CN110845640A - Heparinoid sulfonated citric acid modified chitosan and preparation method thereof - Google Patents
Heparinoid sulfonated citric acid modified chitosan and preparation method thereof Download PDFInfo
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical class OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 title claims abstract description 190
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 145
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 229920001499 Heparinoid Polymers 0.000 title claims abstract description 12
- 239000002554 heparinoid Substances 0.000 title claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 38
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- KEQGZUUPPQEDPF-UHFFFAOYSA-N 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione Chemical compound CC1(C)N(Cl)C(=O)N(Cl)C1=O KEQGZUUPPQEDPF-UHFFFAOYSA-N 0.000 claims abstract description 19
- XTHPWXDJESJLNJ-UHFFFAOYSA-N chlorosulfonic acid Substances OS(Cl)(=O)=O XTHPWXDJESJLNJ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 238000006277 sulfonation reaction Methods 0.000 claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 239000008367 deionised water Substances 0.000 claims description 30
- 229910021641 deionized water Inorganic materials 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 30
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 26
- 238000000502 dialysis Methods 0.000 claims description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- 230000007935 neutral effect Effects 0.000 claims description 13
- 239000000706 filtrate Substances 0.000 claims description 12
- 239000003513 alkali Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- 238000007738 vacuum evaporation Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 2
- 230000006196 deacetylation Effects 0.000 claims 1
- 238000003381 deacetylation reaction Methods 0.000 claims 1
- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 abstract description 9
- 230000010100 anticoagulation Effects 0.000 abstract description 8
- 238000005917 acylation reaction Methods 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 abstract 1
- 238000010992 reflux Methods 0.000 abstract 1
- 238000001291 vacuum drying Methods 0.000 abstract 1
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 6
- 229920000669 heparin Polymers 0.000 description 6
- 229960002897 heparin Drugs 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 238000006266 etherification reaction Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000023555 blood coagulation Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000006473 carboxylation reaction Methods 0.000 description 3
- 230000032050 esterification Effects 0.000 description 3
- 238000005886 esterification reaction Methods 0.000 description 3
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 230000010933 acylation Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000003114 blood coagulation factor Substances 0.000 description 2
- 229940019700 blood coagulation factors Drugs 0.000 description 2
- 230000021523 carboxylation Effects 0.000 description 2
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000002628 heparin derivative Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 230000006181 N-acylation Effects 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 125000001557 phthalyl group Chemical group C(=O)(O)C1=C(C(=O)*)C=CC=C1 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
A heparinoid sulfonated citric acid modified chitosan and its preparation method are provided. Firstly, carrying out alkalization treatment on Chitosan (CS) to obtain alkalized chitosan; adding an ethanol solution containing Citric Acid (CA) into the alkalized chitosan, and performing acylation reaction on the Citric Acid (CA) and the alkalized chitosan to obtain citric acid modified chitosan (CACS); then adding a sulfonation reagent prepared from chlorosulfonic acid and formamide into the citric acid modified chitosan to perform sulfonation reaction, controlling the reaction temperature to be 60-70 ℃, and refluxing for 3-6 h; and after the reaction is finished, filtering, washing and vacuum drying at normal temperature to obtain the sulfonated citric acid modified chitosan (SCACS). According to the invention, the sulfonated citric acid modified chitosan with a heparinoid structure is prepared by sulfonating the citric acid modified chitosan, so that the hydrophilicity of the chitosan is effectively improved, and the anticoagulation property and the protein pollution resistance of the chitosan are improved.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to heparin-like substance sulfonated citric acid modified chitosan and a preparation method thereof.
Background
In the anticoagulant mechanism, heparin is used as a natural anticoagulant in a human body, and a main chain structure of the heparin contains a sulfonic acid group with strong negative charges, so that the heparin can inhibit the formation of prothrombin and interfere the activation of thrombin on blood coagulation factors, has good inhibition effect on both internal and external blood coagulation paths, and can block the progress of blood vessel blockage and thrombosis caused by blood coagulation. However, heparin is apt to cause thrombocytopenia in clinical application, thereby exacerbating the condition of patients. The current research is mainly focused on the modification of polysaccharide molecules such as chitosan, chondroitin, hyaluronic acid and the like with similar molecular structures with heparin. The chitosan is a natural alkaline polysaccharide, and has the advantages of good antibacterial property and biocompatibility, degradability and the like. Meanwhile, the molecular chain of the chitosan contains a large number of active groups such as hydroxyl, amino and the like, so that active site modification is provided for further modification of the chitosan, and the chitosan is a potential ideal raw material for synthesizing heparinoid. However, chitosan has a hemostatic function, and when contacting blood, a blood coagulation phenomenon is easily formed, so that the application of chitosan in the field of biomedicine is limited to a great extent. The invention aims to improve the hydrophilicity and the anticoagulation performance of chitosan, designs modified chitosan with good biocompatibility and provides a preparation method of heparin-like substance sulfonated citric acid modified chitosan.
At present, chemical modification of chitosan mainly introduces specific functional groups by methods of etherification, carboxylation, esterification, N-acylation and the like. The hydroxyethyl ether chitosan with higher solubility under neutral condition is prepared by etherification reaction (chitosan modification and antibacterial property research of textiles finished by the chitosan [ J ]. functional polymer science, 2002,15(2):194-198.) and the like, and the hydrophilicity and the antibacterial property of the hydroxyethyl ether chitosan are greatly improved. Tianing Liu et al (materials science and Engineering C,2017,79:570-580.) introduce a large amount of hydroxyl groups and sulfonic groups into the chitosan structure by etherification modification and sulfonation at the C6-OH position of chitosan, thus improving the hydrophilicity and anticoagulation of chitosan. Research on antibacterial performance of carboxymethyl chitosan-based composite antibacterial agents with different particle sizes [ J ] modern biomedical progress 2006,6(7):1-4.) carboxymethyl chitosan is prepared through a carboxylation reaction, and water solubility of chitosan and antibacterial performance of part of bacteria are improved. Baumann et al (Carbohydrate Research,2001,331(1):43-57.) modified chitosan by esterification to prepare a series of chitosan derivatives, and investigated the effect of the sulfonated substitution site of chitosan on the anticoagulant activity, and found that the prepared sulfonated chitosan had slightly lower anticoagulant properties than heparin. The synthesis [ J ] of water-soluble N- (2-carboxybenzoyl) chitosan, chemical research and application, 2004,16(1): 8-10.) of Wangzhouyu and the like modifies chitosan by maleic anhydride in a homogeneous system taking acetic acid and acetone as media to obtain a water-soluble N-acylated chitosan derivative, confirms the principle of preferential acylation at amino position and provides theoretical basis for acylation reaction sites of chitosan.
Most of the above methods for modifying chitosan introduce hydroxyl, carboxyl, sulfonic group and the like through etherification, carboxylation, esterification, acylation and the like to improve the anticoagulation performance of chitosan, but have the disadvantages of insignificant anticoagulation improvement, harsh reaction conditions, long reaction time and the like. If citric acid and sulfonic acid groups with a multi-carboxyl structure are simultaneously introduced to the chitosan structure, the synergistic effect between different hydrophilic groups can be further utilized, so that the hydrophilicity and the anticoagulation property of the modified chitosan are further increased.
Therefore, the invention designs and prepares the sulfonated citric acid modified chitosan with a heparinoid structure by performing molecular design on the modified chitosan and simultaneously accessing citric acid and sulfonic acid groups with a multi-carboxyl structure on the chitosan structure, and further improves the hydrophilicity and biocompatibility of the chitosan through the synergistic effect of the citric acid structure, the sulfonic acid groups and the hydroxyl groups. The invention has the advantages of simple preparation process and mild and controllable reaction conditions.
Disclosure of Invention
The invention aims to design a heparinoid sulfonated citric acid modified chitosan, which has a chemical structural formula as follows:
in the structure, n is 300-500.
The invention also aims to provide a preparation method of the heparinoid sulfonated citric acid modified chitosan, which comprises the following steps:
(1) alkalization treatment of chitosan
Respectively adding Chitosan (CS) and a NaOH solution with the mass fraction of 30-35% into a container at normal temperature, uniformly stirring, freezing in a refrigerator for 2-10 days, thawing, and filtering to remove alkali liquor to obtain the alkalized chitosan; wherein the mass ratio of the chitosan to the NaOH solution is 1: 3-10;
(2) preparation of citric acid modified chitosan
At normal temperature, dissolving Citric Acid (CA) in absolute ethyl alcohol, adding alkalized chitosan, uniformly stirring, and reacting at 55-65 ℃ for 2-5 hours, wherein the mass ratio of the alkalized chitosan to the citric acid to the absolute ethyl alcohol is 1: 1-3: 10-30; after the reaction is finished, adding deionized water, and uniformly stirring, wherein the mass ratio of the deionized water to the absolute ethyl alcohol is 1-3: 1; standing for layering, taking supernatant, performing vacuum evaporation at 40-60 ℃ to remove ethanol, cooling, and adjusting the pH to be neutral by using dilute hydrochloric acid; and then adding acetone into the neutral solution, wherein the mass ratio of the acetone to the deionized water is (0.5-1.5): 1, generating white precipitate, filtering, washing, and drying in vacuum for 12-24 h at normal temperature to obtain citric acid modified chitosan (CACS);
(3) sulfonation of citric acid modified chitosan
Slowly adding chlorosulfonic acid into formamide at the temperature of 1-5 ℃ to prepare a sulfonation reagent, wherein the mass ratio of chlorosulfonic acid to formamide is 1: 8-15; and then adding the citric acid modified chitosan and a sulfonation reagent into a reactor respectively, heating to 60-70 ℃, and reacting for 2-6 h, wherein the mass ratio of the citric acid modified chitosan to chlorosulfonic acid is 1: 2-5; and after the reaction is finished, adding deionized water for dilution, and then filtering, wherein the mass ratio of the citric acid modified chitosan to the deionized water is 1: 50-80 parts; putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis at normal temperature for 20-24 h, and adjusting the pH to 12-14 by using a NaOH solution with the concentration of 1-5 mol/L; standing for 20-40 min, adjusting the pH to 3-5 by using a hydrochloric acid solution with the concentration of 1-5 mol/L, then putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis for 15-30 h at normal temperature, and evaporating at normal temperature in vacuum to obtain the sulfonated citric acid modified chitosan (SCACS).
The chemical reaction in the preparation process is as follows:
CS+CA→CACS (1)
CACS+HSO3Cl→SCACS
wherein CACS represents citric acid modified chitosan, and the chemical structural formula is as follows:
SCACS represents sulfonated citric acid modified chitosan, and the chemical structural formula is as follows:
in the structure, n is 300-500.
According to the invention, sulfonated citric acid modified chitosan is synthesized by adopting a two-step method, hydrophilic carboxyl and sulfonic group are introduced into the structure of chitosan, so that the hydrophilicity of chitosan is improved to a great extent, and the solubility of chitosan before modification is less than 0.01g/100g of water; after modification, the solubility increased gradually. The solubility of the citric acid modified chitosan is 6-10 g/100g of water, and the solubility of the sulfonated citric acid modified chitosan is 12-20 g/100g of water. The blood compatibility of the modified chitosan is also improved, the hemolysis rate is reduced to 1.3% from 3.5% before modification, the deformation degree and aggregation quantity of platelets are both obviously reduced, no thrombosis is generated, and the results show that the modified chitosan obviously improves the hydrophilicity and the anticoagulation property.
The invention has the following advantages:
(1) hydrophilic carboxyl and sulfonic acid group which can interact with blood coagulation factors are simultaneously connected into the chitosan structure, so that the hydrophilicity and anticoagulation of the chitosan are improved;
(2) the preparation process is simple, and the reaction conditions are mild and controllable.
Drawings
FIG. 1 is a flow chart of the preparation process of the heparinoid sulfonated citric acid modified chitosan of the present invention.
Detailed Description
The invention is further described below with reference to the following figures and examples:
example 1
The preparation process of the invention is shown in figure 1. Respectively adding Chitosan (CS) and a NaOH solution with the mass fraction of 30% into a container at normal temperature, uniformly stirring, freezing in a refrigerator for 10 days, thawing, and filtering to remove alkali liquor to obtain the alkalized chitosan; wherein the mass ratio of the chitosan to the NaOH solution is 1: 5.
At normal temperature, dissolving Citric Acid (CA) in absolute ethyl alcohol, then adding alkalized chitosan, uniformly stirring, and then placing at 55 ℃ for reaction for 3h, wherein the mass ratio of the alkalized chitosan to the citric acid to the absolute ethyl alcohol is 1: 2: 12; and after the reaction is finished, adding deionized water, and uniformly stirring, wherein the mass ratio of the deionized water to the absolute ethyl alcohol is 2: 1; standing for layering, taking supernatant, performing vacuum evaporation at 50 ℃ to remove ethanol, cooling, and adjusting pH to neutral with dilute hydrochloric acid; and then adding acetone into the neutral solution, wherein the mass ratio of acetone to deionized water is 1: 1, generating white precipitate, filtering, washing, and drying in vacuum at normal temperature for 24h to obtain the citric acid modified chitosan (CACS).
Slowly adding chlorosulfonic acid into formamide at 5 ℃ to prepare a sulfonation reagent, wherein the mass ratio of chlorosulfonic acid to formamide is 1: 10; and then adding the citric acid modified chitosan and a sulfonation reagent into a reactor respectively, heating to 65 ℃, and reacting for 4 hours, wherein the mass ratio of the citric acid modified chitosan to the chlorosulfonic acid is 1: 3; and after the reaction is finished, adding deionized water for dilution, and then filtering, wherein the mass ratio of the citric acid modified chitosan to the deionized water is 1: 70; putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis at normal temperature for 24h, and adjusting the pH to 12 by using a NaOH solution with the concentration of 3 mol/L; standing for 40min, adjusting pH to 3 with 3mol/L hydrochloric acid solution, dialyzing the filtrate in dialysis bag with molecular weight cutoff of 7000Da at room temperature for 24 hr, and vacuum evaporating at room temperature to obtain sulfonated citric acid modified chitosan (SCACS).
Example 2
Respectively adding Chitosan (CS) and a NaOH solution with the mass fraction of 30% into a container at normal temperature, uniformly stirring, freezing in a refrigerator for 6 days, thawing, and filtering to remove alkali liquor to obtain the alkalized chitosan; wherein the mass ratio of the chitosan to the NaOH solution is 1: 6.
at normal temperature, dissolving Citric Acid (CA) in absolute ethyl alcohol, then adding alkalized chitosan, uniformly stirring, and then placing at 55 ℃ for reaction for 3h, wherein the mass ratio of the alkalized chitosan to the citric acid to the absolute ethyl alcohol is 1: 2: 15; and after the reaction is finished, adding deionized water, and uniformly stirring, wherein the mass ratio of the deionized water to the absolute ethyl alcohol is 2: 1; standing for layering, taking supernatant, performing vacuum evaporation at 40 ℃ to remove ethanol, cooling, and adjusting pH to neutral with dilute hydrochloric acid; and then adding acetone into the neutral solution, wherein the mass ratio of acetone to deionized water is 0.5: 1, generating white precipitate, filtering, washing, and drying in vacuum at normal temperature for 14h to obtain the citric acid modified chitosan (CACS).
Slowly adding chlorosulfonic acid into formamide at 4 ℃ to prepare a sulfonation reagent, wherein the mass ratio of chlorosulfonic acid to formamide is 1: 8; and then adding the citric acid modified chitosan and a sulfonation reagent into a reactor respectively, heating to 60 ℃, and reacting for 4 hours, wherein the mass ratio of the citric acid modified chitosan to the chlorosulfonic acid is 1: 3; and after the reaction is finished, adding deionized water for dilution, and then filtering, wherein the mass ratio of the citric acid modified chitosan to the deionized water is 1: 50; putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis at normal temperature for 20h, and adjusting the pH to 12 by using a NaOH solution with the concentration of 2 mol/L; standing for 20min, adjusting pH to 5 with 3mol/L hydrochloric acid solution, dialyzing the filtrate in dialysis bag with molecular weight cutoff of 7000Da at room temperature for 18h, and vacuum evaporating at room temperature to obtain sulfonated citric acid modified chitosan (SCACS).
Example 3
Respectively adding Chitosan (CS) and 35% NaOH solution into a container at normal temperature, uniformly stirring, freezing in a refrigerator for 5 days, thawing, and filtering to remove alkali liquor to obtain the alkalized chitosan; wherein the mass ratio of the chitosan to the NaOH solution is 1: 3.
at normal temperature, dissolving Citric Acid (CA) in absolute ethyl alcohol, then adding alkalized chitosan, uniformly stirring, and then placing at 60 ℃ for reaction for 3h, wherein the mass ratio of the alkalized chitosan to the citric acid to the absolute ethyl alcohol is 1: 2: 16; and after the reaction is finished, adding deionized water, and uniformly stirring, wherein the mass ratio of the deionized water to the absolute ethyl alcohol is 3: 1; standing for layering, taking supernatant, performing vacuum evaporation at 45 ℃ to remove ethanol, cooling, and adjusting pH to neutral with dilute hydrochloric acid; and then adding acetone into the neutral solution, wherein the mass ratio of acetone to deionized water is 1.5: 1, generating white precipitate, filtering, washing, and drying in vacuum at normal temperature for 14h to obtain the citric acid modified chitosan (CACS).
Slowly adding chlorosulfonic acid into formamide at 4 ℃ to prepare a sulfonation reagent, wherein the mass ratio of chlorosulfonic acid to formamide is 1: 10; and then adding the citric acid modified chitosan and a sulfonation reagent into a reactor respectively, heating to 65 ℃, and reacting for 4 hours, wherein the mass ratio of the citric acid modified chitosan to the chlorosulfonic acid is 1: 2; and after the reaction is finished, adding deionized water for dilution, and then filtering, wherein the mass ratio of the citric acid modified chitosan to the deionized water is 1: 60, adding a solvent to the mixture; putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis at normal temperature for 22h, and adjusting the pH to 13 by using a NaOH solution with the concentration of 3 mol/L; standing for 25min, adjusting pH to 5 with 3mol/L hydrochloric acid solution, dialyzing the filtrate in dialysis bag with molecular weight cutoff of 7000Da at room temperature for 18h, and vacuum evaporating at room temperature to obtain sulfonated citric acid modified chitosan (SCACS).
Example 4
Respectively adding Chitosan (CS) and 33% NaOH solution into a container at normal temperature, uniformly stirring, freezing in a refrigerator for 10 days, thawing, and filtering to remove alkali liquor to obtain the alkalized chitosan; wherein the mass ratio of the chitosan to the NaOH solution is 1: 10.
at normal temperature, dissolving Citric Acid (CA) in absolute ethyl alcohol, then adding alkalized chitosan, uniformly stirring, and then placing at 65 ℃ for reaction for 5 hours, wherein the mass ratio of the alkalized chitosan to the citric acid to the absolute ethyl alcohol is 1: 3: 25; and after the reaction is finished, adding deionized water, and uniformly stirring, wherein the mass ratio of the deionized water to the absolute ethyl alcohol is 2: 1; standing for layering, taking supernatant, performing vacuum evaporation at 50 ℃ to remove ethanol, cooling, and adjusting pH to neutral with dilute hydrochloric acid; and then adding acetone into the neutral solution, wherein the mass ratio of acetone to deionized water is 1.5: 1, generating white precipitate, filtering, washing, and drying in vacuum at normal temperature for 24h to obtain the citric acid modified chitosan (CACS).
Slowly adding chlorosulfonic acid into formamide at the temperature of 2 ℃ to prepare a sulfonation reagent, wherein the mass ratio of chlorosulfonic acid to formamide is 1: 12; and then adding the citric acid modified chitosan and a sulfonation reagent into a reactor respectively, heating to 70 ℃, and reacting for 6 hours, wherein the mass ratio of the citric acid modified chitosan to the chlorosulfonic acid is 1: 5; and after the reaction is finished, adding deionized water for dilution, and then filtering, wherein the mass ratio of the citric acid modified chitosan to the deionized water is 1: 70; putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis at normal temperature for 24h, and adjusting the pH to 12 by using a NaOH solution with the concentration of 4 mol/L; standing for 30min, adjusting pH to 5 with 2mol/L hydrochloric acid solution, dialyzing the filtrate in dialysis bag with molecular weight cutoff of 7000Da at room temperature for 0h, and vacuum evaporating at room temperature to obtain sulfonated citric acid modified chitosan (SCACS).
Claims (3)
1. A heparinoid sulfonated citric acid modified chitosan is characterized in that: the chitosan structure is grafted with citric acid and sulfonic acid groups, and the chemical structural formula is as follows:
in the structure, n is 300-500.
2. A method for preparing the heparinoid sulfonated citric acid modified chitosan according to claim 1, comprising the steps of:
(1) alkalization treatment of chitosan
Respectively adding chitosan and 30-35% of NaOH solution into a container at normal temperature, wherein the mass ratio of the chitosan to the NaOH solution is 1: 3-10; after uniformly stirring, placing the mixture in a refrigerator for freezing for 2-10 days, thawing, and filtering to remove alkali liquor to obtain the alkalized chitosan;
(2) preparation of citric acid modified chitosan
Dissolving citric acid in absolute ethyl alcohol at normal temperature, adding alkalized chitosan, uniformly stirring, and reacting at 55-65 ℃ for 2-5 hours, wherein the mass ratio of the alkalized chitosan to the citric acid to the absolute ethyl alcohol is 1: 1-3: 10-30; after the reaction is finished, adding deionized water, and uniformly stirring, wherein the mass ratio of the deionized water to the absolute ethyl alcohol is 1-3: 1; standing for layering, taking supernatant, performing vacuum evaporation at 40-60 ℃ to remove ethanol, cooling, and adjusting the pH to be neutral by using dilute hydrochloric acid; and then adding acetone into the neutral solution, wherein the mass ratio of the acetone to the deionized water is 0.5-1.5: 1, generating white precipitate, filtering, washing, and drying in vacuum for 12-24 hours at normal temperature to obtain citric acid modified chitosan;
(3) sulfonation of citric acid modified chitosan
Slowly adding chlorosulfonic acid into formamide at the temperature of 1-5 ℃ to prepare a sulfonation reagent, wherein the mass ratio of chlorosulfonic acid to formamide is 1: 8-15; and then adding the citric acid modified chitosan and a sulfonation reagent into a reactor respectively, heating to 60-70 ℃, and reacting for 2-6 h, wherein the mass ratio of the citric acid modified chitosan to chlorosulfonic acid is 1: 2-5; and after the reaction is finished, adding deionized water for dilution, and then filtering, wherein the mass ratio of the citric acid modified chitosan to the deionized water is 1: 50-80 parts; putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for normal-temperature dialysis for 20-24 h, and adjusting the pH to 12-14 by using a NaOH solution with the concentration of 1-5 mol/L; standing for 20-40 min, adjusting the pH to 3-5 by using a hydrochloric acid solution with the concentration of 1-5 mol/L, then putting the filtrate into a dialysis bag with the molecular weight cutoff of 7000Da for dialysis for 15-30 h at normal temperature, and evaporating at normal temperature in vacuum to obtain the sulfonated citric acid modified chitosan.
3. The method for preparing the heparinoid substance sulfonated citric acid modified chitosan according to claim 2, wherein in the step (1), the viscosity of the chitosan is 100-200 mPa-s, and the deacetylation degree is not less than 95%.
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