CN110218267B - Preparation method and application of iron dextran - Google Patents
Preparation method and application of iron dextran Download PDFInfo
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Abstract
The invention provides a preparation method and application of iron dextranBelongs to the technical field of glucose polymers, and the preparation method comprises the following steps: preparing superfine molecular dextran by using dextran enzyme to perform enzymolysis on medium molecular dextran; with TEMPO/CuBr2Oxidizing the superfine molecular dextran by a NaClO oxidation reaction system to prepare an oxidized dextran solution; and (3) dripping a ferric trichloride solution and an alkali solution into the oxidized dextran solution at the same time, performing a complex reaction to prepare a dextran iron complex solution, and purifying to obtain the dextran iron. The invention also provides the application of the iron dextran in iron supplement preparations. The preparation method of the invention is easy to obtain the ultra-micro molecular dextran with stable quality, small distribution width index and narrow molecular weight distribution range, the preparation method has high selectivity of oxidation reaction, less side reaction and good oxidation effect, and the obtained oxidized dextran has high carboxyl content, good water solubility and strong complexing ability to heavy metal ions.
Description
Technical Field
The invention belongs to the technical field of glucose polymers, and particularly relates to a preparation method and application of iron dextran.
Background
Dextran (dextran) is a high molecular polymer formed by polymerizing a plurality of glucoses, also called dextran, and products with different molecular weights are generated due to different numbers of polymerized glucose molecules, and comprise high molecular dextran (weight average molecular weight 100-200kDa), medium molecular dextran (weight average molecular weight 60-80kDa), low molecular dextran (weight average molecular weight 20-40kDa), small molecular dextran (weight average molecular weight 10-20kDa), micro molecular dextran (weight average molecular weight 7-10kDa) and ultra micro molecular dextran (weight average molecular weight 3-6kDa), dextran iron can be obtained by complexing dextran with iron, dextran iron injection is a well recognized piglet iron supplement injection at home and abroad at present, dextran iron injection with the iron content of 150 mg. m L-1 is injected by one-time intramuscular injection in 1m L within 1-4 d after the birth of a piglet, the dextran iron supplement injection can meet the iron requirement of the whole lactation period of the piglet, the piglet iron anemia is prevented, the piglet death rate is promoted, but the mortality rate of the piglet is reduced, but the dextran iron loss of the currently produced dextran injection is more than the dextran iron loss of the dextran injection, the dextran iron loss of the dextran injection is greatly influenced by the iron loss of the newly prepared by the iron injection, the iron loss of the dextran injection at home and the iron loss of the dextran injection, the dextran injection is the dextran injection, the dextran iron loss of the dextran injection is the important degradation of the dextran injection is the most of the dextran injection is the important degradation of the dextran iron loss of the dextran injection before the dextran of the dextran injection, the dextran injection is the dextran of the dextran injection, the dextran of the dextran injection is the dextran of the.
The invention patent with the grant publication number of CN 104031170B discloses a dextran iron raw material for human intravenous injection and a preparation method thereof, wherein the dextran iron for human intravenous injection is prepared by subjecting dextran to hydrolysis, ultrafiltration or gel column, oxidation, complexation, ultrafiltration refinement, alcohol precipitation or spray drying to obtain the dextran iron raw material which has the weight average molecular weight of 139000-157000 and the molecular weight distribution of less than 2.0 and can meet the requirement of human intravenous injection. The invention patent with the publication number of CN 104829745B discloses a method for preparing iron dextran, which adjusts the pH value of the prepared iron dextran after preparing the complexing solution, so that the dissolving performance of the iron dextran is greatly improved, and the iron content in the prepared iron dextran can reach more than 40 percent; the solubility in cold water can reach more than 4g/100g of cold water; and the content of Na +, Cl-and the like is greatly reduced, and the purity of the product is higher. The invention patent with the publication number of CN 104829745B discloses a new method for generating iron dextran, which mainly comprises four steps: 1) hydrolyzing with hydrochloric acid at high temperature to convert dextran into required low molecular dextran; 2) oxidizing by adopting a method of hydrogen peroxide step-by-step oxidation, and keeping a certain temperature to obtain dextran with a proper structure during complexation; 3) the method of acid-base simultaneous uniform dripping is adopted to prepare iron dextran by controlling temperature and speed, and the product is more stable by high-temperature aging; 4) the final product is subjected to filtration and ultrafiltration desalination concentration by a ceramic membrane.
Disclosure of Invention
The invention aims to provide a preparation method of iron dextran, which is easy to obtain ultra-micro molecular dextran with stable quality, small distribution width index and narrow molecular weight distribution range.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a preparation method of iron dextran comprises the following steps:
s1: preparing superfine molecular dextran by using dextran enzyme to perform enzymolysis on medium molecular dextran;
s2: with TEMPO/CuBr2Oxidizing the superfine molecular dextran by a NaClO oxidation reaction system to prepare an oxidized dextran solution;
s3: and (3) dripping a ferric trichloride solution and an alkali solution into the oxidized dextran solution at the same time, performing a complex reaction to prepare a dextran iron complex solution, and purifying to obtain the dextran iron.
The most important ring in the preparation process of the iron dextran is the preparation of oxidized dextran, namely, dextran molecules must be activated in advance before being complexed with iron salts, namely, terminal hydroxyl groups must be oxidized into carboxyl groups. The quality of the oxidized dextran preparation directly influences the complexation degree of the oxidized dextran and ferric salt, thereby influencing the yield of iron, in particular to the preparation of the oxidized superfine molecular dextran, which has higher requirements than the preparation of the oxidized superfine molecular dextran and the small molecular dextran. In the oxidation reaction system, TEMPO passes through CuBr2After oxidation, the nitrogen carbonyl cation is converted into corresponding nitrogen carbonyl cation and TEMPOH, the nitrogen carbonyl cation becomes an oxidant with strong oxidizing property, C6 primary hydroxyl of the polysaccharide can be quickly oxidized into aldehyde group, other hydroxyl is not affected, and CuBr2Reduced to monovalent copper, and TEMPOH and monovalent copper can be re-oxidized to divalent copper, and then catalytic oxidation cycle is carried out; simultaneously, NaClO and CuBr2The NaBrO generated in the reaction can further oxidize aldehyde groups into carboxyl groups. The oxidation reaction system has the characteristics of high selectivity, mild reaction conditions, low degradation rate, simplicity, high efficiency, energy conservation, environmental protection and the like, and is specifically represented as: the method can selectively oxidize primary hydroxyl on a molecular chain C6 of the ultra-micro dextran into carboxyl under catalysis, does not basically affect secondary hydroxyl on C2 and C3, and can effectively reduce side reaction in the oxidation process; the invention can improve the content of carboxyl in oxidized dextran by using an oxidation reaction system, greatly improve the complexing ability of the oxidized dextran on heavy metal ions, and greatly reduce the number of hydroxyl in the ultra-micro dextran, while the residual hydroxyl is not enough to form a crystal structure with high ordered intermolecular and intramolecular sequences, the occurrence of oxidation can reduce the proportion of a crystalline region, so that the polycrystalline structure is converted to amorphous, the crystal structure is destroyed, the permeation of water molecules is easy, the water solubility of the oxidized dextran is improved, and the carboxyl serving as a hydrophilic group can also improve the water solubility of the oxidized dextran.
Preferably, in S1, the concentration of molecular dextran in the enzymolysis system is 20-50 g/L, and the concentration of dextranase is 60-80U/g substrate.
Preferably, in S1, the enzymolysis temperature is 40-60 deg.C, and the time is 10-20 min.
More preferably, in S1, the medium molecular dextran is pretreated by triethanolamine/sodium p-toluenesulfonate/water system before enzymolysis. The pretreatment can not only destroy the crystal structure of the medium molecular dextran, reduce the strongest binding force among molecules, increase the distance among molecules, make the dextranase more easily enter the interior of the low and medium molecular dextran, and improve the accessibility of an enzymolysis reaction system to the medium molecular dextran; and moreover, more hydroxyl groups can be exposed from the medium molecular dextran, the solubility of the medium molecular dextran in an enzymolysis reaction system is enhanced, the accessibility of the medium molecular dextran in the enzymolysis reaction system, the enzymolysis reaction activity and the reaction uniformity are improved, the enzymolysis reaction is easier to be carried out, and the ultra-micro molecular dextran with stable quality, small distribution width index and narrow molecular weight distribution range is easy to obtain. The pretreatment can also ensure that the crystallinity of the obtained superfine molecular dextran is low, so that the subsequent oxidation is easier to perform, and the oxidation effect of the superfine molecular dextran is improved.
Preferably, in S2, TEMPO is used in an amount of 1g of the ultrafine molecular dextran2-5mmol，CuBr2The dosage of the NaClO is 0.2-0.5mmol, and the dosage of the NaClO is 1-3 mmol. TEMPO, CuBr2And reasonable amount of NaClO, so that the time required by oxidation is short, the generation amount of carboxyl is large, the degradation of oxidized dextran is small, the carboxyl content on the molecular chain of the obtained oxidized dextran is high, and the weight loss rate is low.
Preferably, in S2, the temperature of the oxidation reaction is 40-60 ℃, and the time of the oxidation reaction is 1-3 h.
Preferably, in S3, the alkali solution is sodium hydroxide solution, and the weight ratio of dextran to ferric trichloride to sodium hydroxide is 1:2.2-2.6: 1.2-1.8.
The invention also discloses iron dextran prepared by the preparation method.
Preferably, the weight average molecular weight of the iron dextran is 3000-6000Da, the polydispersity is 1.0-1.5, and the iron content is 38-42 wt%. Can meet the requirements of iron dextran raw materials for injection. The polydispersity is more preferably in the range 1.2 to 1.4, this narrow range of polydispersity not only ensures the uniformity of the molecular weight, but the inventors have also found that it helps to ensure and ensure the uniformity in the release of iron and to exclude the presence of unstable iron.
The invention also discloses application of the iron dextran in iron supplement preparations. The iron supplement preparation is used for preventing or treating iron deficiency anemia in animal or human subjects, and provides a high-quality product for preventing iron deficiency anemia.
Compared with the prior art, the invention has the beneficial effects that: the preparation method can selectively oxidize primary hydroxyl on a molecular chain C6 of the superfine dextran into carboxyl by an oxidation reaction system, reduces side reaction in the oxidation process, improves the content of carboxyl in the oxidized dextran, improves the water solubility of the oxidized dextran, and has strong complexing ability on heavy metal ions; the preparation method can improve the accessibility of the molecular dextran in an enzymolysis reaction system, the enzymolysis reaction activity and the reaction uniformity by pretreatment, is easy to obtain the ultra-micro molecular dextran with stable quality, small distribution width index and narrow molecular weight distribution range, and can also ensure that the obtained ultra-micro molecular dextran has low crystallinity, so that the subsequent oxidation is easier to perform, and the oxidation effect of the ultra-micro molecular dextran is improved; the weight average molecular weight of the iron dextran prepared by the preparation method is 3000-6000Da, the polydispersity is 1.0-1.5, and the iron content is 30-40 wt%, so that the requirement of the iron dextran raw material for injection can be met.
The invention adopts the technical scheme to provide the preparation method and the application of the iron dextran, makes up the defects of the prior art, and has reasonable design and convenient operation.
Drawings
FIG. 1 is a Fourier transform infrared spectrum of oxidized dextran in test example 1 of the present invention;
FIG. 2 shows the content of carboxyl groups in oxidized dextran in test example 1 according to the present invention;
FIG. 3 shows the results of the determination of the water solubility of oxidized dextran in test example 1 of the present invention.
Detailed Description
Exemplary embodiments that embody features and advantages of the invention are described in detail below. It is to be understood that the invention is capable of other and different embodiments and its several details are capable of modification without departing from the scope of the invention, and that the description is intended to be illustrative in nature and not to be construed as limiting the invention.
An embodiment of the invention provides a preparation method of iron dextran, which comprises the following steps:
s1: preparing superfine molecular dextran by using dextran enzyme to perform enzymolysis on medium molecular dextran;
s2: with TEMPO/CuBr2Oxidizing the superfine molecular dextran by a NaClO oxidation reaction system to prepare an oxidized dextran solution;
s3: and (3) dripping a ferric trichloride solution and an alkali solution into the oxidized dextran solution at the same time, performing a complex reaction to prepare a dextran iron complex solution, and purifying to obtain the dextran iron.
The most important ring in the preparation process of the iron dextran is the preparation of oxidized dextran, namely, dextran molecules must be activated before being complexed with iron salt, namelyThe terminal hydroxyl group must be oxidized to a carboxyl group. The quality of the oxidized dextran preparation directly influences the complexation degree of the oxidized dextran and ferric salt, thereby influencing the yield of iron, in particular to the preparation of the oxidized superfine molecular dextran, which has higher requirements than the preparation of the oxidized superfine molecular dextran and the small molecular dextran. In the oxidation reaction system, TEMPO passes through CuBr2After oxidation, the nitrogen carbonyl cation is converted into corresponding nitrogen carbonyl cation and TEMPOH, the nitrogen carbonyl cation becomes an oxidant with strong oxidizing property, C6 primary hydroxyl of the polysaccharide can be quickly oxidized into aldehyde group, other hydroxyl is not affected, and CuBr2Reduced to monovalent copper, and TEMPOH and monovalent copper can be re-oxidized to divalent copper, and then catalytic oxidation cycle is carried out; simultaneously, NaClO and CuBr2The NaBrO generated in the reaction can further oxidize aldehyde groups into carboxyl groups. The oxidation reaction system has the characteristics of high selectivity, mild reaction conditions, low degradation rate, simplicity, high efficiency, energy conservation, environmental protection and the like, and is specifically represented as follows: the method can selectively oxidize primary hydroxyl on a molecular chain C6 of the ultra-micro dextran into carboxyl under catalysis, does not basically affect secondary hydroxyl on C2 and C3, and can effectively reduce side reaction in the oxidation process; the invention can improve the content of carboxyl in oxidized dextran by using an oxidation reaction system, greatly improve the complexing ability of the oxidized dextran on heavy metal ions, and greatly reduce the number of hydroxyl in the ultra-micro dextran, while the residual hydroxyl is not enough to form a crystal structure with high ordered intermolecular and intramolecular sequences, the occurrence of oxidation can reduce the proportion of a crystalline region, so that the polycrystalline structure is converted to amorphous, the crystal structure is destroyed, the permeation of water molecules is easy, the water solubility of the oxidized dextran is improved, and the carboxyl serving as a hydrophilic group can also improve the water solubility of the oxidized dextran.
In one embodiment of the present invention, the preparation method comprises the following steps:
s1: adding medium molecular dextran into water, stirring and heating until the dextran is completely dissolved, cooling, adding dextranase, reacting at constant temperature, heating to 80-90 ℃ to inactivate the dextranase, and then adopting ultrafiltration equipment to intercept or gel column separation to respectively remove the dextran with molecular weight more than 6000Da and less than 3000Da so as to obtain dextran solution with absolute molecular weight of 3000-6000Da, namely the ultra-micro molecular dextran solution;
s2: adding TEMPO and CuBr into a special micro-molecular dextran solution with the mass concentration of 20-30%2And NaClO, after the oxidation is finished, the pH value of the system is adjusted in the oxidation process to keep the pH value of the system at 9.0-10.0;
s3: dripping ferric trichloride solution with mass concentration of 40-50% and alkali solution with mass concentration of 30-40% into oxidized dextran solution simultaneously, controlling the complexing temperature of the system at 40-60 ℃, pH at 3.0-5.0, carrying out complexing reaction for 3-4h, adjusting the final pH value of the product to 8.0-10.0 by using sodium hydroxide, starting heating and keeping at 95-105 ℃, continuously reacting for 1-1.5h, standing for 24h, centrifuging the obtained reaction solution at 3000-6000rpm to remove solid impurities, and filtering out metal and impurity ions by an ultrafiltration membrane with the molecular weight cutoff range of 3000-6000Da to remove metal and impurity ions, after detecting that the iron content and various indexes are qualified, directly diluting or concentrating and blending into iron dextran injection solutions with different proportions, and also carrying out freeze drying or spray drying to obtain the iron dextran.
In one embodiment of the present invention, in S1, the concentration of molecular dextran in the enzymolysis system is 20-50 g/L, such as 22 g/L, 25 g/L, 30 g/L, 35.4 g/L, 40 g/L, 44 g/L, 47 g/L, etc., and the concentration of dextranase is 60-80U/g substrate, such as 62U/g, 66U/g, 70.2U/g, 75.4U/g, 76U/g, 79U/g, etc.
In one embodiment of the present invention, in S1, the enzymolysis temperature is 40-60 ℃, e.g., 42 ℃, 43 ℃, 45 ℃, 48 ℃, 52 ℃, 54 ℃, 56 ℃, 57 ℃, etc., and the time is 10-20min, e.g., 11min, 12min, 13min, 14min, 15.5min, 16min, 17min, 18min, 19min, etc.
In an embodiment of the present invention, in S1, the medium molecular dextran is pretreated by triethanolamine/sodium p-toluenesulfonate/water system before enzymolysis. The pretreatment can not only destroy the crystal structure of the medium molecular dextran, reduce the strongest binding force among molecules, increase the distance among molecules, make the dextranase more easily enter the interior of the low and medium molecular dextran, and improve the accessibility of an enzymolysis reaction system to the medium molecular dextran; and moreover, more hydroxyl groups can be exposed from the medium molecular dextran, the solubility of the medium molecular dextran in an enzymolysis reaction system is enhanced, the accessibility of the medium molecular dextran in the enzymolysis reaction system, the enzymolysis reaction activity and the reaction uniformity are improved, the enzymolysis reaction is easier to be carried out, and the ultra-micro molecular dextran with stable quality, small distribution width index and narrow molecular weight distribution range is easy to obtain. The pretreatment can also ensure that the crystallinity of the obtained superfine molecular dextran is low, so that the subsequent oxidation is easier to perform, and the oxidation effect of the superfine molecular dextran is improved. The weight ratio of triethanolamine to sodium p-toluenesulfonate to water in the triethanolamine/sodium p-toluenesulfonate/water system is 5-10:3-8: 100. The pretreatment process comprises the following steps: adding medium molecular dextran into triethanolamine/sodium p-toluenesulfonate/water system at a solid-to-liquid ratio of 1:10-20, stirring, standing for 6-12h, washing with water to neutral, and drying.
In one embodiment of the present invention, in S2, the amount of TEMPO used per 1g of the ultrafine dextran is 2-5mmol, such as 2.4mmol, 2.6mmol, 3.0mmol, 3.5mmol, 4.1mmol, 4.4mmol, 4.7mmol, etc., CuBr2The amount of (B) is 0.2 to 0.5mmol, for example, 0.24mmol, 0.26mmol, 0.31mmol, 0.33mmol, 0.37mmol, 0.39mmol, 0.43mmol, 0.48mmol, etc., and the amount of NaClO is 1 to 3mmol, for example, 1.2mmol, 1.5mmol, 1.9mmol, 2.1mmol, 2.5mmol, 2.6mmol, 2.8mmol, 2.9mmol, etc. TEMPO, CuBr2And reasonable amount of NaClO, so that the time required by oxidation is short, the generation amount of carboxyl is large, the degradation of oxidized dextran is small, the carboxyl content on the molecular chain of the obtained oxidized dextran is high, and the weight loss rate is low.
In one embodiment of the present invention, in S2, the temperature of the oxidation reaction is 40-60 deg.C, such as 42 deg.C, 43 deg.C, 45 deg.C, 48 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 57 deg.C, etc., and the time of the oxidation reaction is 1-3h, such as 1.5h, 2.0h, 2.5h, etc.
In an embodiment of the invention, in S3, the alkali solution is a sodium hydroxide solution, and the weight ratio of dextran to ferric chloride to sodium hydroxide is 1:2.2-2.6:1.2-1.8, such as 1:2.2:1.3, 1:2.2:1.4, 1:2.2:1.5, 1:2.2:1.6, 1:2.2:1.7, 1:2.3:1.3, 1:2.3:1.4, 1:2.3:1.5, 1:2.3:1.6, 1:2.3:1.7, 1:2.4:1.3, 1:2.4:1.4, 1:2.4:1.5, 1:2.4:1.6, 1:2.4:1.7, 1:2.5:1.3, 1:2.5: 1.5: 1.6, 1:2.5: 1.6: 1.4: 1.7, 1:2.5:1.3, 1:2.5: 1.5: 1.6: 1.5: 1.6, 1.5: 1.6: 1.5: 1.6, 1.6: 1.5: 1.6: 1.5: 1.
An embodiment of the invention provides iron dextran prepared by the preparation method. The weight-average molecular weight of the iron dextran is 3000-6000Da, the polydispersity is 1.0-1.5, and the iron content is 38-42 wt%. Can meet the requirements of iron dextran raw materials for injection. The polydispersity is more preferably in the range 1.2 to 1.4, this narrow range of polydispersity not only ensures the uniformity of the molecular weight, but the inventors have also found that it helps to ensure and ensure the uniformity in the release of iron and to exclude the presence of unstable iron.
An embodiment of the invention provides application of the iron dextran in an iron supplement preparation. The iron supplement preparation is used for preventing or treating iron deficiency anemia in animal or human subjects, and provides a high-quality product for preventing iron deficiency anemia.
The preparation method and the use of iron dextran according to an embodiment of the present invention will be further described with reference to specific examples.
Example 1:
a preparation method of iron dextran comprises the following steps:
s1, adding medium molecular dextran into water to enable the concentration of the molecular dextran to be 20 g/L, stirring and heating until the dextran is completely dissolved, cooling, adding dextranase with the concentration of 60U/g substrate, reacting at a constant temperature of 80 ℃ for 10min, heating to 80 ℃ to inactivate the dextranase, intercepting by ultrafiltration equipment or separating by a gel column, and removing the dextranase with molecular weights of more than 6000Da and less than 3000Da respectively to obtain dextran solution with the absolute molecular weight of 3000 plus 6000Da, namely the ultra-micro molecular dextran solution;
s2: adding TEMPO and CuBr into a special micro-molecular dextran solution with the mass concentration of 20%2And NaClO, the amount of TEMPO is 2mmol and CuBr per 1g of superfine dextran2The dosage of the sodium chloride is 0.2mmol, the dosage of the NaClO is 1mmol, the oxidation reaction is finished for 1h at the temperature of 40 ℃ to prepare oxidized dextran solution, and the pH value of the system is adjusted in the oxidation process to keep the pH value of the system at 9.0-10.0;
s3: dropping ferric trichloride solution with mass concentration of 40% and sodium hydroxide solution with mass concentration of 30% into oxidized dextran solution at the same time, wherein the weight ratio of dextran to ferric trichloride to sodium hydroxide is 1:2.2:1.2, controlling the complexing temperature of the system at 40 ℃ and the pH value at 3.0 in the dropping process, carrying out complexing reaction for 3h, adjusting the final pH value of the product to 8.0 by using sodium hydroxide, starting to heat up to 95 ℃, continuously reacting for 1h, standing for 24h, centrifuging the obtained reaction solution at 3000rpm to remove solid impurities, and filtering out metal and impurity ions by an ultrafiltration membrane with the molecular weight cutoff range of 3000Da and 6000Da, and directly diluting or concentrating and blending the obtained reaction solution into iron injection solutions with different proportions after detecting that the iron content and various indexes are qualified, or carrying out freeze drying or spray drying to obtain the iron dextran. The weight average molecular weight of the iron dextran is 5428Da, the polydispersity is 1.42, and the iron content is 38.9 wt%.
Example 2:
a preparation method of iron dextran comprises the following steps:
s1, adding medium molecular dextran into water to enable the concentration of the molecular dextran to be 50 g/L, stirring and heating until the dextran is completely dissolved, cooling, adding dextranase with the concentration of 80U/g substrate, reacting at a constant temperature of 90 ℃ for 20min, heating to 90 ℃ to inactivate the dextranase, intercepting by adopting ultrafiltration equipment or separating by using a gel column, and respectively removing the dextranase with molecular weights of more than 6000Da and less than 3000Da to obtain a dextran solution with the absolute molecular weight of 3000 plus 6000Da, namely an extra-micro molecular dextran solution;
s2: adding TEMPO and CuBr into 30% ultra-micro molecular dextran solution2And NaClO, the amount of TEMPO is 5mmol and CuBr per 1g of superfine dextran2The dosage of the sodium chloride is 0.5mmol, the dosage of NaClO is 3mmol, the oxidation reaction is finished for 3 hours at the temperature of 60 ℃ to prepare oxidized dextran solution, and the oxidation reaction is carried out on the dextran solutionAdjusting the pH value of the system to maintain the pH value of the system at 9.0-10.0;
s3: dropping a ferric trichloride solution with mass concentration of 50% and a sodium hydroxide solution with mass concentration of 40% into an oxidized dextran solution at the same time, wherein the weight ratio of dextran to ferric trichloride to sodium hydroxide is 1:2.6:1.8, controlling the complexing temperature of the system at 60 ℃, the pH value at 5.0 and carrying out complexing reaction for 4 hours in the dropping process, adjusting the final pH value of the product to 10.0 by using sodium hydroxide, starting to heat and keep at 105 ℃, continuously reacting for 1.5 hours, standing for 24 hours, centrifuging the obtained reaction solution at 6000rpm to remove solid impurities, carrying out ultrafiltration on the ultrafiltration membrane with the intercepted molecular weight range of 3000 and 6000Da to remove metal and impurity ions, detecting the iron content and various indexes, directly diluting or blending and concentrating the reaction solution into iron dextran injection solutions with different proportions, and carrying out freeze drying or spray drying to obtain the iron dextran. The weight average molecular weight of the iron dextran is 5526Da, the polydispersity is 1.37, and the iron content is 39.7 wt%.
Example 3:
a preparation method of iron dextran comprises the following steps:
s1, adding medium molecular dextran into water to enable the concentration of the molecular dextran to be 35 g/L, stirring and heating until the dextran is completely dissolved, cooling, adding dextranase with the concentration of 70U/g substrate, reacting at a constant temperature of 85 ℃ for 16min, heating to 85 ℃ to inactivate the dextranase, intercepting by ultrafiltration equipment or separating by a gel column, and removing the dextrans with molecular weights of more than 6000Da and less than 3000Da respectively to obtain a dextran solution with an absolute molecular weight of 3000 plus 6000Da, namely an extra-micro molecular dextran solution;
s2: adding TEMPO and CuBr into a superfine molecular dextran solution with the mass concentration of 26%2And NaClO, the amount of TEMPO is 4mmol and CuBr per 1g of superfine dextran2The dosage of the sodium chloride is 0.3mmol, the dosage of the NaClO is 2mmol, the oxidation reaction is finished for 2 hours at the temperature of 50 ℃ to prepare oxidized dextran solution, and the pH value of the system is adjusted in the oxidation process to keep the pH value of the system at 9.0-10.0;
s3: dropping 45% ferric trichloride solution and 32% sodium hydroxide solution into oxidized dextran solution simultaneously, wherein the weight ratio of dextran to ferric trichloride to sodium hydroxide is 1:2.5:1.5, the complexing temperature of the system is controlled at 50 ℃, the pH value is 4.0 during dropping, the final pH value of the product is adjusted to 90.0 by sodium hydroxide, the temperature is raised to 100 ℃, the reaction is continued for 1h, then the reaction solution is kept still for 24h, the obtained reaction solution is centrifuged at 5000rpm to remove solid impurities, and metal and impurity ions are ultra-filtered by an ultrafiltration membrane with the molecular weight cutoff range of 3000 and 6000Da, after detecting that the iron content and various indexes are qualified, the dextran solution can be directly diluted or concentrated and prepared into iron injection solutions with different proportions, and can also be freeze-dried or spray-dried to obtain the dextran iron. The weight average molecular weight of the iron dextran is 4852Da, the polydispersity is 1.24, and the iron content is 41.2 wt%.
Example 4:
the difference from example 3 is that: pretreatment is carried out before enzymolysis of medium-molecule dextran as follows: adding medium molecular dextran into triethanolamine/sodium p-toluenesulfonate/water system with solid-to-liquid ratio of 1:10-20 and weight ratio of 5-10:3-8:100, stirring, standing for 6-12h, washing with water to neutral, and drying. The weight average molecular weight of the iron dextran is 4822Da, the polydispersity is 1.14, and the iron content is 42.0 wt%.
Compared with example 3, the dextran iron obtained in example 4 has smaller polydispersity and higher iron content, which indicates that the pretreatment in example 4 can improve the accessibility of the middle molecular dextran in an enzymolysis reaction system, the enzymolysis reaction activity and the reaction uniformity, and is easy for the enzymolysis reaction to be carried out, so as to obtain the ultra-micro molecular dextran with stable quality, small distribution width index and narrow molecular weight distribution range. And the pretreatment can also ensure that the crystallinity of the obtained superfine molecular dextran is low, so that the subsequent oxidation is easier to perform, and the oxidation effect of the superfine molecular dextran is improved.
Comparative example 1:
the difference from example 3 is that: in S2, the oxidation reaction system is CuBr2/NaClO。
Comparative example 2:
the difference from example 3 is that: in S2, the oxidation reaction system is TEMPO/NaClO.
Comparative example 3:
the difference from example 3 is that: in S2, the oxidation reaction system is TEMPO/CuBr2。
Test example 1:
1. analysis of carboxyl groups in oxidized dextran
1.1 Fourier transform Infrared Spectroscopy
Determining carboxyl groups in oxidized dextran by Fourier transform infrared spectroscopy, pressing into a sheet for detection by taking KBr as a solid diluent, and specifically comprising the following steps: firstly, precisely weighing 1mg of samples of dry dextran and oxidized dextran in a mortar respectively, uniformly grinding under an infrared lamp, precisely weighing 100mg of dry KBr, adding the dry KBr (the ratio of the KBr to the samples is 100:1), grinding for about 30min, and completely mixing the dry dextran and the oxidized dextran; and then taking a proper amount of sample to be detected, tabletting in a mould for 30s, and preparing into a thin sheet to be detected. The background was previously scanned with a blank KBr sample, which was then scanned with a fourier transform infrared spectrometer. The result is shown in figure 1, and in the infrared spectrum of the ultra-micro molecular dextran, 3450cm-1The broader peak is hydrogen bond O-H stretching vibration peak between dextran molecules, 2915cm-1Is saturated C-H stretching vibration peak at 1164cm on sugar ring-1Is the C-O absorption peak on the sugar ring, 1025cm-1The existence of α -1, 6-glycosidic bond in the dextran long chain can be proved by the peak in the figure, all the peaks are 3397cm-1The broader peak at (a) was calibrated for the standard. The Fourier transform infrared spectrogram of the oxidized product is quite similar to the original dextran, but the oxidized dextran is 1615cm-1An absorption peak which is not found in the original figure appears at a wavelength around the wavelength, and the absorption peak is an absorption peak of a carbonyl group in a carboxyl group, and the generation of the carboxyl group is confirmed by the peak, and 1164cm-1Sugar ring C-O absorption peak and 1025cm-1The peak at (A) still exists, which also indicates that the product after oxidation still retains a large amount of α -D-glucopyranose units and still has a certain degree of polymerization, and example 3 shows a spectrum of 1615cm-1The peak intensity of the nearby absorption peak is obviously stronger than that of comparative examples 1-3, namely the oxidation degree of example 3 is better than that of comparative examples 1-3, which shows that the oxidation reaction system can selectively catalyze and oxidize primary hydroxyl on a special micro-molecular dextran molecular chain C6 into carboxyl, secondary hydroxyl on C2 and C3 is not affected basically, and side reaction in the oxidation process can be effectively reduced; the invention can improve the carboxyl content in the oxidized dextran by using an oxidation reaction system, greatly improves the complexing capability of the oxidized dextran on heavy metal ions, and greatly reduces the number of hydroxyl in the ultra-micro molecular dextran. Example 4 spectrum 1615cm-1The peak intensity of the nearby absorption peak is obviously stronger than that of the embodiment 3, that is, the oxidation degree of the embodiment 4 is better than that of the embodiment 3, which shows that the pretreatment of the embodiment 4 can also enable the medium molecular dextran to expose more hydroxyl groups, and the obtained superfine molecular dextran has low crystallinity, so that the subsequent oxidation is easier to perform, and the oxidation effect of the superfine molecular dextran is improved.
1.2 carboxyl group content in oxidized dextran
The carboxyl content of oxidized dextran with low molecular mass is determined by a titration method, 5g of oxidized dextran is precisely weighed, the oxidized dextran is dissolved for about 30min in hydrochloric acid solution containing 0.1 mol/L in 30m L under the condition of stirring, a dialysis bag is used for dialysis for 16h, pure water is used for washing for multiple times until chloride ions are completely removed, the dialyzed sample is dried for 48h at 40 ℃, the dried sample is added into a 600m L beaker and dissolved in 250m L pure water, the sample is boiled for 15min until the chloride ions are completely dissolved, the sample is titrated with 0.1 mol/L sodium hydroxide solution, phenolphthalein is used as an indicator, a blank control is dissolved in 30m L pure water, the same processing is carried out, and the mass fraction of carboxyl is calculated as follows:
in the formula:
concentration/(mol/L) of C-NaOH standard solution;
the volume of V-NaOH standard solution consumed at the end of titration/m L;
m-mass of sample/g.
The results are shown in FIG. 2, and the content of carboxyl groups in the oxidized dextran obtained in example 3 is higher than that in comparative examples 1-3, which shows that the degree of oxidation of example 3 is better than that in comparative examples 1-3; in example 4, the content of carboxyl groups in the oxidized dextran is higher than that in example 4, which shows that the degree of oxidation of example 4 is better than that of example 3.
1.3 determination of the Water solubility of oxidized dextran
Weighing oxidized dextran 20mg and dissolving in 0.4m L, oscillating to fully dissolve the oxidized dextran, then centrifuging for 2min at 13400rpm, pouring out supernatant liquor, drying, precisely weighing the dried sample, and dividing the mass by the mass of the sample to obtain water solubility, as shown in fig. 3, the water solubility of the oxidized dextran obtained in example 3 is better than that of comparative examples 1-3, which illustrates that the oxidation process of example 3 greatly reduces the number of hydroxyl groups in the superfine dextran, the residual hydroxyl groups are not enough to form a crystal structure with high intermolecular and intramolecular order, the occurrence of oxidation reduces the proportion of a crystalline region, the polycrystalline structure is converted to amorphous form, the crystal structure is destroyed, the permeation of water molecules is easy, the water solubility of the oxidized dextran is improved, and the water solubility of the oxidized dextran can be improved by using carboxyl groups as hydrophilic groups, the water solubility of the oxidized dextran obtained in example 4 is better than that of example 4, and the pretreatment of example 4 can make the obtained superfine dextran have low crystallinity and make the oxidized dextran obtain more carboxyl groups.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (9)
1. A preparation method of iron dextran is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing superfine molecular dextran by using dextran enzyme to perform enzymolysis on medium molecular dextran; pretreating dextran with triethanolamine/sodium p-toluenesulfonate/water system before enzymolysis; the weight ratio of triethanolamine to sodium p-toluenesulfonate to water in the triethanolamine/sodium p-toluenesulfonate/water system is 5-10:3-8: 100; the pretreatment process comprises the following steps: adding medium molecular dextran into triethanolamine/sodium p-toluenesulfonate/water system at a solid-to-liquid ratio of 1:10-20, stirring, standing for 6-12h, washing with water to neutral, and drying;
s2: oxidizing the superfine molecular dextran by using a TEMPO/CuBr2/NaClO oxidation reaction system to prepare an oxidized dextran solution;
s3: and (3) dripping a ferric trichloride solution and an alkali solution into the oxidized dextran solution at the same time, performing a complex reaction to prepare a dextran iron complex solution, and purifying to obtain the dextran iron.
2. The method of claim 1, wherein the dextran enzyme concentration in the enzymolysis system is 20-50 g/L, and the dextran enzyme concentration is 60-80U/g substrate in S1.
3. The method for preparing iron dextran according to claim 1, wherein: in the S1, the enzymolysis temperature is 40-60 ℃ and the time is 10-20 min.
4. The method for preparing iron dextran according to claim 1, wherein: in the S2, the dosage of TEMPO is 2-5mmol, the dosage of CuBr2 is 0.2-0.5mmol, and the dosage of NaClO is 1-3mmol per 1g of the superfine molecular dextran.
5. The method for preparing iron dextran according to claim 1 or 4, wherein: in the S2, the temperature of the oxidation reaction is 40-60 ℃, and the time of the oxidation reaction is 1-3 h.
6. The method for preparing iron dextran according to claim 1, wherein: in the S3, the alkali solution is a sodium hydroxide solution, and the weight ratio of oxidized dextran to ferric trichloride to sodium hydroxide is 1:2.2-2.6: 1.2-1.8.
7. A method of preparing iron dextran according to any one of claims 1 to 6.
8. The iron dextran according to claim 7, wherein: the weight-average molecular weight of the iron dextran is 3000-6000Da, the polydispersity is 1.0-1.5, and the iron content is 38-42 wt%.
9. Use of iron dextran according to claim 7 or 8 in iron supplement formulations.
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