Preparation method of sulfated boletus nigrum polysaccharide and application of sulfated boletus nigrum polysaccharide in surface modification of medical materials

Abstract

The invention discloses a preparation method of sulfated boletus nigrum polysaccharide and an application of the sulfated boletus nigrum polysaccharide in surface modification of a medical material, and the sulfated boletus nigrum polysaccharide is applied to surface modification of the medical material by adopting a sulfur trioxide pyridine method to modify a polysaccharide molecule with a sulfuric acid group to achieve the purpose of sulfating the polysaccharide. When the sulfated boletus nigrum polysaccharide is subjected to actual biological activity detection, the oxidation resistance, the anticoagulant property, the antibacterial property and the anti-inflammatory property of the sulfated boletus nigrum polysaccharide are improved compared with those of the boletus nigrum polysaccharide before sulfating modification, and after the surface of a medical material is modified, the medical material is more stable.

Description

Technical Field

[0001] The invention relates to the technical field of structural modification of polysaccharides, and in particular to a preparation method of sulfated boletus polysaccharide and application thereof in surface modification of medical materials. Background Art

[0002] Boletus edulis polysaccharide is one of the main active ingredients in Boletus edulis, and has biological activities such as immunomodulatory function, lowering blood lipids, lowering blood sugar and anti-tumor activity. However, the biological activity of Boletus edulis polysaccharide is relatively single.

[0003] In recent years, with the changes in people's living habits and dietary structure, cardiovascular diseases such as coronary heart disease and coronary atherosclerosis have become important factors affecting human health. Metal vascular stent implantation is currently the main method for treating cardiovascular diseases, but when metal stents are implanted in the body, they may cause vascular intima damage, induce intima and smooth muscle cell proliferation, complement activation leading to thrombosis and other problems, which have affected the mid- and long-term treatment of intra-arterial stent implantation. Summary of the invention

[0004] The purpose of the present invention is to provide a sulfated boletus polysaccharide having anti-oxidation, anti-coagulation, anti-bacterial, anti-inflammatory and other biological activities, and an application method thereof in surface modification of medical materials.

[0005] In order to achieve the above technical purpose, the technical solution of the present invention is:

[0006] A method for preparing sulfated boletus polysaccharide comprises the following steps:

[0007] (1) First, the dried brown yellow boletus polysaccharide is dissolved in anhydrous dimethyl sulfoxide to make the brown yellow boletus polysaccharide fully dissolved in the anhydrous dimethyl sulfoxide.

[0008] (2) adding sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution, reacting at a temperature of 50°C to 60°C for 2h to 3h, wherein the ratio of sulfur trioxide-pyridine complex to brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution is 1:15-20.

[0009] (3) After the reaction was completed, the mixture was cooled to room temperature, neutralized with NaOH solution to neutrality, and dialyzed with ultrapure water (Mw cutoff = 3500 Da) for 2 to 3 days.

[0010] (4) The dialyzate is concentrated under reduced pressure at a temperature below 50° C., and then freeze-dried to obtain sulfated boletus polysaccharide.

[0011] An application of sulfated boletus polysaccharide in surface modification of medical materials comprises the following steps:

[0012] (1) Dissolve medical polyhydroxyalkanoate particles in chloroform, and after stirring and dissolving, pour the solution onto a polytetrafluoroethylene plate (D=10 cm), evaporate overnight at room temperature, and after the chloroform solvent evaporates completely, make a polyhydroxyalkanoate membrane (PHA membrane) into a square membrane with a side length of 1.0 cm, mix and stir and wash in a 40-50% ethanol solution, then repeatedly wash with distilled water, and place in a desiccator after vacuum drying for use.

[0013] (2) Add the prepared PHA membrane to an ethylenediamine / isopropanol solution and react at 40°C to 50°C, then rinse the membrane repeatedly with distilled water, and after vacuum drying, soak the amino-treated membrane in an HCl solution for 30 min to 40 min, and then rinse it repeatedly with ultrapure water and dry it in an oven at 45°C to 55°C to obtain an amino-treated polyhydroxyalkanoate membrane with a positive charge on the surface, which is stored in a desiccator for later use.

[0014] (3) Sulfated boletus polysaccharide and ultrapure water are mixed to form a sulfated boletus polysaccharide solution, chitosan is dissolved in 2% acetic acid solution to form a chitosan solution, an amino-polyhydroxyalkanoate membrane with a positive charge on the surface is immersed in the sulfated boletus polysaccharide solution for 10 min to 30 min, the membrane is taken out and gently rinsed with ultrapure water, and then the membrane is immersed in the chitosan solution for 10 min to 30 min, the membrane is taken out and gently rinsed with ultrapure water, and a PHA multilayer membrane with a surface covered with sulfated boletus polysaccharide is obtained.

[0015] When the sulfated brown boletus polysaccharide of the present invention is tested for actual biological activity, the antioxidant, anticoagulant, antibacterial and anti-inflammatory properties of the sulfated brown boletus polysaccharide are improved compared with the brown boletus polysaccharide before sulfate modification. After modifying the surface of the medical material, the medical material is made more stable. DETAILED DESCRIPTION

[0016] Embodiment 1:

[0017] A method for preparing sulfated boletus polysaccharide comprises the following steps:

[0018] (1) First, 300 mg of dried Boletus edulis polysaccharide was dissolved in 90 mL of anhydrous dimethyl sulfoxide and stirred at room temperature for 1 h to allow the Boletus edulis polysaccharide to be fully dissolved in the anhydrous dimethyl sulfoxide.

[0019] (2) adding sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution, and reacting at 50° C. for 2 h; wherein the ratio of sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution is 1:15.

[0020] (3) The mixture was cooled to room temperature, neutralized with 1 mol / L NaOH solution to neutrality, and dialyzed with ultrapure water (Mw cutoff = 3500 Da) for 2 d.

[0021] (4) The dialyzate is concentrated under reduced pressure at a temperature below 50° C., and then freeze-dried to obtain sulfated boletus polysaccharide.

[0022] The application of sulfated boletus polysaccharide in surface modification of medical materials comprises the following steps:

[0023] (1) 3.0 g of medical polyhydroxyalkanoate particles were dissolved in 30 mL of chloroform. After stirring and dissolving, the solution was poured into a polytetrafluoroethylene plate (D = 10 cm) and evaporated overnight at room temperature. After the chloroform solvent was completely evaporated, the polyhydroxyalkanoate membrane (PHA membrane) was made into a square membrane with a side length of 1.0 cm, mixed and stirred in a 40% ethanol solution for 2 h, and then repeatedly washed with distilled water. After vacuum drying, it was placed in a desiccator for use.

[0024] (2) The prepared PHA membrane was added to a 0.09 g / mL ethylenediamine / isopropanol solution, reacted at 40°C for 4 min, and then repeatedly rinsed with distilled water, vacuum dried and stored in a desiccator for later use. The amino-treated membrane was soaked in a 0.012 mol / L HCl solution for 30 min, and then repeatedly rinsed with ultrapure water and dried in an oven at 45°C to obtain an amino-treated polyhydroxyalkanoate membrane with a positive charge on the surface, which was stored in a desiccator for later use.

[0025] (3) Sulfated brown boletus polysaccharide and ultrapure water were mixed to form a 2 mg / mL sulfated brown boletus polysaccharide solution, and chitosan was dissolved in a 2% acetic acid solution to form a 2 mg / mL solution. The PHA membrane was immersed in the sulfated Pseudomonas solution for 10 minutes, and then the membrane was taken out and gently rinsed with ultrapure water; the membrane was immersed in the chitosan solution for 10 minutes, and then the membrane was taken out and gently rinsed with ultrapure water. The membrane was alternately assembled in the two solutions to prepare a multilayer membrane with an amino polyhydroxyalkanoate ester covered with sulfated brown boletus polysaccharide on the surface.

[0026] Embodiment 2:

[0027] A method for preparing sulfated boletus polysaccharide comprises the following steps:

[0028] (1) First, 300 mg of dried Boletus edulis polysaccharide was dissolved in 90 mL of anhydrous dimethyl sulfoxide and stirred at room temperature for 1 h to allow the Boletus edulis polysaccharide to be fully dissolved in the anhydrous dimethyl sulfoxide.

[0029] (2) adding sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution, and reacting at 55° C. for 2.5 hours; wherein the ratio of sulfur trioxide-pyridine complex to brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution is 1:18.

[0030] (3) The mixture was cooled to room temperature, neutralized with 1 mol / L NaOH solution to neutrality, and dialyzed with ultrapure water (Mw cutoff = 3500 Da) for 2.5 d.

[0031] (4) The dialyzate is concentrated under reduced pressure at a temperature below 50° C., and then freeze-dried to obtain sulfated boletus polysaccharide.

[0032] The application of sulfated boletus polysaccharide in surface modification of medical materials comprises the following steps:

[0033] (1) 3.0 g of medical polyhydroxyalkanoate particles were dissolved in 35 mL of chloroform. After stirring and dissolving, the solution was poured onto a polytetrafluoroethylene plate (D=10 cm) and evaporated overnight at room temperature. After the chloroform solvent was completely evaporated, the polyhydroxyalkanoate membrane (PHA membrane) was made into a square membrane with a side length of 1.0 cm, mixed and stirred in a 45% ethanol solution for 2 h, and then repeatedly washed with distilled water. After vacuum drying, it was placed in a desiccator for use.

[0034] (2) The prepared PHA membrane was added to a 0.09 g / mL ethylenediamine / isopropanol solution, reacted at 44°C for 4 min, and then repeatedly rinsed with distilled water, vacuum dried and stored in a desiccator for later use. The amino-treated membrane was soaked in a 0.012 mol / L HCl solution for 35 min, and then repeatedly rinsed with ultrapure water and dried in an oven at 50°C to obtain an amino-treated polyhydroxyalkanoate membrane with a positive charge on the surface, which was stored in a desiccator for later use.

[0035] (3) Sulfated brown boletus polysaccharide and ultrapure water were mixed to form a 2 mg / mL sulfated brown boletus polysaccharide solution, and chitosan was dissolved in a 2% acetic acid solution to form a 2 mg / mL solution. The PHA membrane was immersed in the sulfated Pseudomonas solution for 20 minutes, and then the membrane was taken out and gently rinsed with ultrapure water; the membrane was immersed in the chitosan solution for 20 minutes, and then the membrane was taken out and gently rinsed with ultrapure water. The membrane was alternately assembled in the two solutions to prepare a multilayer membrane with an amino polyhydroxyalkanoate ester covered with sulfated brown boletus polysaccharide on the surface.

[0036] Embodiment 3:

[0037] A method for preparing sulfated boletus polysaccharide comprises the following steps:

[0038] (1) First, 300 mg of dried Boletus edulis polysaccharide was dissolved in 90 mL of anhydrous dimethyl sulfoxide and stirred at room temperature for 1 h to allow the Boletus edulis polysaccharide to be fully dissolved in the anhydrous dimethyl sulfoxide.

[0039] (2) adding sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution, and reacting at 60° C. for 3 h; wherein the ratio of sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution is 1:20.

[0040] (3) The mixture was cooled to room temperature, neutralized with 1 mol / L NaOH solution to neutrality, and dialyzed with ultrapure water (Mw cutoff = 3500 Da) for 3 d.

[0041] (4) The dialyzate is concentrated under reduced pressure at a temperature below 50° C., and then freeze-dried to obtain sulfated boletus polysaccharide.

[0042] The application of sulfated boletus polysaccharide in surface modification of medical materials comprises the following steps:

[0043] (1) 3.0 g of medical polyhydroxyalkanoate particles were dissolved in 40 mL of chloroform. After stirring and dissolving, the solution was poured onto a polytetrafluoroethylene plate (D = 10 cm) and evaporated overnight at room temperature. After the chloroform solvent was completely evaporated, the polyhydroxyalkanoate membrane (PHA membrane) was made into a square membrane with a side length of 1.0 cm, mixed and stirred in a 50% ethanol solution for cleaning for 2 h, and then repeatedly washed with distilled water. After vacuum drying, it was placed in a desiccator for use.

[0044] (2) The prepared PHA membrane was added to a 0.09 g / mL ethylenediamine / isopropanol solution, reacted at 50°C for 4 min, and then the membrane was repeatedly rinsed with distilled water, vacuum dried and stored in a desiccator for later use. The amino-treated membrane was soaked in a 0.012 mol / L HCl solution for 40 min, and then repeatedly rinsed with ultrapure water and dried in an oven at 55°C to obtain an amino-treated polyhydroxyalkanoate membrane with a positive charge on the surface, which was stored in a desiccator for later use.

[0045] (3) Sulfated brown boletus polysaccharide and ultrapure water were mixed to form a 2 mg / mL sulfated brown boletus polysaccharide solution, and chitosan was dissolved in a 2% acetic acid solution to form a 2 mg / mL solution. The PHA membrane was immersed in the sulfated Pseudomonas solution for 30 minutes, and then the membrane was taken out and gently rinsed with ultrapure water; the membrane was immersed in the chitosan solution for 30 minutes, and then the membrane was taken out and gently rinsed with ultrapure water. The membrane was alternately assembled in the two solutions to prepare a multilayer membrane with an amino polyhydroxyalkanoate ester covered with sulfated brown boletus polysaccharide on the surface.

[0046] The antioxidant capacity of the sulfated boletus polysaccharide was reflected by measuring: ABTS free radical scavenging ability, superoxide anion free radical scavenging ability, and hydroxyl free radical scavenging ability.

[0047] Fresh rabbit blood and anticoagulant were prepared to prepare platelet plasma, which was mixed with sulfated boletus polysaccharide solutions of different concentrations, and thrombin was added to measure the coagulation time to determine its anticoagulant ability.

[0048] The antibacterial capacity was determined by the broth microdilution method, and the minimum inhibitory concentration and minimum bactericidal concentration against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were determined.

[0049] The MTT method was used to determine the toxic effects of polysaccharide samples on RAW264.7 cells to determine their anti-inflammatory ability.

[0050] When the sulfated brown boletus polysaccharide of the present invention is tested for actual biological activity, the antioxidant, anticoagulant, antibacterial and anti-inflammatory properties of the sulfated brown boletus polysaccharide are improved compared with the brown boletus polysaccharide before sulfate modification. After modifying the surface of the medical material, the medical material is made more stable.

[0051] The above embodiments do not limit the present invention in any way, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing sulfated boletus polysaccharide, characterized in that: The following steps are involved: (1) dissolving the dried brown yellow boletus polysaccharide in anhydrous dimethyl sulfoxide, so that the brown yellow boletus polysaccharide is fully dissolved in the anhydrous dimethyl sulfoxide; (2) adding sulfur trioxide-pyridine complex to the brown yellow boletus polysaccharide-anhydrous dimethyl sulfoxide solution, and reacting at 50°C to 60°C for 2h to 3h; (3) after the reaction is completed, cooling the mixture to room temperature, neutralizing it with NaOH solution to neutrality, and dialyzing it with ultrapure water for 2d to 3d; (4) concentrating the dialyzate under reduced pressure at a temperature below 50°C, and then freeze-drying it to obtain sulfated brown yellow boletus polysaccharide.

2. The method for preparing sulfated boletus polysaccharide according to claim 1, characterized in that: The ratio of sulfur trioxide-pyridine complex to boletus polysaccharide-anhydrous dimethyl sulfoxide solution is 1:15-20.

3. The use of the sulfated boletus polysaccharide according to claim 1 or 2 in surface modification of medical materials, characterized in that: The following steps are involved: (1) Dissolve the medical polyhydroxyalkanoate particles in chloroform, and after stirring and dissolving, pour the solution into a polytetrafluoroethylene plate and evaporate it overnight at room temperature. After the chloroform solvent is completely evaporated, mix and stir the polyhydroxyalkanoate membrane in a 40-50% ethanol solution, wash it repeatedly with distilled water, and place it in a desiccator after vacuum drying for later use; (2) adding the prepared polyhydroxyalkanoate membrane to an ethylenediamine / isopropanol solution, reacting at 40°C to 50°C, then repeatedly washing the membrane with distilled water, vacuum drying, soaking the amination membrane in an HCl solution for 30min to 40min, then repeatedly washing with ultrapure water, and drying in an oven at 45°C to 55°C to obtain an amination polyhydroxyalkanoate membrane with a positive charge on the surface, which is stored in a desiccator for later use; (3) Sulfated brown boletus polysaccharide and ultrapure water are mixed to form a sulfated brown boletus polysaccharide solution, chitosan is dissolved in 2% acetic acid solution to form a chitosan solution, and an amino polyhydroxyalkanoate membrane with a positive charge on the surface is immersed in the sulfated brown boletus polysaccharide solution for 10 min to 30 min, the membrane is taken out and gently rinsed with ultrapure water, and then the membrane is immersed in the chitosan solution for 10 min to 30 min, the membrane is taken out and gently rinsed with ultrapure water, to obtain a multilayer membrane with an amino polyhydroxyalkanoate covered with sulfated brown boletus polysaccharide on the surface.