weight of 5562 Da;

Figure 1-7 is a purity diagram of a weight average molecular weight of 60.588 Da;

Figure 1-8 shows the purity of the weight average molecular weight of 70,398 Da;

Figure 1-9 shows the purity of the weight average molecular weight of 79,825 Da;

Figure 1-10 is a purity diagram of a weight average molecular weight of 90,627 Da;

Figure 1-11 shows the purity of the weight average molecular weight of 100,156 Da;

Figure 2 shows the molecular weight test report of black sea cucumber-sea sea cucumber glycosaminoglycan and depolymerized sea cucumber glycosaminoglycan.

Figure 2-1 Test report on the molecular weight of glycosaminoglycans in the natural fraction of black sea cucumber;

Figure 2-2 Molecular weight test report of weight average molecular weight of 21,254 Da

Figure 2-3 Molecular weight test report of weight average molecular weight of 31,887 Da

Figure 2-4 Molecular weight test report of weight average molecular weight of 40,682 Da

Figure 2-5 Molecular weight test report of weight average molecular weight of 48,769 Da

Figure 2-6 shows the molecular weight test report of weight average molecular weight of 5562 Da;

Figure 2-7 Molecular weight test report of weight average molecular weight of 60.588 Da;

Figure 2-8 Molecular weight test report of weight average molecular weight of 70,398 Da;

Figure 2-9 Molecular weight test report of weight average molecular weight of 79,825 Da; Figure 2-10 Molecular weight test report of weight average molecular weight of 90,627 Da;

Figure 2-11 Molecular weight test report of weight average molecular weight of 100,156 Da;

Fig. 3 is a linear relationship between the anticoagulant dose and the clotting time of the sea cucumber glycosaminoglycan in the black sea cucumber.

Figure 3-1 DHG-1 in vitro anticoagulation experiment results.

Figure 3-2 DHG-2 in vitro anticoagulant test results.

detailed description

The method for extracting the glycosaminoglycan from sea cucumber refers to the sea cucumber glycosaminoglycan extracted from sea cucumber, and after degrading and depolymerizing, a glycosaminoglycan is produced, and the depolymerized sea cucumber glycosaminoglycan having the desired molecular weight is collected. Methods for extracting sea cucumber glycosaminoglycans from sea cucumber body walls are well known to those skilled in the art, such as Chinese patent ZL200910305363.5.

Example 1

Extraction of sea cucumber glycosaminoglycan:

Weigh 0.5kg of black sea cucumber, and soak for 16h. The sea cucumber body wall was drained with water, minced, weighed and hydrated to 4 kg, placed in a 60 ° C water bath, added with 6 mol / L sodium hydroxide to adjust the pH to 8.2 ± 0.2, added 20 ml hydrolyzed protease Alcalase (Novozymes (Shenyang) Biotechnology Co., Ltd.) Stir, digest for 4 hours, inactivate at 85 °C for 10 minutes, and cool. Centrifuge at 5 ° C, collect the supernatant, add 6mol / L hydrochloric acid to adjust the pH to 2.5 ± 0.2, refrigerate at 4 ° C for 2 hours, centrifuge, collect the supernatant, add 6mol / L sodium hydroxide to adjust the pH to 7.5 ± 0.2, 0.8 volume of ethanol was added and allowed to stand at 4 ° C for 12 h.

Centrifuge, collect and weigh the precipitate, add 10 times the weight of distilled water, heat to 85 °C ± 2 ° C, after complete dissolution, add 6mol / L sodium hydroxide to adjust the pH to 9.0 ± 0.2, add calcium chloride to the solution The concentration of calcium chloride reached 3% (w/v), the temperature was raised to 92 ° C for 15 minutes, cooled to room temperature, centrifuged at 4 ° C, the supernatant was collected, and the pH was adjusted to 11.0 ± 0.1 with a saturated sodium carbonate solution, and centrifuged. The supernatant was collected, adjusted to pH 6.0 ± 0.1 with 6 mol / L hydrochloric acid, 1 volume of ethanol was added, and refrigerated at 4 ° C for 12 h;

The frozen liquid was centrifuged, and the precipitate was collected and weighed. Two times of distilled water was added, and the mixture was fully dissolved by heating. Potassium acetate was added to make a final concentration of 2 mol/L, and allowed to stand at 4 ° C for 12 hours. After centrifugation, the precipitate was collected and weighed, and 2 volumes of distilled water was added thereto, and heated to fully dissolve it, and potassium acetate was added thereto to have a final concentration of 2 mol/L, and allowed to stand at 4 ° C for 12 hours. After centrifugation, the precipitate was washed three times with a cold 2 mol/L potassium acetate solution, and then washed successively with 80% ethanol, 95% ethanol, and absolute ethanol. After the ethanol was evaporated, it was dried at 80 ° C, and weighed to obtain a crude product A.

The crude product A was added with 0.05 mol/L and pH 6.0 in HAc-NaAc buffer to prepare a 2% solution column, and the solution was passed. After the cellulose chromatography column, washed with 1.5 column volume of 0.4 mol / L NaCl of HAc-NaAc buffer CpH6.0 ± 0.1), and then Imol / L NaCl of HAc-NaAc buffer CpH6.0 ± 0. i; > Elution, the eluent was collected according to the change speed of the UV detector at 220 nm, placed in a 60 ° C water bath, adjusted to pH 11 ± 0.1 with NaOH, added 3% hydrogen peroxide by volume, kept for 4 hours, cooled, After centrifugation, the supernatant was collected, adjusted to pH 7.2 ± 0.1 with HC1, added with 1 time ethanol, and allowed to stand at 4 ° C for 12 h.

After centrifugation, the precipitate was collected and washed successively with 80% ethanol, 95% ethanol, and absolute ethanol to obtain a crude product B.

The crude product B is dissolved in 5% solution with distilled water, and concentrated to 1/2 of the original volume with an ultrafiltration membrane with a molecular weight cut off of 10,000. The water is added to the original volume, and then ultrafiltered to 1/2 volume, and water is added again. The filtrate is freeze-dried to obtain sea cucumber glycosaminoglycan,

The sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Figure 1-1), and the depolymerized sea cucumber sugar obtained by the example. The amine glycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 109,401 Da, and the D value was 1.27 (see Figure 2-1).

Example 2

Preparation of depolymerized sea cucumber glycosaminoglycan

Example 2-1

The pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 42 hours. The solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of absolute ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 20,000 Da to 22,000 Da, a value of < 1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-2), and the depolymerization obtained by the example was obtained. The sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 21254, and the D value was 1.29 (see Figure 2-2 for the map).

Example 2-2

The sea cucumber glycosaminoglycan pure product in the above Example 1 was formulated into a 5% solution with 5% acetic acid, and 30% dioxygen was added. Water allowed the concentration of hydrogen peroxide in the solution to be 3%, and controlled depolymerization at 40 °C for 30 h. The solution was neutralized to neutral with 0.1 mol/min of sodium hydroxide, and 3 times by volume of absolute ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 22,100 Da to 35,000 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-3), and the depolymerization obtained by the example was obtained. The weight fraction of the sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 31887, and the D value was 1.37 (see Figure 2-3). Example 2-3

The pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 20 hours. The solution was neutralized to neutrality with 0.1 mol of hydrazine sodium hydroxide, and ethanol was precipitated by adding 3 volumes of absolute ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 35,100 Da to 45,000 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product having a purity of 99.0% (see Figure 1-4), and the depolymerization obtained by the example was obtained. The sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 40682, and the D value was 1.35 (see Figure 2-4). Examples 2-4

The above-mentioned sea cucumber glycosaminoglycan was prepared into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 12 hours. The solution was neutralized to neutral with 0.1 mol/min of sodium hydroxide, and 3 times by volume of absolute ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 45,100 Da to 53,000 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-5), and the depolymerization obtained by the example. Sea cucumber glycosaminoglycan through gel column (TSK gel G4000PWXL, TOSOH) chromatographic analysis showed that the product has a weight average molecular weight of 48,769 and a D value of 1.38 (see Figure 2-5 for the spectrum). Example 2-5

The pure sea cucumber glycosaminoglycan in Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 8 hours and 20 minutes. The solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of absolute ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 54,506 Da to 57,450 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-6), and the depolymerization obtained by the example. The weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 54,962 Da, and the D value was 1.37 (see Figure 2-6).

Example 2-6

The pure sea cucumber glycosaminoglycan in Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 °C for 5 hours and 30 minutes. The solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of absolute ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 57,500 Da to 65,400 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Fig. 1-7), and the depolymerization obtained by the example was obtained. The sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 60,588 Da, and the D value was 1.36 (see Figure 2-7).

Example 2-7

The pure sea cucumber glycosaminoglycan in Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 3 h 30 min. The solution was neutralized to neutral with 0.1 mol/l sodium hydroxide, and 3 times volume of absolute ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain depolymerized sea cucumber sugar amine. The crude product of glycans.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 65,500 Da to 75,400 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Fig. 1-8 for the map), and the depolymerization obtained by the example Chromatographic analysis of sea cucumber glycosaminoglycan by gel column (TSK gel G4000PWXL, TOSOH) showed that the weight average molecular weight of the product was 70,398 Da, and the D value was 1.31 (see Figure 2-8 for the map).

Example 2-8

The pure sea cucumber glycosaminoglycan in Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 2 h 25 min. The solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and 3 times by volume of absolute ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 75,500 Da to 86,000 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Fig. 1-9 for the map), and the depolymerization obtained by the example Chromatographic analysis of sea cucumber glycosaminoglycan by gel column (TSK gel G4000PWXL, TOSOH) showed that the product had a weight average molecular weight of 79,825 Da and a D value of 1.34 (see Figure 2-9 for the map).

Example 2-9

The pure sea cucumber glycosaminoglycan of Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization at 40 ° C for 40 min. The solution was neutralized to neutral with 0.1 mol/min of sodium hydroxide, and 3 times by volume of absolute ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. The glycosaminoglycan has a molecular weight of 86,050 Da to 96,000 Da, a value of <1.5, and a purity of 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example can be obtained by the differential refractive index detector (RID-10A, Shimadzu). 99.0% pure product (see Figure 1-10), the depolymerized sea cucumber glycosaminoglycan obtained by this example was analyzed by gel column (TSK gel G4000PWXL, TOSOH) to find the weight average molecular weight of the product 90,627 Da, D The value is 1.32 (see Figure 2-10 for the map)

Example 2-10

The pure sea cucumber glycosaminoglycan in Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 40 min. The solution was neutralized to neutral with 0.1 mol/1 sodium hydroxide, and ethanol was precipitated by adding 3 volumes of absolute ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.

The crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber. Glycosaminoglycans have molecular weights ranging from 96,050 Da to 102,000 Da. The value is < 1.5 and the purity is 98% or more.

The depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Fig. 1-11), and the depolymerization obtained by the example was obtained. The sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 100,156 Da, and the D value was 1.29 (see Figure 2-11).

Example 3

40.0 g of sea cucumber glycosaminoglycan or depolymerized sea cucumber glycosaminoglycan obtained above was added to 80 g of mannitol, 1000 ml of water for injection was dissolved, and ultrafiltration, filling and lyophilization were carried out to obtain 1000 bottles of sea cucumber glycosaminoglycan for injection. Or lyophilized powder injection of depolymerized sea cucumber glycosaminoglycan.

Example 4

Pharmacodynamic experiment of sea cucumber glycosaminoglycan and depolymerized sea cucumber glycosaminoglycan

4.1 in vitro anticoagulation experiment

4.1.1 Test materials

Test sample:

Name: Depolymerized sea cucumber glycosaminoglycan, the following abbreviations: DHG; DHG-1 (Example 2-6), DHG-2 (Example 2-7); Preparation: After precision absorption, diluted with physiological saline for injection Required concentration.

Test animal

Line: Rabbit; Source: Shanghai Chenxing Experimental Rabbit Co., Ltd.; Gender: Male; Weight: 1850 g; Animal Certificate No.: SCXK (Shanghai) 2008-0010. 4.1.2 Test equipment

Platelet aggregation clotting factor analyzer (model LG-PABER Beijing Shidi Scientific Instrument Co.;).

4.1.3 Experimental methods

On the day of the experiment, add rabbit plasma 80μ1, 0.9% sodium chloride solution 10μ1 to the sample cell, preheat for 180s, add 1μ calcium chloride solution 10μ1, mix immediately to avoid air bubbles, use platelet aggregation factor analyzer Start the calculation time and record the condensation time of each sample cell, which is blank.

Precisely weigh the reference solution and dilute it to a different concentration with a 0.9% sodium chloride solution, which is a different concentration of the sample solution DHG-1 (10.0 g/ml~200.(^g/ml), DHG-2 ( 8.0 g/ml~120.(^g/ml).

The plasma coagulation time of each sample solution was determined by using 样品μΙ different concentrations of sample solution instead of 10μ10.9% sodium chloride solution. Each concentration was measured in parallel 4 times and averaged.

4.1.4 Experimental results

The experimental results show that the final concentration of the sample is in the DHG-1 (10.0 g/ml~140. (^g/ml), DHG-2 (8.0 g/ml~ 120.0μ _§ /ιη1) dose range, and the clotting time is prolonged with the dose. The clotting time is prolonged and the increasing trend is moderated. Therefore, the depolymerized sea cucumber glycosaminoglycan composition is safer and controllable for anticoagulation.

DHG-1 in vitro anticoagulant test results

4.2 Effects of subcutaneous injection of depolymerized sea cucumber glycosaminoglycan and natural sea cucumber glycosaminoglycan on rat coagulation system

4.2.1 Test materials

Test sample: Name: Natural sea cucumber glycosaminoglycan (Example 1), depolymerized sea cucumber glycosaminoglycan (Example 2); Preparation: After precision extraction, it was diluted with physiological saline for injection to the desired concentration.

4.2.2 Test animals

Line: SD rat; Source: Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.; Gender: Male; Weight: 180-200 g; Animal certificate number: SCXK (Shanghai) 2008-0016; Feeding: Animals are fed in positive pressure Purify and ventilate the animal room, room temperature 23±1 °C, humidity 50~70%, artificial lighting simulates day and night changes, free to eat and drink.

4.2.3 Test equipment

Automatic coagulation analyzer Sysmex CA-1500.

4.2.4 Experimental methods

SD rats were divided into 10 groups, the negative control group (subcutaneous injection of 0.5 ml of normal saline), and the two dose groups (10, 20 mg/kg) were administered subcutaneously in a volume of 0.5 ml. Different time periods after subcutaneous injection (0.5h, 1.0h, 2.0h, 4h, 6h, 8h, 12h) Determination of prothrombin time (PT), activated partial thromboplastin time (ΑΡΤΤ) and coagulation by abdominal aortic blood collection Enzyme time (ΤΤ) values, see Tables 2 and 4.

Animals in each group were anesthetized with 3% cytotoxic intraperitoneal injection (O.lml/lOOg body weight) at 10 min before surgery, and fixed intra-abdominal surgery with supine 3.2% sodium citrate anticoagulation vacuum blood collection tube.

4.2.5 Test results

Depolymerization of sea cucumber glycosaminoglycan and natural sea cucumber glycosaminoglycan 10mg/kg and 20mg/kg have significant effects on APTT, TT, and ΡΤ. Different weight average molecular weight depolymerized sea cucumber glycosaminoglycans have anticoagulant activity with time. Incremental, the anticoagulant elongation rate reaches the peak time between 2-8h, and the weight average molecular weight is smaller and the peak time specific gravity averages the molecular weight to reach the peak time earlier. The anticoagulant effect of the natural molecular segment of the sea cucumber glycosaminoglycan after subcutaneous injection of 10 mg/kg reached a peak at 4 h; at 20 mg/kg, it peaked at 4 h. Depolymerization of sea cucumber sugar aminoglycan 10mg/kg, 20mg/kg dose of different molecular segments onset time and the peak time of arrival, subcutaneous injection of depolymerized sea cucumber glycosaminoglycans have a significant effect on APTT, making APTT prolonged more than Range of 150% - 250%, see Table 2-1, 2-2, 2-3, 2-4.

U OOO/ lOZ lD/lDd Project clotting time (s)

(10mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h

21,254Da 46.0±1.2 49.4±1.6 50.2±1.2 57.1±1.5 50.4±1.6 47. 1.2 44.2 0.9

31,887Da 50.5±1.0 54.1 0.8 55.4±1.1 61.9 1.2 52.8 0.9 50.8±1.5 45.9 1.6

40,682Da 52.4 0.7 55.9±0.9 57.9 0.7 64.9 1.4 55.2±1.6 51.8±1.0 47.2 1.3

48,769Da 55.4±1.5 59.4±1.3 60.7±1.0 68.1±1.7 58.3±1.2 55.6±1.4 50.1±1.0

5662Da 57.5±1.4 61. 1.7 63.0±1.6 69.1±1.8 60.0±1.6 57.0±1.4 50.8 1.2 ττ 60,588Da 60.0±1.2 63.8±1.3 66.9±1.2 77.0 0.9 62.3±1.7 58.6±1.8 53.2 1.7

70,398Da 58.8 0.9 63.0 1.8 64.5±1.5 75.9 1.7 61.6±1.4 57.5±1.7 52.2 1.1

79,825Da 54.2±1.5 57.7±1.6 59.2±1.0 66.3±1.3 57.0±1.5 54.4±1.5 48.6 1.2

90,627Da 49. 1.1 51.3±1.5 52.2 0.9 59.4 1.6 51.8 0.8 48.5±1.1 45.3±1.4

100,156Da 44.8 0.7 48.6±0.9 49.4 0.8 53.8 1.0 48.8±1.5 45.6±1.3 43.7 1.2 Natural 44.3±1.0 47.5±1.4 48.7±1.3 53.3±0.6 47.3±1.7 44.3±1.1 42.8 1.5 Blank 40.3±1.1 40.9 0.8 39.8 0.5 40.1± 0.8 40.6 0.6 41.0 0.7 40.5±0.9 Table 2-2 Prolongation rate of clotting time in rats

Experimental results of subcutaneous injection of anticoagulation in rats

Blank 15.0±0.6 15.3±0.6 15.2 0.5 15.2 0.4 14.9 0.3 15.1±0.7 14.8 0.2 Project clonal time (S)

Ττ (10mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h

21,254Da 41.7 0.6 42.1±0.7 43.8 0.3 45.1±0.6 43.5 0.4 42.5±0.8 41.8 0.5

31,887Da 42.6 0.8 42.7 0.3 44.7 0.4 46.3±0.5 43.9 0.2 42.9 0.6 42.2±0.9

40,682Da 43.1 0.2 43.0 0.6 45.6 0.5 47.4 0.3 45.0 0.7 43.5±0.5 42.6 0.4

48,769Da 44.2 0.6 44.8 0.9 46.8 1.0 50.6 0.5 46.9 0.3 44.6 0.8 43.6 0.1

5562Da 44.8±1.0 45.1±0.7 47.8 1.3 51.5±1.6 47.8 0.9 46.0 0.9 44.2 0.7

60,588Da 48.0±0.6 50.3±0.8 54.5±0.9 54.3±1.2 50.5 0.7 47.9 1.1 45.9 0.8

70,398Da 47.1 0.8 48.5±1.0 51.7 0.7 52.9 0.9 48.9 0.5 46.6 0.6 45.2 0.4

79,825Da 43.4 0.4 43.8 0.8 46.3±0.6 48.1±0.5 46.2 0.8 44.1±0.6 43.3±1.1

90,627Da 42. 1.2 42.6 1.6 44.3±1.2 45.8 1.5 44. 1.6 42.6 1.2 42. 0.9

100,156Da 41.5 0.7 41.7 0.5 43.7 0.8 44.6 0.4 43.0±0.6 42.0 0.5 41.6 0.3 Natural 41.3±1.2 41.1±0.5 42.6 0.5 44.1±0.6 42.3±1.0 41.4 0.8 41.3 0.5 Blank 40.5 0.9 39.8 0.4 40.9 0.6 41.2 0.8 41.0±0.6 40.5± 0.8 39.9 0.2

Table 2-4 Prolongation rate of clotting time in rats

61

T .t7000/M0ZN3/X3d 100, 156Da 2. 41% 4. 72% 6. 85% 8. 21% 4. 95% 3. 64% 2. 74% Natural 1. 95% 3. 28% 4. 14% 7. 09% 3. 29% 2. 17% 1. 92%

4.3 Effect of depolymerizing sea cucumber glycosaminoglycan on arteriovenous catheter thrombosis model in rats

4.3.1 Test materials

Test sample - Name: Natural molecular segment sea cucumber glycosaminoglycan and depolymerized sea cucumber glycosaminoglycan (Example 1; Example 2); Preparation: After precision pipetting, it was diluted with physiological saline for injection to the desired concentration. Control sample: Name: Heparin; Source: National Pharmaceutical Group Chemical Reagent Co., Ltd.; Batch number: F20091029; Content: 150U/mg; Preparation: After precision weighing, dissolve and dilute to the desired concentration with physiological saline for injection.

Test animals: strain: SD rat; Source: Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.; Gender: Male; Weight: 180-220 g; Animal certificate number: SCXK (Shanghai) 2008-0016; Feeding: Animal breeding In the positive pressure purification and ventilation animal room, room temperature 23±1 °C, humidity 50~70%, artificial lighting simulation changes day and night, free to eat and drink.

4.3.2 Test equipment

BS 110 s electronic balance, produced by SARTORIUS, with a minimum weight of 0.1 mg.

4.3.3 Test method

SD rats in each group were divided into different drug-administered groups, the negative control group (physiological saline 1 ml/kg), and the positive control low molecular weight heparin group (2 mg/kg). All drugs were administered subcutaneously in a volume of 0.5 ml.

Each group of animals was anesthetized with 10% chloral hydrate (100~400 mg/kg) 10 min before surgery, and then fixed on the back, the neck skin was cut, and the left carotid artery and the right external jugular vein were separated. The tube is connected, and a 7cm long 4th surgical thread is placed in the tube. The blood flow was opened for 15 minutes after administration for 2 hours, and then the silk thread was taken out and weighed, minus the weight of the silk thread, which is the wet weight of the thrombus. The mean and standard deviation of thrombus wet weight of each test group were calculated and compared with the saline group by t-test. The thrombus wet weight inhibition rate of each test group was calculated according to the following formula: Thrombosis rate _{(%) =} thrombus wet weight (dissolved fine-blood sputum (fine) _x recommended

Thrombosis wet weight (solvent group)

4.3.4 Test results

See Table 3-1. Positive drugs and test drugs can significantly inhibit thrombus formation after drug administration. The test drug has a significant inhibitory effect on thrombosis.

Table 3-1 Effects of rat arteriovenous catheter thrombosis model Group n dose (mg/kg) Thrombosis weight (mg) Thrombosis inhibition rate (%) Blank 10 0.5ml 65.5±2.8

Low molecular weight heparin 40. _3±7 .3«

4 38.47%

21,254Da 10 16 50.2±6.9* 23.36%

31,887Da 10 16 44.6±8.2" 31.91%

40,682Da 10 16 41.8±7.5** 36.18%

48,769Da 10 16 39.3±9.4" 40.00%

5562Da 10 16 37.2±8.7" 43.21%

60,588Da 10 16 35.8±10.1" 45.34%

70,398Da 1 1 16 36.4±6.8" 44.43%

79,825Da 10 16 42.1±9.5" 35.73% natural 10 16 48.9±9.2* 25.34% compared with the negative group: * P<0.05, ** P<0.01

4.4 Effects of subcutaneous injection of sea cucumber glycosaminoglycan composition depolymerized by different molecular weight fractions on rat coagulation system

4.4.1 Test materials

Test sample:

Name: Depolymerized sea cucumber glycosaminoglycan 60,588Da, 70,398Da _; natural sea cucumber glycosaminoglycan; Source: Shanghai Kairun Biomedical Co., Ltd.; Preparation: After precision absorption, diluted with physiological saline for injection to the required concentration. 4.4.2 Test animals

Line: SD rat; Source: Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.; Gender: Male; Weight: 180-220 g; Animal certificate number: SCXK (Shanghai) 2008-0016; Feeding: Animals are fed in positive pressure Purify and ventilate the animal room, room temperature 23±1 °C, humidity 50~70%, artificial lighting simulates day and night changes, free to eat and drink.

4.4.3 Test equipment

Automatic coagulation analyzer Sysmex CA-1500

4.4.4 Experimental method

SD rats in each group were divided into different drug-administered groups, negative control group (subcutaneous injection of normal saline 0.5ml), depolymerized sea cucumber glycosaminoglycans of different molecular weights (60, 588Da, 70, 398Da), natural sea cucumber sugar amine The glycan composition was administered by subcutaneous injection at a dose ratio of 1:1 (10 mg/kg), and the blank was injected with a volume of 0.5 ml of physiological saline. The plasma activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (ΤΤ) values were determined by subcutaneous aortic blood sampling at different time points after subcutaneous injection. See Table 5.

Each group of animals was anesthetized with 3% tachyin inoculation (O.lml/lOOg body weight) at 10 minutes before surgery, supine After the fixed abdominal surgery, the blood was collected by a disposable 3.2% sodium citrate anticoagulation vacuum blood collection tube.

4.4.5 Test results

The experimental results show that subcutaneous injection of depolymerized sea cucumber glycosaminoglycan composition with different molecular weight fraction has significantly prolonged APTT TT activity, and can overcome the slow or short duration of onset of single molecular weight. See Table 4-1 for experimental data. 2

Anticoagulant test results of subcutaneous injection of rats with different molecular fractions

Table 4-2 Prolongation rate of clotting time in rats with anticoagulated subcutaneous injection of different molecular segments Project for a while

(10mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h

APTT 60,588Da 10mg/kg + natural lOmg/kg

38.82% 52.94% 91.95% 188.00% 121.85% 84.46% 9.27%

70,398Da 10mg/kg + natural lOmg/kg

13.16% 26.14% 50.34% 104.00% 62.25% 32.43% 10.60% Item

(lOmg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h

PT 60,588Da 10mg/kg + natural lOmg/kg

2.67% 7.95% 15.44% 23.65% 12.67% 8.05% 3.95%

70,398Da 10mg/kg + natural lOmg/kg

5.33% 7.95% 14.77% 21.62% 6.00% 4.03% 0.00% Item

(lOmg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h ττ 60,588Da 10mg/kg + natural lOmg/kg

33.67% 45.43% 48.03% 61.86% 44.61% 33.83% 23.23%

70,398Da 10mg/kg + natural lOmg/kg

21.36% 33.76% 31.77% 48.17% 24.31% 19.01% 11.87%

Claims

Claim

1. One or more of black sea cucumber glycosaminoglycans having a weight average molecular weight greater than 20,000 Da or a black sea cucumber glycosaminoglycan of a natural molecular segment, for use in the preparation of a medicament for preventing and treating arterial thromboembolic diseases.

2. The use according to claim 1, wherein the thromboembolic disease comprises atherothrombotic disease, venous thromboembolic disease, hypercoagulable state, thrombus formed after surgery or prevention of surgery The formation of blood clots.

3. The use according to claim 1, wherein the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 20,000 Da or the sea cucumber glycosaminoglycan of the natural molecular segment means that the weight average molecular weight is greater than 20,000 Da depolymerized sea cucumber glycosaminoglycans of any weight average molecular weight or natural molecular segment of sea cucumber glycosaminoglycan, or a weight average molecular weight of more than 20,000 Da depolymerized sea cucumber glycosaminoglycans of any weight average molecular weight or natural molecular segment A multi-stage mixture of sea cucumber glycosaminoglycans.

4. The use according to claim 1, wherein the weight average molecular weight of the depolymerized sea cucumber glycosaminoglycan is: 20,000 Da~22, 300 Da, 29,250 Da~32,500 Da, 39,050 Da~42, 100 Da, 45,800 Any of Da~50,000Da, 54,506Da~57,450Da, 57,500Da~65,400Da, 65,500Da~75,400Da, 75,500Da~86,000Da, 86,050Da~96,000Da, 96,050Da~102,000Da.

5. The use according to claim 1, wherein the weight average molecular weight of the depolymerized sea cucumber glycosaminoglycan is: 20,000 Da~22, 300 Da, 29,250 Da~32,500 Da, 39,050 Da~42, 100 Da, 45,800 Any of Da~50,000Da, 54,600Da~57,00Da, 57,600Da~65,000Da, 65,600Da~75,300Da, 75,600Da~85,500Da, 86,200Da~95,500Da, 96,100Da~101,500Da.

The use according to any one of claims 1 to 5, characterized in that the medicament comprises a therapeutically effective amount of the depolymerized sea cucumber glycosaminoglycan and a pharmaceutically acceptable carrier.

7. Use according to claim 6, characterized in that the medicament is an injection for intravenous or subcutaneous injection or a lyophilized powder injection.