Low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres and their preparation method and anticoagulant application

Abstract

Low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres, their preparation method and anticoagulant application belong to the field of development and utilization of marine biological resources. To solve the problem of preparing sea cucumber polysaccharide microspheres with anticoagulant effects and capable of targeting pulmonary thromboembolism sites, the key points are as follows: adding α-amylase and pectinase to the sea cucumber acidic mucopolysaccharide solution, enzymatically catalyzing and degrading the sea cucumber acidic mucopolysaccharide solution under ultrasonic action to obtain an enzymolysis solution; centrifuging and filtering the enzymolysis solution, and then passing it through a ceramic membrane with a molecular weight cut-off of 5000-8000 Da to obtain a low-molecular-weight sea cucumber acidic mucopolysaccharide solution; adding the low-molecular-weight sea cucumber acidic mucopolysaccharide solution with a solution percentage concentration of 15-20% to Antarctic krill oil, stirring evenly, then adding glutaraldehyde for curing, washing, and then performing vacuum drying to obtain low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres. The effect is that they have significant anticoagulant and antithrombotic activities, have an obvious alleviating effect on pulmonary thromboembolism, and have low bleeding side effects.

Description

Technical Field

[0001] The present invention belongs to the field of development and utilization of marine biological resources, and particularly relates to a preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres and their application in the treatment of thrombosis. Background Art

[0002] Thrombus and the vascular embolism caused by it are still one of the main risk factors endangering life and health in modern times. Myocardial infarction, abnormal cardiac blood flow, stroke, lower limb arterial embolism, lower limb arterial thrombosis, lower limb deep vein thrombosis, pulmonary embolism, etc. caused by it are often accompanied by diseases such as atherosclerosis, inflammation, and tumors, posing a great threat to the lives of patients. Among them, pulmonary embolism (PE), as the third leading cause of death in humans after malignant tumors and myocardial infarction, has 500,000 new cases in the United States every year, and its fatality rate is as high as 10%. Although there is no clear epidemiological data in China, pulmonary embolism is usually also complicated in a large number of reports on cardio-pulmonary vascular diseases. The most common embolus in pulmonary embolism is venous thrombus originating from the lower limbs and pelvis, which causes embolism by circulating to the lungs.

[0003] Currently, the main treatments for pulmonary embolism are thrombolysis and anticoagulation. Among them, the commonly used thrombolytic drugs include streptokinase (SK), urokinase (VK), or alteplase (rt-PA). Streptokinase has antigenicity and may cause severe allergic reactions; urokinase not only degrades fibrin in thrombi but also degrades fibrinogen and coagulation factors such as thrombin in the blood, greatly increasing the risk of bleeding; although alteplase has a lower bleeding risk, there are still problems such as low recanalization rate, risk of intracranial hemorrhage, and short half-life requiring continuous intravenous injection. In addition, anticoagulation therapy is very effective and important in combating pulmonary embolism. Traditional anticoagulant drugs, such as unfractionated heparin (UFH) and low molecular weight heparin (LMWH), have high anticoagulant intensity and fast speed and are currently widely used in the clinical treatment of thrombus patients. However, the onset of heparin depends on the expression of antithrombin in plasma, which is prone to bleeding, and there is still no good solution to heparin-induced thrombocytopenia (HIT) and osteoporosis; vitamin K antagonists (warfarin) have a slow onset and simultaneously inhibit two vitamin K-dependent anticoagulant proteins with relatively shorter half-lives, namely protein C (half-life 8h) and protein S (half-life 60h), and a transient hypercoagulable state may occur in the body before effective anticoagulation is achieved. Novel anticoagulant drugs such as rivaroxaban directly inhibit FXa or thrombin without relying on antithrombin in plasma and have a rapid onset, but their complication incidence rate is much higher than expected. The above-mentioned thrombus treatment drugs usually cannot act centrally on the local thromboembolic site, and the dosage and other aspects during use need to be very precisely controlled to avoid massive blood loss caused by the imbalance of the coagulation system and fibrinolytic system in non-thrombus sites. Therefore, developing novel anticoagulant active ingredients with high anticoagulant activity and low bleeding risk and an anticoagulant active ingredient targeted delivery system for pulmonary thromboembolism has great practical significance, which can reduce the risk of systemic bleeding, reduce the dosage of the drugs used, and enhance the treatment effect of pulmonary thromboembolism. There is no relevant content published in the current existing technology. Summary of the Invention

[0004] In order to solve the problem of preparing sea cucumber polysaccharide microspheres with anticoagulant effect and capable of targeting pulmonary thromboembolic sites, in a first aspect, a preparation method of low molecular weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application includes

[0005] S1. Adding α-amylase and pectinase to the sea cucumber acidic mucopolysaccharide solution, and enzymatically catalyzing and degrading the sea cucumber acidic mucopolysaccharide solution under ultrasonic action to obtain an enzymatic hydrolysate;

[0006] S2. Centrifuging and filtering the enzymatic hydrolysate, and then passing it through a ceramic membrane with a molecular weight cut-off of 5000 - 8000 Da to obtain a low molecular weight sea cucumber acidic mucopolysaccharide solution;

[0007] S3. Add the low-molecular-weight sea cucumber acidic mucopolysaccharide solution with a solution percentage concentration of 15-20% to Antarctic krill oil, stir evenly, then add glutaraldehyde for solidification, wash and then perform vacuum drying to obtain low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres.

[0008] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the ratio of α-amylase to pectinase in step S1 is 1-3:1, the enzyme-substrate ratio is 3-4%, the enzymatic hydrolysis temperature is 30-60°C, and the enzymatic hydrolysis time is 3-5 h.

[0009] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the ultrasonic power in step S1 is 150-200 W, and the ultrasonic action time is 60-100 min.

[0010] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the volume ratio of the low-molecular-weight sea cucumber acidic mucopolysaccharide solution to the Antarctic krill oil in step S3 is 4-5:1, and the volume percentage of glutaraldehyde in the mixed solution of the sea cucumber acidic mucopolysaccharide solution and Antarctic krill oil is 0.2-0.5%.

[0011] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the stirring speed in step S3 is 1500-2500 r / min, the reaction temperature is 40-50°C, the stirring time is 60-120 min, and the solidification time is 15-30 min.

[0012] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the washing is performed 2-3 times with isopropanol.

[0013] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the particle size range of the low-molecular-weight sea cucumber polysaccharide microspheres prepared in step S3 is 2-10 microns.

[0014] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the method for preparing the sea cucumber acidic mucopolysaccharide solution in step S1 includes

[0015] S10. Prepare crude sea cucumber polysaccharide;

[0016] S101. Chop, homogenize, enzymatically hydrolyze, and centrifuge and precipitate dried sea cucumbers to obtain a first supernatant;

[0017] S102. Add 95% ethanol to the first supernatant for alcohol precipitation, then centrifuge, wash the precipitate, and perform spray drying to obtain crude sea cucumber polysaccharide.

[0018] S11. Dissolve the crude sea cucumber polysaccharide in an NaCl solution, and then separate and purify it through a DEAE-cellulose anion exchange column to obtain the sea cucumber acidic mucopolysaccharide solution.

[0019] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, preferably, the dried sea cucumber in step S101 is one or any combination of Apostichopus japonicus, Thelenota ananas, Stichopus hermanni, Bolbometopon muricatum, and Holothuria leucospilota;

[0020] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the dried sea cucumber in step S101 is one or a combination of Apostichopus japonicus and Stichopus hermanni;

[0021] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the mass ratio of the compound protease used in the enzymatic hydrolysis in step S101 is alkaline protease: papain: trypsin = 2-5: 1-3: 1-3;

[0022] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the mass ratio of the compound protease to the dried sea cucumber in step S101 is 1: 10-15;

[0023] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the enzymatic hydrolysis reaction temperature in step S101 is 50°C-60°C, the enzymatic hydrolysis pH is 8.0-9.0, and the enzymatic hydrolysis time is 3-6 h;

[0024] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the static temperature for alcohol precipitation in step S102 is 4-8°C, and the static time is 24-48 h;

[0025] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the ethanol used for precipitate washing in step S102 is ethanol with a volume concentration greater than 75%;

[0026] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the concentration of the NaCl solution in step S11 is 2-4 mol / L;

[0027] For the preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to some embodiments of the present application, the DEAE-cellulose anion exchange column in step S11 is gradient eluted with NaCl phosphate buffer solutions of 0.5, 1.0, 1.4, 1.8, and 2.5 mol / L, and the elution fractions of 1.4 and 1.8 mol / L are collected to obtain the sea cucumber acidic mucopolysaccharide solution.

[0028] The low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres targeting the pulmonary thromboembolism site according to some embodiments of the present application in the second aspect are obtained by the preparation method described in any one of the above.

[0029] The application of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres prepared by the preparation method described in any one of some embodiments of the present application in the third aspect in the preparation of anticoagulant devices.

[0030] Advantages of the present invention:

[0031] In the first aspect, the present invention uses an ultrasonic-assisted enzymatic hydrolysis technique to degrade macromolecular sea cucumber polysaccharide. The degradation process and the molecular weight of the degraded molecules are easy to control, the reaction conditions are relatively mild, and a large amount of reaction reagents do not need to be added. Compared with the disadvantages of wide molecular weight distribution and poor homogeneity in chemical degradation, it has been greatly improved.

[0032] In the second aspect, the ultrasonic-assisted enzymatic hydrolysis technique described in the present invention uses a composite enzyme to generate a high-frequency oscillation effect on the solution system under ultrasonic action, greatly increasing the contact frequency between the enzyme and the substrate, improving the degradation effect of sea cucumber polysaccharide, and the sulfate group content in the finally obtained low-molecular-weight sea cucumber polysaccharide has no obvious decrease, retaining the anticoagulant activity to the greatest extent and reducing the risk of bleeding.

[0033] In the third aspect, the present invention uses Antarctic krill oil and low-molecular-weight sea cucumber polysaccharide to prepare oil-water microsphere particles by an emulsification cross-linking method, which has good dispersibility, high particle size uniformity, and the microspheres are round, smooth and regular.

[0034] In the fourth aspect, compared with other tissues and organs, the microsphere particles of the present invention have strong lung targeting. The low-molecular-weight sea cucumber polysaccharide microspheres are mechanically filtered by the pulmonary capillary bed after intravenous injection and concentrated in the pulmonary thromboembolism site, playing a role in reducing platelet aggregation and inhibiting the activities of thrombin, FXa and FXIII, so as to achieve the purpose of relieving pulmonary embolism. The existence of the targeting effect reduces the drug delivery to other non-thrombotic sites. Therefore, it not only improves the remission speed and effect of pulmonary thromboembolism, but also greatly reduces the probability of bleeding in other parts of the body.

[0035] From the above, the sea cucumber polysaccharide microspheres prepared by the preparation method of low-molecular-weight sea cucumber polysaccharide microspheres provided by the present invention have significant anticoagulant and antithrombotic activities, have an obvious relieving effect on pulmonary thromboembolism, and have low bleeding side effects. And the production method is simple and easy to operate, suitable for industrial preparation scenarios, and has broad application prospects in the prevention and treatment of future thrombotic diseases. Brief Description of the Drawings

[0036] Figure 1 Indicates the APTT values of each experimental group. Detailed Embodiments

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only possible technical implementations of the present invention, not all possible implementations. Those skilled in the art can fully combine the embodiments of the present invention to obtain other embodiments without creative labor, and these embodiments are also within the protection scope of the present invention.

[0038] In recent years, acidic mucopolysaccharides from various sea cucumbers have attracted increasing attention from thrombus researchers due to their anticoagulant activity. Compared with mucopolysaccharides from terrestrial vertebrates, sea cucumber polysaccharides have higher repeat sequence regularity and higher sulfate group content, both of which have been verified to have a great relationship with the anticoagulant activity of polysaccharides. A number of studies have shown that sea cucumber polysaccharides act on the endogenous coagulation pathway, inhibit the activation of thrombin and FXa and the activity of FXIII to reduce the production of insoluble fibrin, and reduce platelet aggregation by inhibiting the production of thromboxane TXA 2 and regulating the content of VWF. The inventors found that sea cucumber polysaccharides from different sea cucumber species have different thrombin and FXa inhibitory activities due to the slight differences in molecular weight, the distribution of fucose branches, and the number and position of sulfate group substitutions.

[0039] The inventors further found that lower molecular weight sea cucumber polysaccharides with a molecular weight less than 80 kDa, although having a slightly reduced anticoagulant activity compared to macromolecular sea cucumber polysaccharides when the side chain sulfate ester groups do not change significantly, have a greatly reduced bleeding risk and have great application potential in the future treatment and prevention of thrombotic diseases.

[0040] The preparation method of the low molecular weight sea cucumber polysaccharide lung-targeted microspheres of the present invention comprises the following technological steps:

[0041] Step 1. Chop, homogenize, enzymatically hydrolyze, and centrifuge and precipitate dried sea cucumbers to obtain a first supernatant. Among them, preferably, the dried sea cucumbers in Step 1 are Apostichopus japonicus, Thelenota ananas, Stichopus hermanni, Holothuria scabra, or Holothuria leucospilota, and more preferably, the dried sea cucumbers are Apostichopus japonicus or Stichopus hermanni. Among them, the complex protease used for enzymatic hydrolysis is a complex protease with a mass ratio of alkaline protease: papain: trypsin = 2-5: 1-3: 1-3, and the mass ratio of the complex protease to the input sea cucumber raw material (dried sea cucumbers) is 1: 10-15. Preferably, the enzymatic hydrolysis reaction temperature is 50°C-60°C, the enzymatic hydrolysis pH is 8.0-9.0, and the enzymatic hydrolysis time is 3-6 h.

[0042] Step 2. Add 95% ethanol to the first supernatant for alcohol precipitation, centrifugation, precipitate washing, and spray drying to obtain crude sea cucumber polysaccharides. Among them, the static temperature for alcohol precipitation is 4-8°C, and the static time is 24-48 h. Ethanol with a volume concentration greater than 75% is used for precipitate washing.

[0043] Step 3. First, dissolve the crude sea cucumber polysaccharide with an NaCl solution, and then separate and purify it through a DEAE-cellulose anion exchange column to obtain acidic sea cucumber mucopolysaccharide. Among them, the concentration of the NaCl solution used to dissolve the crude sea cucumber polysaccharide is 2 - 4 mol / L, and NaCl phosphate buffer solutions (0.05 mol / L Na 2 HPO 4 and NaH 2 PO 4 , pH 7.8) with concentrations of 0.5, 1.0, 1.4, 1.8, and 2.5 mol / L are used for gradient elution. Collect the elution fractions at 1.4 and 1.8 mol / L as the acidic sea cucumber mucopolysaccharide solution.

[0044] Step 4. Add α-amylase and pectinase to the acidic sea cucumber mucopolysaccharide solution and carry out enzymatic catalytic degradation under ultrasonic action. After centrifugal filtration, the enzymatic hydrolysate is ultrafiltered and desalted through a ceramic membrane to obtain a low-molecular-weight acidic sea cucumber mucopolysaccharide solution. Among them, the molecular weight cut-off of the ceramic membrane used is 5000 - 8000 Da. The ratio of α-amylase to pectinase used is 1 - 3:1, the enzyme-substrate ratio is 3 - 4%, the enzymatic hydrolysis temperature is 30 - 60 °C, and the enzymatic hydrolysis time is 3 - 5 h. The ultrasonic power used is 150 - 200 W, and the action time is 60 - 100 min.

[0045] Step 5. After adjusting the concentration of the low-molecular-weight acidic sea cucumber mucopolysaccharide solution, slowly add it to the stirring Antarctic krill oil, then add glutaraldehyde for curing. After washing 2 - 3 times with isopropanol, carry out vacuum drying to obtain low-molecular-weight acidic sea cucumber mucopolysaccharide microspheres. Among them, the concentration of the low-molecular-weight acidic sea cucumber mucopolysaccharide solution is adjusted to 15 - 20%, and the concentration is the volume percentage of the mixed solution.

[0046] Among them, the volume ratio of the low-molecular-weight acidic sea cucumber mucopolysaccharide solution to Antarctic krill oil is 4 - 5:1. The stirring speed is 1500 - 2500 r / min, the reaction temperature is 40 - 50 °C, and the emulsification and stirring time is 60 - 120 min. The volume percentage of added glutaraldehyde to the mixed solution is 0.2 - 0.5%, and the curing time is 15 - 30 min. The particle size range of the prepared low-molecular-weight sea cucumber polysaccharide microspheres is 2 - 10 microns.

[0047] The preparation method of the above-mentioned low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres of the present invention obtains sea cucumber polysaccharide through steps such as chopping, enzymatic hydrolysis, ethanol precipitation, and purification by anion exchange column from sea cucumbers. Then, low-molecular-weight sea cucumber polysaccharide is obtained through ultrasonic-assisted enzymatic hydrolysis and ultrafiltration concentration. Finally, low-molecular-weight sea cucumber polysaccharide microspheres with lung targeting are prepared by the emulsion cross-linking method. The prepared sea cucumber polysaccharide microspheres have extremely high lung targeting after intravenous injection, can effectively relieve pulmonary thromboembolism, and at the same time greatly reduce the adverse effects of bleeding in other parts of the body, improving the safety of thrombus treatment, and having very broad application prospects in the prevention and treatment of future thrombotic diseases.

[0048] Example 1: A preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres specifically includes the following steps:

[0049] Step 101: Chop 1 Kg of Apostichopus japonicus, add 10 times the volume of double-distilled water, homogenize, add 10 g each of alkaline protease, papain, and trypsin, adjust the pH to 8.5, and carry out enzymatic hydrolysis at 50 °C for 4 hours.

[0050] Step 102: Add 95% ethanol to the above enzymatic hydrolysate, let it stand at 4 °C for 24 hours, centrifuge at 4000 r / min for 10 min, discard the supernatant, take the precipitate, wash the precipitate once with 90% and 80% ethanol respectively, and dry it in an oven at 50 °C for 4 hours to obtain about 80 g of crude sea cucumber polysaccharide.

[0051] Step 103: After the crude sea cucumber polysaccharide obtained in Step 2 is fully dissolved with 3 mol / L NaCl solution, gradient elution is carried out with 0.5, 1.0, 1.4, 1.8, 2.5 mol / L NaCl phosphate buffer solution, and the elution fractions of 1.4 and 1.8 mol / L are collected as sea cucumber acidic mucopolysaccharide solution. Among them, the pH of the phosphate buffer solution is 7.8, which is prepared from 0.05 mol / L Na 2 HPO 4 and NaH 2 PO 4 preparation.

[0052] Step 104: Add 5 g each of α-amylase and pectinase to the sea cucumber acidic mucopolysaccharide solution obtained in Step 103, adjust the temperature to 40 °C, carry out ultrasonic treatment at a power of 150 W for 60 min, and the enzymatic catalytic degradation time is 4 hours. The obtained enzymatic hydrolysate is subjected to ultrafiltration desalination and concentration treatment through a ceramic membrane with a molecular weight cut-off of 5000 Da to obtain a low-molecular-weight sea cucumber acidic mucopolysaccharide solution.

[0053] Step 105: Adjust the concentration of the low-molecular-weight sea cucumber acidic mucopolysaccharide solution described in Step 104 to 20%, slowly add it to 5 times the volume of Antarctic krill oil, with an emulsification stirring speed of 2500 r / min, a temperature of 50 °C, and an emulsification time of 120 min. After emulsification, add 0.02% glutaraldehyde for curing, and then wash it 3 times with isopropanol and obtain low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres (LMW-SCPM) by vacuum drying.

[0054] Example 2: A preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres, specifically including the following steps:

[0055] Step 101: Chop 1 Kg of Apostichopus japonicus, add 12 times the volume of double-distilled water, homogenize, add 20 g of alkaline protease, 5 g of papain and 5 g of trypsin, adjust the pH to 9, and carry out enzymatic hydrolysis at 60 °C for 6 hours.

[0056] Step 102: Add 95% ethanol to the above enzymatic hydrolysate, let it stand at 6 °C for 36 hours, centrifuge at 3000 r / min for 15 min, discard the supernatant, take the precipitate, wash the precipitate once with 90% and 80% ethanol respectively, and dry it in an oven at 50 °C for 4 hours to obtain about 85 g of crude sea cucumber polysaccharide.

[0057] Step 103: The crude sea cucumber polysaccharide obtained in Step 2 is fully dissolved with 4 mol / L NaCl solution, and then gradient elution is carried out with 0.5, 1.0, 1.4, 1.8, 2.5 mol / L NaCl phosphate buffer solution, and the elution fractions of 1.4 and 1.8 mol / L are collected as sea cucumber acidic mucopolysaccharide solution. Among them, the pH of the phosphate buffer solution is 7.8, which is prepared from 0.05 mol / L Na 2 HPO 4 and NaH 2 PO 4 Preparation.

[0058] Step 104: Add 5 g of α-amylase and pectinase respectively to the sea cucumber acidic mucopolysaccharide solution obtained in Step 103, adjust the temperature to 30 °C, and carry out ultrasonic treatment at a power of 100 W for 60 min, and the enzymatic catalytic degradation time is 5 hours. The obtained enzymatic hydrolysate is ultrafiltered and desalted through a ceramic membrane with a cut-off molecular weight of 6000 Da and concentrated to obtain a low-molecular-weight sea cucumber acidic mucopolysaccharide solution.

[0059] Step 105: Adjust the concentration of the low-molecular-weight sea cucumber acidic mucopolysaccharide solution described in Step 104 to 15%, slowly add it to 5 times the volume of Antarctic krill oil, with an emulsification stirring speed of 2000 r / min, a temperature of 40 °C, and an emulsification time of 120 min. After emulsification, add 0.02% glutaraldehyde for curing, and then wash it 3 times with isopropanol and obtain low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres (LMW-SCPM) through vacuum drying.

[0060] Example 3: A preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres, specifically including the following steps:

[0061] Step 101: Chop 1 Kg of Apostichopus japonicus, add 15 times the volume of double-distilled water, homogenize, add 10 g of alkaline protease, 5 g of papain, and 15 g of trypsin, adjust the pH to 8.0, and carry out enzymatic hydrolysis at 55 °C for 3 hours.

[0062] Step 102: Add 95% ethanol to the above enzymatic hydrolysis solution, let it stand at 4 °C for 24 hours, centrifuge at 4000 r / min for 10 min, discard the supernatant, take the precipitate, wash the precipitate once with 90% ethanol and once with 80% ethanol, and dry it in an oven at 50 °C for 4 hours to obtain about 77 g of crude sea cucumber polysaccharide.

[0063] Step 103: After the crude sea cucumber polysaccharide obtained in Step 2 is fully dissolved with 2 mol / L NaCl solution, gradient elution is carried out with 0.5, 1.0, 1.4, 1.8, 2.5 mol / L NaCl phosphate buffer solution, and the elution fractions of 1.4 and 1.8 mol / L are collected as sea cucumber acidic mucopolysaccharide solution. Among them, the pH of the phosphate buffer solution is 7.8, which is prepared from 0.05 mol / L Na 2 HPO 4 and NaH 2 PO 4 Preparation.

[0064] Step 104: Add 2.5 of α-amylase and 7.5 g of pectinase to the sea cucumber acidic mucopolysaccharide solution obtained in Step 103, adjust the temperature to 60 °C, and carry out ultrasonic treatment at a power of 150 W for 60 min, with an enzymatic catalytic degradation time of 3 hours. The obtained enzymatic hydrolysis solution is ultrafiltered and desalted through a ceramic membrane with a molecular weight cut-off of 8000 Da and concentrated to obtain a low-molecular-weight sea cucumber acidic mucopolysaccharide solution.

[0065] Step 105: Adjust the concentration of the low-molecular-weight sea cucumber acidic mucopolysaccharide solution described in Step 104 to 20%, slowly add it to 4 times the volume of Antarctic krill oil, with an emulsification stirring speed of 2000 r / min, a temperature of 45 °C, and an emulsification time of 90 min. After emulsification, add 0.02% glutaraldehyde for curing, and then wash it 3 times with isopropanol and obtain low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres (LMW-SCPM) through vacuum drying.

[0066] Experimental example:

[0067] Experiment 1: Detection of the molecular weight of degraded sea cucumber polysaccharide

[0068] The molecular weight of the sea cucumber polysaccharide solution obtained after ultrasonic enzymatic hydrolysis was detected. The sample was prepared into a 1 mg / mL solution with a mobile phase (0.1 mol / L K2SO4 solution), and after filtration through a 0.22 μm membrane, it was detected by high-performance gel filtration chromatography. The standard curve was plotted using the relationship between the elution time and the weight-average molecular weight of dextran standards (weight-average molecular weights 6000, 10000, 50000, 150000, 270000 Da), and the weight-average molecular weight of the sample was calculated according to the standard curve. The results are shown in the following table:

[0069] Table 1 Molecular weight of degraded sea cucumber polysaccharide

[0070]

[0071] As can be seen from Table 1, the weight-average molecular weight of the sea cucumber polysaccharide obtained after ultrasonic enzymatic hydrolysis has been greatly reduced compared with the previous crude sea cucumber polysaccharide, and the degradation effect is good.

[0072] Experiment 2: Sulfate group content of sea cucumber polysaccharide microspheres

[0073] The sea cucumber acidic mucopolysaccharide solution and the low-molecular-weight sea cucumber acidic mucopolysaccharide solution obtained in the examples were vacuum dried to obtain sea cucumber acidic mucopolysaccharide powder (SCP) and low-molecular-weight sea cucumber acidic mucopolysaccharide powder (LMW-SCP). 2 mg each of the low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres (LMW-SCPM) was taken and added to an ampoule bottle, 1 mL of 2 mol / L trifluoroacetic acid solution was added, and after reacting at 110 °C for 8 h, it was dried, redissolved with ultrapure water and then dried again. After repeating 3 times, it was detected by ion chromatography, and K 2 SO 4 was used as the standard solution for injection detection, and the standard curve was plotted. The sulfate group content of the sample was calculated according to the standard curve. The results are shown in the following table:

[0074] Table 2 Changes in sulfate group content in sea cucumber polysaccharide

[0075]

[0076] It can be seen from Table 2 that the sulfate content of the low-molecular-weight sea cucumber acidic mucopolysaccharide obtained after ultrasonic-assisted enzymatic degradation and the low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres after microsphere treatment did not show a significant decrease compared with that of the sea cucumber acidic mucopolysaccharide.

[0077] Experiment 3: Antiplatelet aggregation activity of sea cucumber polysaccharide microspheres

[0078] Blood was taken from the abdominal artery of rats. After centrifugation at 1000 r / min for 10 min, the upper suspension was removed to obtain platelet-rich plasma. After the remaining blood was centrifuged at 3000 r / min for 10 min, the supernatant was taken and the platelet count in the platelet-rich plasma was adjusted to about 5×10⁸ / ml. A blank group, an SCP group (10 μg / mL), an LMW-SCP group (10 μg / mL), an LMW-SCPM group (10 μg / mL), and a positive group (standard heparin 4 μg / mL) were set up in the experiment. 100 μL of SCP, LMW-SCP, LMW-SCPM, and standard heparin were added to 1 ml of platelet-rich plasma respectively, and collagen (final concentration 10 μg / mL) and adrenaline (final concentration 3 μg / mL) were added during incubation at 37°C for 5 min to induce platelet aggregation, and the maximum platelet aggregation rate within 5 min was detected. The results are shown in the following table:

[0079] Table 3 Platelet aggregation rates of different experimental groups

[0080]

[0081] The results in Table 3 show that although the antiplatelet aggregation activities of sea cucumber polysaccharide, low-molecular-weight sea cucumber polysaccharide, and low-molecular-weight sea cucumber polysaccharide microspheres are lower than that of standard heparin, they still show sufficient effects in relieving platelet aggregation. In particular, the antiplatelet aggregation activity of the low-molecular-weight sea cucumber polysaccharide after microsphere treatment has been improved to a certain extent.

[0082] Experiment 4: In vitro anticoagulant effect of sea cucumber polysaccharide microspheres

[0083] Blood was taken from the veins of rats and placed in a plastic tube or siliconized glass tube containing 1 / 10 volume of 0.109 mol / L sodium citrate anticoagulant solution, and gently inverted and mixed. After centrifugation at 3000 r / min for 15 minutes, the upper layer liquid was collected and centrifuged at 3000 r / min for 15 min, and the supernatant was taken for a coagulation activity experiment. 90 μL of plasma was added with 10 μL of SCP, LMW-SCP, LMW-SCPM, and the standard heparin of the positive control respectively to make the final concentration reach 4 μg / mL, and incubated at 37°C for 2 min. After adding 25 μl of APTT detection reagent, it was incubated at 37°C for 5 min, and then mixed with 0.025 M CaCl 2 solution, and the clotting time was recorded with an automatic coagulation analyzer. The APTT values of each experimental group are as Figure 1 shown. FromFigure 1 It can be shown that sea cucumber polysaccharide, low molecular weight sea cucumber polysaccharide, and low molecular weight sea cucumber polysaccharide microspheres all exhibited significant APTT prolongation effects and possessed ideal anticoagulant activities.

[0084] Experiment 5: Lung targeting efficiency of low molecular weight sea cucumber polysaccharide microspheres

[0085] Thirty-six rats were randomly divided into 2 groups, with 18 rats in each group. One group was the LMW-SCP injection group, and the other group was the LMW-SCPM group. The rats were fasted for 12 h before administration. LMW-SCP injection and LMW-SCPM were respectively injected into the tail vein at a dose of 4 mg / kg. At 0.5, 2, 4, 8, 12, and 24 h after administration (6 rats at each time point, 3 rats in each group), the rats were decapitated. After draining all the blood, samples of the heart, liver, spleen, lung, and kidney were obtained. Then, 2-fold volume of normal saline was added, and the above samples were made into homogenates using a tissue homogenizer. 200 μL of the tissue homogenate was precisely pipetted into a 1.5 mL centrifuge tube, 600 μL of methanol was added, vortexed for 2 min, centrifuged at 10 000 r / min for 5 min. 500 μL of the supernatant was precisely pipetted, dried under nitrogen, re-dissolved in 200 μL of methanol, vortexed for 1 min, 20 μL was taken for injection, and the content of sea cucumber polysaccharide was determined by HPLC. According to the method of Gupta, the targeting efficiency (Te) was used to evaluate the targeting of LMW-SCPM. Te = (AUC) target / (AUC) non-target. The results are shown in the following table:

[0086] Table 4 AUC and targeting efficiency of LMW-SCP and LMW-SCPM in different tissues

[0087]

[0088] Judging from the fact that a Te value greater than 1 indicates that the pharmaceutical preparation has selectivity for the target organ over the non-target organ, and the greater the Te value, the stronger the selectivity. It can be seen from Table 4 that the low molecular weight sea cucumber acidic mucopolysaccharide microspheres have strong lung tropism.

[0089] Experiment 6: Therapeutic effect of low molecular weight sea cucumber polysaccharide microspheres on rat pulmonary embolism

[0090] 60 mice were randomly divided into 6 groups, namely blank control group, model group, SCP group, LMW-SCP group, LMW-SCPM group and standard heparin group, with 10 mice in each group. Except for the blank control group, the mice in each group were injected with collagen (1.5 mg / kg) and adrenaline (0.5 mg / kg) through the tail vein to construct a mouse pulmonary embolism model. The blank control group mice were injected with an equal volume of normal saline through the tail vein. 2 minutes later, the mice in the SCP group, LMW-SCP group, LMW-SCPM group and standard heparin group were injected with drugs through the tail vein at a dose of 0.3 mg / kg. The survival of mice with acute pulmonary embolism within 15 minutes was observed, the survival time and number of mice with pulmonary embolism were recorded, and the survival rate was calculated. The results are as follows:

[0091] Table 5 Survival rate of mice with acute pulmonary embolism

[0092]

[0093]

[0094] The results in Table 5 show that the effect of low molecular weight sea cucumber acidic mucopolysaccharide treated with microspheroidization on relieving pulmonary thromboembolism in mice is more significant than that of standard heparin and sea cucumber acidic mucopolysaccharide that has not been treated with microspheroidization. The obvious thrombus in the pulmonary capillaries of mice in the model group was greatly improved in the LMW-SCPM group. At the same time, during the experiment, it was found that the bleeding rate of sea cucumber polysaccharide microspheres was also lower than that of the SCP group, and was far lower than that of the standard heparin group.

[0095] The above experimental results show that the sea cucumber polysaccharide microspheres prepared by the preparation method of the low molecular weight sea cucumber acidic mucopolysaccharide microspheres provided by the present invention can not only effectively relieve pulmonary thromboembolism, but also have a good inhibitory effect on overall venous thrombosis, and the probability of systemic bleeding after ingestion is low, and it is a potential high-quality thrombosis prevention and treatment ingredient.

[0096] Finally, it should be noted that the above examples are only some specific embodiments of the present invention, and all derivatives that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres, characterized in that, it includes S1. Adding α-amylase and pectinase to the sea cucumber acidic mucopolysaccharide solution, and enzymatically catalyzing and degrading the sea cucumber acidic mucopolysaccharide solution under ultrasonic action to obtain an enzymatic hydrolysate; S2. Centrifuging and filtering the enzymatic hydrolysate, and then passing it through a ceramic membrane with a molecular weight cut-off of 5000-8000 Da to obtain a low-molecular-weight sea cucumber acidic mucopolysaccharide solution; S3. Adding the low-molecular-weight sea cucumber acidic mucopolysaccharide solution with a volume percentage concentration of 15%-20% to Antarctic krill oil, stirring evenly, then adding glutaraldehyde for curing, washing and then performing vacuum drying to obtain low-molecular-weight sea cucumber acidic mucopolysaccharide microspheres.

2. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 1, characterized in that, the volume ratio of the low-molecular-weight sea cucumber acidic mucopolysaccharide solution to the Antarctic krill oil in step S3 is 4-5:1, and the volume percentage of glutaraldehyde in the mixed solution is 0.2%-0.5%, wherein the mixed solution is a mixed solution of sea cucumber acidic mucopolysaccharide solution and Antarctic krill oil.

3. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 2, characterized in that, the stirring speed in step S3 is 1500-2500 r / min, the reaction temperature is 40-50 °C, the stirring time is 60-120 min, and the curing time is 15-30 min.

4. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 2, characterized in that, the washing is performed by washing with isopropanol 2-3 times.

5. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 2, characterized in that, the particle size range of the low-molecular-weight sea cucumber polysaccharide microspheres prepared in step S3 is 2-10 microns.

6. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to any one of claims 2-5, characterized in that, the method for preparing the sea cucumber acidic mucopolysaccharide solution in step S1 includes S10. Preparing sea cucumber crude polysaccharide; S101. Chopping, homogenizing, enzymatically hydrolyzing, and centrifugally precipitating dried sea cucumbers to obtain a first supernatant; S102. Adding 95% ethanol to the first supernatant for alcohol precipitation, then centrifuging, washing the precipitate, and spray drying to obtain sea cucumber crude polysaccharide; S11. Dissolving the sea cucumber crude polysaccharide with NaCl solution, and then separating and purifying it through a DEAE-cellulose anion exchange column to obtain the sea cucumber acidic mucopolysaccharide solution.

7. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, characterized in that, the dried sea cucumbers in step S101 are one or any combination of Apostichopus japonicus, Thelenota ananas, Stichopus hermanni, Holothuria scabra, and Holothuria leucospilota.

8. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 7, characterized in that, the dried sea cucumbers in step S101 are one or a combination of Apostichopus japonicus and Stichopus hermanni.

9. The preparation method of low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that in the enzymatic hydrolysis in step S101, the mass ratio of the compound protease used is alkaline protease: papain: trypsin = (2-5):(1-3):(1-3).

10. The preparation method of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that the mass ratio of the compound protease in step S101 to the dried sea cucumber is 1:(10-15).

11. The preparation method of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that in the enzymatic hydrolysis reaction in step S101, the temperature is 50°C - 60°C, the pH is 8.0 - 9.0, and the time is 3 - 6 h.

12. The preparation method of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that in the alcohol precipitation in step S102, the static temperature is 4 - 8°C, and the static time is 24 - 48 h.

13. The preparation method of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that the precipitation washing in step S102 is carried out with ethanol having a volume concentration greater than 75%.

14. The preparation method of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that the concentration of the NaCl solution in step S11 is 2 - 4 mol / L.

15. The preparation method of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres according to claim 6, It is characterized in that the DEAE-cellulose anion exchange column in step S11 is eluted with NaCl phosphate buffer solutions of 0.5, 1.0, 1.4, 1.8, 2.5 mol / L in a gradient manner, and the elution fractions of 1.4 and 1.8 mol / L are collected to obtain the sea cucumber acidic mucopolysaccharide solution.

16. A low-molecular-weight sea cucumber polysaccharide lung-targeted microsphere targeting the pulmonary thromboembolic site, It is characterized in that it is obtained by the preparation method described in any one of claims 1 - 15.

17. Use of the low-molecular-weight sea cucumber polysaccharide lung-targeted microspheres prepared by the preparation method described in any one of claims 1 - 15 in the preparation of anticoagulant devices.

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