Low-molecular-weight sulfuric acid galactan derivative, preparation method thereof and application of low-molecular-weight sulfuric acid galactan derivative in preparation of anticoagulant and / or antithrombotic drugs

Abstract

The invention discloses a low-molecular-weight sulfuric acid galactan derivative, a preparation method thereof and application of the low-molecular-weight sulfuric acid galactan derivative in preparation of anticoagulant and / or antithrombotic drugs. The low-molecular-weight sulphated galactan derivative is prepared by taking red algae-derived polysaccharide as a raw material through degradation, sulfation modification and purification, and anticoagulant activity research on the low-molecular-weight sulphated galactan derivative shows that the low-molecular-weight sulphated galactan derivative has remarkable in-vivo and in-vitro anticoagulant activity, and can be used for preparing the low-molecular-weight sulphated galactan derivative. Moreover, the compound has a relatively strong selective inhibition effect on an endogenous blood coagulation pathway, is low in bleeding risk and can be used for preventing and / or treating thrombotic related diseases. The polypeptide has the potential of being developed into in-vivo and in-vitro anticoagulant / antithrombotic drugs, and has a wide market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a low molecular weight sulfated galactan derivative and a preparation method thereof and application thereof in the preparation of anticoagulant and / or antithrombotic drugs. Background Art

[0002] Cardiovascular and cerebrovascular diseases are a general term for cardiovascular and cerebrovascular diseases, which are characterized by high morbidity, high disability rate, high recurrence rate and high mortality rate. The number of people who die from cardiovascular and cerebrovascular diseases each year accounts for a quarter of the total global death toll, and has become a major disease that seriously threatens human health. Abnormal coagulation function is the main pathological factor in the formation of the pathological mechanism of cardiovascular and cerebrovascular diseases - vascular embolism. According to clinical manifestations, thrombi are mainly divided into arterial thrombosis and venous thrombosis. Among them, venous thromboembolism is a chronic disease that includes deep vein thrombosis and pulmonary embolism. It is the third largest cardiovascular disease after acute myocardial infarction and stroke. According to statistics, it affects the lives and health of nearly 10 million people worldwide on average each year.

[0003] At present, anticoagulant drugs represented by heparin, vitamin K antagonist warfarin and new oral anticoagulants have been widely used in the prevention and treatment of venous thromboembolism. The mechanism of action of these drugs is mainly to inhibit the activity of thrombin (FⅡa) or FⅩa in the common coagulation pathway, and some drugs lack anticoagulant antagonists, which will increase the risk of bleeding and also bring some other serious side effects such as heparin-induced thrombocytopenia. Therefore, it is urgent to study and develop safer and more effective anticoagulant and antithrombotic drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a low molecular weight sulfated galactan derivative and a preparation method thereof and use thereof in the preparation of anticoagulant and / or antithrombotic drugs. The present invention proves through experiments that the low molecular weight sulfated galactan derivative has a strong selective inhibitory effect on the intrinsic coagulation pathway and has a technical prospect of being developed into a drug for the treatment and prevention of thrombotic diseases.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The present invention provides a low molecular weight sulfated galactan derivative and a pharmaceutically acceptable salt thereof. The low molecular weight sulfated galactan derivative has a weight average molecular weight of 3-15 kDa, a mass percentage content of β-D-galactose of 10%-45%, a mass percentage content of 3,6-endoether-α-D-galactose of 15%-30%, and a mass percentage content of sulfate groups of 15%-50%. The structural formula thereof is shown in general formula (I): , (I)

[0007] Where R1=H or SO3 - ; R2 = H or SO3 - ; R3 = H or SO3 - ; n represents an integer from 5 to 50.

[0008] Furthermore, the low molecular weight sulfated galactan derivative comprises at least one of the following repeating structural units: , Repeating structural unit A Repeating structural unit B , Repeating structural unit C Repeating structural unit D , Repeating structural unit E Repeating structural unit F , Repeating structural unit G.

[0009] The present invention provides a method for preparing a low molecular weight sulfated galactan derivative, the preparation method comprising the following steps:

[0010] (1) Degradation: Using sulfated galactan and its derivative polysaccharides from red algae as raw materials, the polysaccharide raw materials are degraded by physical degradation (microwave degradation), chemical degradation (acid degradation, oxidation degradation, Fenton degradation), enzymatic degradation, or a combination of two or more degradation methods to prepare low molecular weight galactan containing both β-1,3-D-galactose (D-Gal) residues (G) and α-1,4-D-3,6-ether galactose (DA) residues;

[0011] (2) Sulfation modification: The low molecular weight galactan is modified with a sulfating agent, so that the C2, C4 and C6 hydroxyl groups of the D-Gal sugar residue contain sulfate groups to varying degrees, and the C2 hydroxyl group of the DA sugar residue contains sulfate groups to varying degrees, thereby preparing a crude low molecular weight sulfated galactan derivative;

[0012] (3) Purification: The crude low molecular weight galactan derivative is dissolved in pure water and then purified by ultrafiltration or dialysis to obtain a purified low molecular weight sulfated galactan derivative.

[0013] Furthermore, the red algae include but are not limited to seaweed of the genus Eucheuma, Chondrus, Kappaphycus, Sugihara, Ginkgo, and Rhodophyta.

[0014] Furthermore, in step (1)

[0015] Acid degradation method: Dissolve galactan sulfate in a 0.02-0.2 mol / L dilute acid solution with a mass ratio concentration of 0.5%-10%, heat at 40-70°C for 0.5h-7h, add alkali solution for neutralization, concentrate the solution, desalt and dry to obtain low molecular weight galactan sulfate;

[0016] Fenton degradation method: prepare galactan sulfate with a mass ratio concentration of 0.5%-10%, adjust the pH value to weak acidity, add an appropriate amount of H2O2 after preheating to a final concentration of 0.1~5%, then add an appropriate amount of FeCl2 to make the final concentration in the solution 0.1~0.5%, heat the reaction at 35-60℃ for 0.5h-6h, concentrate the solution, desalt and dry to obtain low molecular weight galactan sulfate.

[0017] Furthermore, in the (1) degradation step, the mass-to-liquid ratio of the raw material to water is 1:100; the dilute acid solution is a dilute sulfuric acid solution with a final concentration of 0.05 mol / L; the degradation time is 1-7 h; and the degradation temperature is 60-70 °C.

[0018] Furthermore, the sulfating agent includes but is not limited to a mixture of one or more of sulfur trioxide-pyridine, sulfur trioxide-triethylamine, and chlorosulfonic acid-pyridine.

[0019] Furthermore, the sulfation method used in step (2) is the sulfur trioxide-pyridine method, the molar ratio of sulfur trioxide-pyridine to the repeating disaccharide unit in the polysaccharide is 2:1-10:1; the molar ratio of DMF to the repeating disaccharide unit in the polysaccharide is 245:1-250:1; the reaction time is 1-6 h; and the reaction temperature is 30-90°C.

[0020] Furthermore, the graded purification method is a combination of one or more methods of a gel chromatography column based on the molecular exclusion principle or a nanofiltration membrane or ultrafiltration membrane or a dialysis bag with a molecular weight cutoff range of 500-1000Da.

[0021] The present invention also provides the use of the low molecular weight sulfated galactan derivative or a pharmaceutically acceptable salt thereof in the preparation of anticoagulant and / or antithrombotic drugs.

[0022] The present invention also provides the use of the low molecular weight sulfated galactan derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating thrombosis-related diseases.

[0023] Furthermore, the compound prevents and treats thrombotic diseases through intravenous injection, subcutaneous injection, inhalation, transdermal administration and the like; the thrombotic diseases include but are not limited to venous thrombosis, arterial thrombosis, ischemic heart disease, and ischemic cerebrovascular disease.

[0024] The low molecular weight sulfated galactan derivative exerts an anticoagulant effect by inhibiting the FIXa-FVIIIa complex in the endogenous coagulation pathway, can significantly prolong the partial prothrombin time (APTT) both in vivo and in vitro, has a weaker effect on the prothrombin time (PT) and the thrombin time (TT), and has no effect on FIB.

[0025] Furthermore, the low molecular weight sulfated galactan derivative has a weaker side effect of causing tail bleeding in mice.

[0026] The present invention also provides an anticoagulant and / or antithrombotic drug, which contains the low molecular weight sulfated galactan derivative or a pharmaceutically acceptable salt thereof.

[0027] Furthermore, the pharmaceutically acceptable salt includes but is not limited to sodium salt, potassium salt or calcium salt.

[0028] Compared with the prior art, the present invention has the following advantages: the low molecular weight sulfated galactan derivative is prepared by degradation, sulfation modification and purification of red algae-derived polysaccharide as raw material, and the anticoagulant activity study shows that the low molecular weight sulfated galactan derivative has significant anticoagulant activity in vitro and in vivo, and has a strong selective inhibitory effect on the FVIIIa-FIXa complex in the endogenous coagulation pathway, and has no significant inhibitory effect on the coagulation factor FXa and thrombin (FIIa) in the common coagulation pathway, and has a low risk of bleeding, and can be used for the prevention and / or treatment of thrombotic related diseases. It has the potential to be developed into an anticoagulant / antithrombotic drug in vitro and in vivo, and has a broad market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 IR spectra of the low molecular weight sulfated galactan κ-CGN-2 and the low molecular weight sulfated galactan derivative S-κ-CGN-4 in Example 2 of the present invention.

[0030] Figure 2 The NMR spectrum of the low molecular weight sulfated galactan derivative S-κ-CGN-4 in Example 2 of the present invention is 1 H-NMR (500MHz) spectrum.

[0031] Figure 3 The NMR spectrum of the low molecular weight sulfated galactan derivative S-κ-CGN-4 in Example 2 of the present invention is 13 C-NMR (125MHz) spectrum.

[0032] Figure 4The effects of the low molecular weight sulfated galactan derivative S-κ-CGN-4 in Example 5 of the present invention on APTT, TT, PT and FIB content in mice; wherein, A: APTT; B: TT; C: PT; D: FIB content, n=8, compared with the blank group, *p<0.05, **p<0.01; compared with the enoxaparin sodium group, #p<0.05, ##p<0.01.

[0033] Figure 5 This is the bleeding risk evaluation of the low molecular weight sulfated galactan derivative S-κ-CGN-4 in Example 6 of the present invention in mice; wherein, n=8, compared with the blank group, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0034] The technical scheme of the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0035] Example 1: Preparation of low molecular weight sulfated galactan derivatives

[0036] 1. Preparation method:

[0037] (1) Degradation: Accurately weigh 0.5 g of galactan sulfate raw material from red algae, add 25 mL of distilled water to dissolve it into a gelatinous state under 80 °C water bath conditions, then add 25 mL of 0.1 mol / L sulfuric acid to make the final concentration of sulfuric acid in the system 0.05 mol / L. Place it in a 60 °C water bath and react for 1.25 h, 1.5 h, 2 h, 2.5 h and 5.5 h, then take it out, cool it to room temperature, add barium carbonate to adjust the pH to 8-9, centrifuge and take the supernatant, perform vacuum concentration and freeze-drying, and low molecular weight galactans with molecular weights of approximately 12 kDa, 10 kDa, 8 kDa, 6 kDa and 2 kDa can be prepared. The samples are recorded as κ-CGN-12 k, κ-CGN-10 k, κ-CGN-8 k, κ-CGN-6 k and κ-CGN-2 k.

[0038] (2) Sulfate derivatives:

[0039] Experiment 1: Weigh 1 g of low molecular weight sulfated galactan (κ-CGN-2 k), dry it thoroughly, place it in a three-necked flask filled with 25 mL of N,N-dimethylformamide (DMF), and stir it in an oil bath at 60 °C for 30 min. According to the molar ratio of sulfur trioxide pyridine complex to the repeating disaccharide unit in low molecular weight galactan of 2:1; 6:1; 8:1 and 10:1, fully dissolve it in 25 mL of DMF to prepare the sulfation reagent. After the preparation is successful, add the sulfation reagent to the three-necked flask and react at 60 °C for 3 h. After the reaction is completed, add 3 times the volume of anhydrous ethanol, stand it overnight, centrifuge and discard the supernatant, redissolve the precipitate with water, and adjust the pH to 8-9 with 2 mol / L NaOH solution. Use a dialysis bag with a molecular weight cutoff of 500 Da to dialyze and desalt until the conductivity of the effluent is less than 3 μs / cm 3 The dialysis was stopped at 4 ℃ and 8 ℃, and the dialyzate was concentrated under reduced pressure and freeze-dried to obtain low molecular weight galactan derivatives. The samples were recorded as S-κ-CGN-1, S-κ-CGN-2, S-κ-CGN-3, and S-κ-CGN-4.

[0040] Experiment 2: Weigh 1 g of low molecular weight sulfated galactan (κ-CGN-12 k, dried overnight at 60 °C) and place it in a three-necked flask filled with 25 mL of DMF and stir it in an oil bath at 60 °C for 30 min. Add a certain amount of sulfur trioxide-pyridine dissolved in 25 mL of DMF (the molar ratio of the sulfation reagent to the repeating disaccharide unit of carrageenan is controlled at 6:1), and react at 30 °C, 50 °C, 60 °C and 80 °C for 3 h. After the reaction, add 3 times the volume of anhydrous ethanol, stand overnight, centrifuge and discard the supernatant, redissolve the precipitate with water, and adjust the pH to 8-9 with 2 mol / L NaOH solution. Use a dialysis bag with a molecular weight cutoff of 500 Da to dialyze and desalt until the conductivity of the effluent is less than 3 μs / cm 3 The dialysis was stopped at 4 ℃ and the dialyzate was concentrated under reduced pressure and freeze-dried to obtain low molecular weight sulfated galactan derivatives. The samples were recorded as S-κ-CGN-6, S-κ-CGN-9, S-κ-CGN-8, and S-κ-CGN-7.

[0041] Experiment 3: Weigh 1 g of low molecular weight sulfated galactan (κ-CGN-6k and κ-CGN-8k) respectively, dry thoroughly, place in a three-necked flask containing 25 mL of N,N-dimethylformamide (DMF), and stir in an oil bath at 60 °C for 30 min. Prepare the sulfation reagent by fully dissolving the sulfur trioxide pyridine complex in 25 mL of DMF according to the molar ratio of 6:1 to the repeating disaccharide unit in the low molecular weight galactan. After the preparation is successful, add the sulfation reagent to the three-necked flask and react at 60 °C for 3 h. After the reaction is completed, add 3 times the volume of anhydrous ethanol, stand overnight, centrifuge and discard the supernatant, redissolve the precipitate with water, and adjust the pH to 8-9 with 2 mol / L NaOH solution. Use a dialysis bag with a molecular weight cutoff of 500 Da to dialyze and desalt until the conductivity of the effluent is less than 3 μs / cm 3 The dialysis was stopped at 4 ℃ and the dialyzate was concentrated under reduced pressure and freeze-dried to obtain low molecular weight galactan derivatives. The samples were named S-κ-CGN-5 and S-κ-CGN-10.

[0042] Example 2: Physical and chemical properties and structural characterization of low molecular weight sulfated galactan derivatives

[0043] 1. Analysis of physical and chemical properties

[0044] The low molecular weight sulfated galactan derivative prepared in Example 1 was measured for relative weight average molecular weight by HPGPC method; the sulfate content of the sample was measured by ion chromatography; and the 3,6-ether-α-D-galactose and β-D-galactose contents of the sample were measured by chemical colorimetry. The relative weight average molecular weight, sulfate, 3,6-ether-α-D-galactose and β-D-galactose contents of the low molecular weight sulfated galactan derivative prepared in the present invention are shown in Table 1.

[0045] The molecular weight of the obtained low molecular weight sulfated galactan derivative is 3-15 kDa; the sulfate content is 15%-50%; the 3,6-endoether-α-D-galactose content is 15%-30%; and the β-D-galactose content is 10-45%.

[0046] Table 1 Physical and chemical properties of a series of oligomeric and low molecular weight sulfated galactan derivatives

[0047]

[0048] 2. Structural characterization of low molecular weight sulfated galactan derivatives

[0049] The structures of low molecular weight sulfated galactan derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy, respectively, taking S-κ-CGN-4 as an example.

[0050] (1) Infrared spectroscopy analysis: Figure 1 As shown in the infrared spectrum of S-κ-CGN-4, the −1 The broad peaks are the stretching vibration peaks of OH; 2954 and 2901 cm −1 The peak at 1061 cm is the stretching vibration peak of CH; −1 The peaks at 1251 and 931 cm are the stretching vibration peaks of CO in COH; −1 They represent the stretching vibration peaks of S=O in -O-SO3 and COC in 3,6-endo-α-D-galactose. In addition, in the fingerprint region 843 cm −1 The peak at 1251 cm is the stretching vibration peak of COS in the sulfate group at the C4 position of β-D-galactose. By comparing with the spectrum of unsulfonated low molecular weight sulfated galactan, it can be found that S-κ-CGN-3 has a peak at 1251 cm −1 The absorption peak intensity at 856 and 817 cm −1 New absorption peaks appeared at , indicating that the sulfate content in S-κ-CGN-4 increased and sulfate substitution occurred at the C2 and C6 positions of 4-sulfate-β-D-galactose.

[0051] Table 2 Infrared spectrum analysis of low molecular weight sulfated galactan derivative S-κ-CGN-4

[0052]

[0053] (2) Nuclear magnetic resonance spectroscopy analysis: low molecular weight sulfated galactan derivative S-κ-CGN-4 1 H-NMR Spectrum and 13 The C-NMR spectra are shown in Figure 2 and Figure 3 As shown, α-(1-3)-4-sulfate-β-D-galactose is represented by G, and β-(1-4)-3,6-endoether-α-D-galactose is represented by DA. 13 The C-NMR spectrum shows that in the sugar ring end group signal region, the anomeric carbon source signal is 95.6 ppm.

[0054] The C signal of DA is at 92ppm, and the terminal group signal after sulfate substitution at C2 of DA is at 103-104ppm. The signals at 103-104ppm are the terminal group carbon signals after sulfate substitution at 4 and 6 of G, and the signals at 101.69ppm are the terminal group carbon signals after sulfate substitution at 2 and 4 of G. For the attribution of NMR spectrum signals, the compound mainly underwent sulfate substitution at C2, C4 and C6 of G, and sulfate substitution at C2 of DA. The specific signal attribution is shown in Table 3. In summary, the sulfate group substitution positions of the low molecular weight sulfated galactan derivative sample S-κ-CGN-4 mainly occurred at C2 and C6 of 4-sulfate-β-D-galactose and C2 of 3,6-ether-α-D-galactose.

[0055] Table 3 Attribution of NMR spectra signals of low molecular weight sulfated galactan derivative S-κ-CGN-4

[0056]

[0057] Example 3: In vitro anticoagulant activity assay of low molecular weight sulfated galactan derivatives

[0058] 1. Experimental methods:

[0059] (1) Prepare solutions of different concentrations of the test sample with physiological saline, take 354 μL of sheep plasma, add 6 μL of the test sample solution of different concentrations, and accurately prepare a series of test plasma of different test sample concentrations. Use a coagulometer and follow the operating procedures of the activated partial thromboplastin time (APTT) kit (Shanghai Sun Biotechnology Co., Ltd.) to measure the APTT, and each sample is repeated three times. Calculate the APTT extension rate of samples with different concentrations, fit the extension rate and different concentrations with a binary linear equation, and calculate the corresponding APTT concentration (EC 2.0 ×APTT and doubled TT concentration (EC 2.0 ×TT). Enoxaparin sodium was used as a positive control.

[0060] (2) Prepare different concentrations of the test sample with physiological saline, take 354 μL of Wistar rat plasma, add 6 μL of the test sample solution of different concentrations, accurately prepare a series of test plasma of different test sample concentrations, use a coagulometer, follow the operating procedures of the activated partial thromboplastin time (APTT) kit and the thrombin time (TT) kit, and perform three replicates for each sample. Calculate the APTT and TT extension rates of samples with different concentrations, fit the extension rates and different concentrations with a binary linear equation, and calculate the corresponding APTT multiple concentration (EC 2.0 ×APTT and doubled TT concentration (EC 2.0 ×TT). Enoxaparin sodium was used as a positive control.

[0061] 2. Experimental results:

[0062] The results of the test are shown in Table 4, which proves that the oligomeric series of low molecular weight galactan sulfate derivatives can prolong the APTT of sheep plasma and have an anticoagulant effect. The anticoagulant activity was further evaluated using rat plasma. As shown in Table 5, the low molecular weight galactan sulfate derivatives can significantly prolong the APTT of rat plasma, but compared with the positive control, it can be found that the low molecular weight galactan sulfate derivatives have a very weak effect on prolonging TT. This shows that it has a weak effect on the common coagulation pathway, a low risk of bleeding, and has the potential to be developed into a safer anticoagulant drug.

[0063] Table 4. In vitro anticoagulant activity test results of different samples in sheep plasma

[0064]

[0065] Note: >160 means that the extension rate is greater than 50% when the concentration is 160 μg / mL; >>160 means that the extension rate is less than 50% when the concentration is 160 μg / mL.

[0066] Table 5. In vitro anticoagulant activity test results of different samples in rat plasma

[0067]

[0068] Note: >160 means that the extension rate is greater than 50% when the concentration is 160 μg / mL; >>160 means that the extension rate is less than 50% when the concentration is 160 μg / mL.

[0069] Example 4: Effects of low molecular weight sulfated galactan derivatives on coagulation factors

[0070] 1. Inhibitory effect of low molecular weight galactan derivatives on FⅡa

[0071] Weigh appropriate amount of S-κ-CGN-4 and S-κ-CGN-10 and prepare a 1 mg / mL stock solution with pure water. Divide into blank control (water) group, positive control (enoxaparin sodium) group, and sample group. Measured using HYPHEN-BioMed FⅡa factor kit. According to the kit instructions, prepare 40 μL of each concentration sample, add 40 μL of diluted ATⅢ solution to start the reaction, incubate in a 37℃ incubator for 2 min, add 40 μL of diluted FⅡa solution, incubate in a 37℃ incubator for 2 min, add 40 μL of diluted chromogenic substrate CS-01, incubate in a 37℃ incubator for 2 min, finally add 80 μL of prepared 20% acetic acid solution to terminate the reaction, and record the OD value at a wavelength of 405 nm. Calculate the IC of sample inhibition of FⅡa 50 value.

[0072] 2. Determination of the inhibitory activity of low molecular weight sulfated galactan derivatives on coagulation factor FⅩa

[0073] Experimental method: Weigh an appropriate amount of S-κ-CGN-4 and S-κ-CGN-10 and prepare a 1 mg / mL stock solution with pure water. Divide into a blank control group (add water), a positive control (enoxaparin sodium) group, and a sample group. The HYPHEN-BioMed FXa factor kit was used for determination. According to the kit instructions, prepare 40 μL of a series of concentration samples, add 40 μL of diluted ATⅢ solution to start the reaction, incubate in a 37 ℃ incubator for 2 min, add 40 μL of diluted FⅩa solution, incubate in a 37 ℃ incubator for 2 min, add 40 μL of diluted chromogenic substrate CS-11, incubate in a 37 ℃ incubator for 2 min, and finally add 80 μL of prepared 20% acetic acid solution to terminate the reaction, and record the OD value at a wavelength of 405 nm. Calculate the IC of the sample to inhibit FⅩa 50 value.

[0074] 3. Determination of the inhibitory activity of low molecular weight sulfated galactan derivatives on endogenous coagulation factor X enzyme (FIXa-FVIIIa complex)

[0075] Weigh appropriate amount of S-κ-CGN-4 and S-κ-CGN-10 and prepare a 1 mg / mL stock solution with pure water. Divide into blank control group (add water), positive control (enoxaparin sodium) group, and sample group. HYPHEN-BioMed FIXa factor kit was used for determination. Add the prepared test solution, 25 μL of FIXa standard solution, and 50 μL of human FⅩ, FⅧ and fibrin polymerization inhibitor solution in sequence to start the reaction, incubate in a 37 ℃ incubator for 2 min, add 50 μL of the prepared activator thrombin-calcium ion-phospholipid solution, incubate in a 37 ℃ incubator for 3 min, add 50 μL of the diluted colorimetric substrate SⅩa-11, incubate in a 37 ℃ incubator for 2 min, and finally add 50 μL of the prepared 20% acetic acid solution to terminate the reaction, and record the OD value at 405 nm. The IC of samples for FⅨa 50 The value is calculated.

[0076] The above experimental results are shown in Table 6. The inhibitory effect of low molecular weight galactan sulfate derivatives on FⅡa and FXa in the common coagulation pathway mediated by ATⅢ is significantly lower than that of enoxaparin sodium, and the effect is extremely weak. The inhibitory effect of low molecular weight galactan sulfate derivatives on FIXa-VIIIa complex in the intrinsic coagulation pathway mediated by ATⅢ is significantly higher than that of enoxaparin sodium group. It shows that its anticoagulant activity mainly exerts its effect by inhibiting the coagulation factor FIXa-FVIIIa complex in the intrinsic coagulation pathway.

[0077] Table 6. Inhibitory effects of low molecular weight sulfated galactan derivatives on coagulation factors

[0078]

[0079] Note: >100 means that when the concentration is 100 μg / mL, the inhibition rate is less than 50%.

[0080] Example 5: Determination of the anticoagulant activity of low molecular weight sulfated galactan derivatives in vivo

[0081] Experimental methods: Male Kunming mice (18-20 g) were randomly divided into 5 groups, namely, normal saline group, enoxaparin sodium group (clinically effective dose 4 mg / kg), S-κ-CGN-4 low-dose group (4 mg / kg), S-κ-CGN-4 medium-dose group (8 mg / kg) and S-κ-CGN-4 high-dose group (16 mg / kg). One hour after subcutaneous injection, blood was collected by removing the eyeballs, and the blood was immediately mixed with 3.8% sodium citrate solution at a ratio of 9:1 (V / V), gently shaken, and centrifuged at 4 ℃, 3000 rpm for 15 min to separate the test plasma. The test plasma was tested for four coagulation indexes (APTT, TT, PT, FIB).

[0082] Experimental results: Figure 4 It was found that when the concentration of low molecular weight galactan sulfate derivatives was 4, 8 and 16 mg / kg, the APTT in mice was significantly prolonged, and the ability to prolong APTT in mice at a concentration of 8 mg / kg was comparable to that of the positive control enoxaparin sodium. At the same experimental efficacy dose, low molecular weight galactan sulfate derivatives and enoxaparin sodium did not significantly affect PT and FIB content in mice. Unlike enoxaparin sodium, low molecular weight galactan sulfate derivatives had no significant effect on TT in mice at doses of 4 mg / kg and 8 mg / kg, and the prolongation of TT was significantly weaker than that of enoxaparin sodium at a dose of 16 mg / kg, indicating that it mainly exerts its anticoagulant effect by acting on the intrinsic coagulation pathway, while its effect on the activity of thrombin (factor IIa) and the conversion of fibrinogen to fibrin in the common coagulation pathway is weak.

[0083] Example 6: Assessment of bleeding risk of low molecular weight sulfated galactan derivatives in mice

[0084] Experimental method: Kunming mice (18-20 g) were divided into normal saline group, enoxaparin sodium group (clinically effective dose 4 mg / kg), enoxaparin sodium group (10 times the effective dose, 40 mg / kg), S-κ-CGN-4 (8 mg / kg) and S-κ-CGN-4 (10 times the effective dose, 80 mg / kg). One hour after subcutaneous injection, the tail tip of the mice was cut off at 5 mm. When blood flowed out, a stopwatch was used to start timing. A blood drop was detected at the tail tip with filter paper every 30 s until no blood flowed out. This period of time was recorded as the bleeding time of the mice.

[0085] Experimental results: Figure 5 It can be seen that low molecular weight galactan sulfate derivatives and enoxaparin sodium did not significantly prolong the tail bleeding time of mice at low doses, but at high doses, the bleeding time caused by low molecular weight galactan sulfate derivatives was significantly lower than that of enoxaparin sodium. This indicates that the risk of bleeding side effects in the low molecular weight galactan sulfate derivative group is lower than that in the enoxaparin sodium group.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A low molecular weight sulfated galactan derivative and a pharmaceutically acceptable salt thereof, characterized in that: The weight average molecular weight of the low molecular weight sulfated galactan derivative is 3-15 kDa, the mass percentage of β-D-galactose is 10%-45%, the mass percentage of 3,6-endoether-α-D-galactose is 15%-30%, and the mass percentage of sulfate groups is 15%-50%. Its structural formula is shown in general formula (I): ， （I） Where R1=H or SO3 - ; R2 = H or SO3 - ; R3 = H or SO3 - ; n represents an integer from 5 to 50.

2. The low molecular weight sulfated galactan derivative according to claim 1, characterized in that: The low molecular weight sulfated galactan derivative comprises at least one of the following repeating structural units: ， Repeating structural unit A Repeating structural unit B ， Repeating structural unit C Repeating structural unit D ， Repeating structural unit E Repeating structural unit F ， Repeating structural unit G.

3. The method for preparing the low molecular weight sulfated galactan derivative according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Degradation: Using sulfated galactan and its derivative polysaccharides from red algae as raw materials, the polysaccharide raw materials are degraded by one or a combination of two or more degradation methods such as microwave degradation, acid degradation, oxidative degradation, Fenton degradation, and enzymatic degradation to prepare low molecular weight galactan containing both β-1,3-D-galactose (D-Gal) residues and α-1,4-D-3,6-endogalactose (DA) residues; (2) Sulfation modification: The low molecular weight galactan is modified with a sulfating agent so that the C2, C4 and C6 hydroxyl groups of the D-Gal sugar residue contain sulfate groups, and the C2 hydroxyl group of the DA sugar residue contains sulfate groups, thereby preparing a crude low molecular weight sulfated galactan derivative; (3) Purification: The crude low molecular weight galactan sulfate derivative is dissolved in pure water and then purified by ultrafiltration or dialysis to obtain a purified low molecular weight galactan sulfate derivative.

4. The preparation method according to claim 3, characterized in that: In step (1) Acid degradation method: Dissolve galactan sulfate in a 0.02-0.2 mol / L dilute acid solution with a mass ratio concentration of 0.5%-10%, heat at 40-70°C for 0.5h-7h, add alkali solution for neutralization, concentrate the solution, desalt and dry to obtain low molecular weight galactan sulfate; Fenton degradation method: prepare galactan sulfate with a mass ratio concentration of 0.5%-10%, adjust the pH value to weak acidity, add an appropriate amount of H2O2 after preheating to a final concentration of 0.1~5%, then add an appropriate amount of FeCl2 to make the final concentration in the solution 0.1~0.5%, heat the reaction at 35-60℃ for 0.5h-6h, concentrate the solution, desalt and dry to obtain low molecular weight galactan sulfate.

5. The preparation method according to claim 3, characterized in that: The sulfating agent is selected from at least one of sulfur trioxide-pyridine, sulfur trioxide-triethylamine, and chlorosulfonic acid-pyridine.

6. The preparation method according to claim 3, characterized in that: The graded purification method is a combination of one or more methods including a gel chromatography column based on the molecular exclusion principle or a nanofiltration membrane or an ultrafiltration membrane or a dialysis bag with a molecular weight cutoff range of 500-1000Da.

7. Use of the low molecular weight sulfated galactan derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of anticoagulant and / or antithrombotic drugs, functional products, and medical devices.

8. The use according to claim 7, characterized in that: The low molecular weight sulfated galactan derivative exerts an anticoagulant effect by acting on the intrinsic coagulation pathway.

9. The use according to claim 7, characterized in that: The drug dosage form is injection, inhalation preparation, aerosol, cream, ointment, gel, patch.

10. An anticoagulant and / or antithrombotic drug, characterized in that: It contains the low molecular weight sulfated galactan derivative according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

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