CN116421622A - Succinylated pullulan chelated iron preparation as well as preparation method and application thereof - Google Patents

Succinylated pullulan chelated iron preparation as well as preparation method and application thereof Download PDF

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CN116421622A
CN116421622A CN202310310554.0A CN202310310554A CN116421622A CN 116421622 A CN116421622 A CN 116421622A CN 202310310554 A CN202310310554 A CN 202310310554A CN 116421622 A CN116421622 A CN 116421622A
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pullulan
iron
succinyl
preparation
succinylated
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杨文智
刘新硕
李海鹰
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Hebei University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0018—Pullulan, i.e. (alpha-1,4)(alpha-1,6)-D-glucan; Derivatives thereof

Abstract

The invention provides a succinylated pullulan chelate iron preparation and a preparation method and application thereof, belonging to the technical field of medical materials. The polysaccharide chelated iron preparation is obtained by utilizing the complexation reaction of free hydroxyl and carboxyl of succinylated pullulan polysaccharide chains and iron ions, and then dialyzing and freeze-drying, and the polysaccharide chelated iron preparation obtained by the invention proves the successful synthesis of the ST-PU chelated iron preparation through infrared, is extremely easy to redissolve and has the effect of improving IDA mouse anemia. Meanwhile, the preparation method has the advantages of wide sources of main raw materials, low cost, mild synthesis conditions, simple production route, simple process flow, strong operability and easier control of production quality, and has price advantage and clinical use value compared with the existing similar products.

Description

Succinylated pullulan chelated iron preparation as well as preparation method and application thereof
Technical Field
The invention relates to the technical field of medical materials, in particular to a succinylated pullulan chelate iron preparation and a preparation method and application thereof.
Background
Iron is the most abundant essential trace element in human body, widely distributed in various tissues and organs of the body, participates in various physiological reactions, is an important component for metabolism and biological processes, synthesizes hemoglobin and myoglobin for conveying oxygen, and plays an important role in mitochondria and various enzyme functions. Iron deficiency occurs when dietary iron intake is limited, when there is insufficient or depleted iron stored in the body. Iron deficiency can be divided into three stages depending on severity: iron reduction (ID), iron-deficiency erythropoiesis (IDE), and iron-deficiency anemia (IDA).
Iron nutrition enhancers have evolved to the third generation, the first generation being inorganic acid salts such as ferrous sulfate, ferric pyrophosphate, etc.; the second generation is organic salts such as ferrous succinate, ferrous fumarate and the like; the third generation of novel iron supplement comprises amino acid iron, heme iron, polysaccharide iron, polypeptide iron compound, iron-enriched yeast, nano material iron supplement and the like. The amino acid chelated iron is characterized in that carboxyl in amino acid molecules forms coordination bonds with iron ions through lone pair electrons, a stable annular structure is generated, the intramolecular charge tends to be neutral, and the solubility in the pH environment of the human digestive system is good. The antioxidant activity of the polysaccharide iron (III) chelate shows stronger hydroxyl radical and superoxide radical scavenging activity than that of polysaccharide, and the nutrition and health care functions are comprehensively revealed along with the gradual deep research of polysaccharide substances, and Fe is used 3+ As an iron element donor, the compound preparation with polysaccharide as a ligand becomes a novel oral iron supplement. Polysaccharide iron (III) complexes, such as iron dextran, are reported at home and abroad, have the advantages incomparable with common iron supplements, and the complexes are strong in stability, high in iron content and higher in bioavailability, and can promote iron absorption. Meanwhile, the polysaccharide iron (III) complex can also play the health care role of polysaccharide, and is an iron supplementing oral preparation with great potential. Therefore, the development and preparation of simple, cheap and effective polysaccharide iron is one development direction of commercial iron supplements.
Disclosure of Invention
In view of the above, the invention aims to provide a succinylated pullulan polysaccharide chelated iron preparation, and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
a preparation method of a succinylated pullulan chelated iron preparation, which comprises the following steps:
s1, dissolving pullulan, 4-dimethylaminopyridine and succinic anhydride in dimethyl sulfoxide, and stirring for reaction while heating to obtain succinyl pullulan;
s2, preparing sodium citrate and succinyl pullulan obtained in the step S1 into succinyl pullulan aqueous solution, stirring and heating, dripping an iron ion solution and an alkaline regulator solution at the same time, stopping dripping after the reaction reaches saturation, and continuing to heat in a water bath to obtain a reaction solution containing succinyl pullulan chelated iron;
s3, centrifuging the reaction solution obtained in the step S2, taking a supernatant, concentrating the supernatant, precipitating with alcohol, standing, performing suction filtration, dissolving the obtained precipitate in water, dialyzing, and lyophilizing to obtain a crude succinyl pullulan chelate iron product;
s4, dissolving the crude succinyl pullulan chelate iron, dialyzing for desalting, and freeze-drying to obtain a pure succinyl pullulan chelate iron product.
Preferably, the weight ratio of the pullulan to the 4-dimethylaminopyridine to the succinic anhydride in the S1 is (18-22): (2-4): (10-14).
Preferably, the molecular weight of the succinyl pullulan sugar in the S1 is 100 kDa-200 kDa, and the substitution degree of the succinyl is 71% -78%.
Preferably, the weight ratio of the sodium citrate to the succinyl pullulan in the S2 is (1-2): (1-2); the concentration of succinyl pullulan in the succinyl pullulan aqueous solution is 0.01 g/mL-0.02 g/mL.
Preferably, the concentration of the iron ion solution in the S2 is 1.5 mol/L-2.5 mol/L, and the concentration of the alkaline regulator solution is 1.5 mol/L-2.5 mol/L; the adding amount and the adding speed of the iron ion solution and the alkaline regulator solution are controlled to maintain the pH value of the reaction system at 7-9, and precipitate is formed in the reaction system and is immediately dissolved; the reaction in S2 is saturated until the reddish brown precipitate in the solution is no longer dissolved.
Preferably, the stirring and heating in S2 is performed while stirring and heating to 60 to 80 ℃.
Preferably, the dialysis is deionized water dialysis for 24-60 hours; the freeze-drying is carried out for 22-26 h at-58-62 ℃ and then for 22-26 h at-58-62 ℃ under the vacuum degree of 0.1 Pa.
Preferably, the centrifugation in the S3 is 3500 r/min-4500 r/min for 4 min-6 min; the alcohol precipitation in the step S3 is absolute alcohol precipitation with the volume of 3 times of the volume of the solution obtained after the addition and the concentration; and (3) standing at room temperature for 22-26 h.
The invention also provides the succinylated pullulan chelate iron preparation prepared by the preparation method of the succinylated pullulan chelate iron preparation in the scheme.
The invention also provides an application of the succinylated pullulan chelate iron preparation in the aspect of iron supplementing medicines.
The beneficial technical effects are as follows: the invention provides a succinylated pullulan polysaccharide chelated iron preparation, a preparation method and application thereof, wherein the succinylated pullulan polysaccharide chain free hydroxyl and carboxyl are used for carrying out complexation reaction with iron ions, and then the polysaccharide chelated iron preparation is obtained after dialysis and freeze-drying. Meanwhile, the preparation method has the advantages of wide sources of main raw materials, low cost, mild synthesis conditions, simple production route, simple process flow, strong operability and easier control of production quality, and has price advantage and clinical use value compared with the existing similar products.
Drawings
FIG. 1 shows succinyl pullulan (ST-PU) and succinyl pullulan chelated iron (ST-PU-Fe) 3+ ) And an infrared spectrum of the sample prepared in example 1; wherein line a represents pullulanPolysaccharide, line b represents succinyl pullulan, and line c represents the sample prepared in example 1;
FIG. 2 is an ultraviolet spectrum of the product prepared in example 1 and a control; wherein line a represents the sample prepared in example 1; line b represents succinyl pullulan (ST-PU);
FIG. 3 is a graph showing the conventional change of mouse blood, which is an improvement experiment of iron deficiency anemia in mice; wherein, fig. 3a is a graph of change in mouse HGB, fig. 3b is a graph of change in mouse RBC, and fig. 3c is a graph of change in mouse HCT;
FIG. 4 is a graph showing the change of serum iron in mice, a mice iron deficiency anemia improvement experiment;
FIG. 5 is a graph showing the change in total iron binding force of serum of mice in an improvement experiment of iron deficiency anemia of mice;
FIG. 6 shows a graph of changes in mouse transferrin for an improvement experiment in mouse iron deficiency anemia;
FIG. 7 is a graph showing changes in iron content in heart, liver, spleen and kidney of mice used in an iron deficiency anemia improvement test;
FIG. 8 is a graph showing changes in organ indices of heart, liver, spleen and kidney of mice used in an iron deficiency anemia improvement test;
FIG. 9 shows a graph of changes in mouse liver catalase, a mouse iron deficiency anemia improvement experiment;
FIG. 10 shows a graph of changes in mouse liver methane dicarboxylic acid aldolase for an iron deficiency anemia improvement experiment in mice;
FIG. 11 shows a graph of changes in mouse liver superoxide dismutase, which is an improvement experiment of iron deficiency anemia in mice.
Detailed Description
The invention provides a preparation method of a succinylated pullulan chelated iron preparation, which comprises the following steps:
s1, dissolving pullulan, 4-dimethylaminopyridine and succinic anhydride in dimethyl sulfoxide, and stirring for reaction while heating to obtain succinyl pullulan;
s2, preparing sodium citrate and succinyl pullulan obtained in the step S1 into succinyl pullulan aqueous solution, stirring and heating, dripping an iron ion solution and an alkaline regulator solution at the same time, stopping dripping after the reaction reaches saturation, and continuing to heat in a water bath to obtain a reaction solution containing succinyl pullulan chelated iron;
s3, centrifuging the reaction solution obtained in the step S2, taking a supernatant, concentrating the supernatant, precipitating with alcohol, standing, performing suction filtration, dissolving the obtained precipitate in water, dialyzing, and lyophilizing to obtain a crude succinyl pullulan chelate iron product;
s4, dissolving the crude succinyl pullulan chelate iron, dialyzing for desalting, and freeze-drying to obtain a pure succinyl pullulan chelate iron product.
According to the invention, pullulan, 4-dimethylaminopyridine and succinic anhydride are dissolved in dimethyl sulfoxide, and the mixture is heated and stirred for reaction, so that succinyl pullulan is obtained.
In the invention, the weight ratio of the pullulan to the 4-dimethylaminopyridine to the succinic anhydride is preferably (18-22): (2-4): (10 to 14), more preferably 20:3:12; the addition amount of the dimethyl sulfoxide is preferably 1.4-1.6 mL of dimethyl sulfoxide per gram of pullulan; the stirring reaction while heating is preferably magnetic stirring at 45-55 ℃. The molecular weight of the succinyl pullulan sugar is preferably 100-200 kDa, and the substitution degree of the succinyl is 71-78%. Pullulan (Pu) is an extracellular linear polysaccharide produced by fermentation of aureobasidium pullulans, and is polymerized from maltotriose repeating units connected by alpha-1, 4 glycosidic bonds through alpha-1, 6 glycosidic bonds. The pullulan has good water solubility and biological safety, and has wide development prospect in the food-grade medicine industry. The pullulan can also be chemically derived into succinyl pullulan (ST-PU), wherein the ST-PU is a macromolecular polymer with anions, the polysaccharide performance is improved through chemical modification, the ST-PU can be used as a drug carrier with slow release function, can be effectively complexed with metal ions, and has the functions of excellent water solubility, biocompatibility, biodegradability and the like. The succinyl pullulan with proper molecular weight and substitution degree of succinyl is obtained by reasonably controlling the types, the adding proportion and the reaction conditions of the raw materials, and the number of succinyl groups in a pullulan unit has important influence on the follow-up chelate iron reaction.
The method prepares the sodium citrate and the obtained succinyl pullulan into succinyl pullulan aqueous solution, and dropwise adds the iron ion solution and the alkaline regulator solution while stirring and heating the solution, stops dropwise adding after the reaction reaches saturation, and continues to heat in water bath to obtain reaction solution containing succinyl pullulan chelated iron.
In the present invention, the weight ratio of the sodium citrate to the succinyl pullulan is preferably (1-2): (1-2), more preferably 1:2; the concentration of succinyl pullulan in the aqueous solution of succinyl pullulan is preferably 0.01g/mL to 0.02g/mL. According to the invention, the sodium citrate is a catalyst for chelation reaction, and researches show that as the weight ratio of the sodium citrate to the succinyl pullulan increases, the final yield of the succinyl pullulan chelated iron is increased and then decreased.
In the present invention, the iron ion solution is preferably an iron chloride solution, an iron sulfate solution or an iron nitrate solution, more preferably an iron chloride solution; the concentration of the iron ion solution is preferably
1.5mol/L to 2.5mol/L, more preferably 2.0mol/L; the alkaline regulator solution is preferably sodium hydroxide solution or potassium hydroxide solution, more preferably sodium hydroxide solution; the concentration of the alkaline regulator is preferably 1.5mol/L to 2.5mol/L, more preferably 2.0mol/L; the addition amount and the addition speed of the iron ion solution and the alkaline regulator solution are preferably controlled so that the pH value of the reaction system is maintained at 7-9, and a precipitate is formed in the reaction system and is immediately dissolved; the reaction is saturated until the reddish brown precipitate in solution is no longer dissolved. The invention realizes the regulation and control of the pH value of the reaction system by controlling the addition amount and the addition speed of the iron ion solution and the alkaline regulator solution, and ensures that the iron ions in the reaction system are fully chelated with succinyl pullulan.
In the invention, the stirring and heating are preferably carried out while stirring to 60-80 ℃; the continuous water bath heating is preferably carried out for 1 to 3 hours at the temperature of 60 to 80 ℃.
The invention takes supernatant after centrifuging the obtained reaction liquid, concentrates the supernatant, carries out alcohol precipitation, stands still, pumps and filters, and the obtained precipitate is dissolved by adding water, dialyzed and freeze-dried to obtain succinyl pullulan polysaccharide chelated iron crude product.
In the present invention, the concentration is preferably 1/4 to 1/3 of the original supernatant volume by using a rotary evaporator; the alcohol precipitation is preferably carried out by adding absolute ethanol with the volume of 3 times of the volume of the solution obtained after concentration; the standing is preferably carried out at room temperature for 22-26 hours; the suction filtration is preferably carried out under the vacuum degree of 0.1MPa by a suction filtration pump on the solution after standing; the sediment is dissolved by adding water, preferably, the sediment obtained by suction filtration is dissolved according to the proportion of adding 1 mL-2 mL deionized water into each 0.1g sediment; the dialysis is preferably deionized water dialysis for 24-60 hours; the freeze-drying is preferably performed by pre-freezing at the temperature of minus 58 ℃ to minus 62 ℃ for 22 to 26 hours, and then freeze-drying at the vacuum degree of 0.1Pa and the temperature of minus 58 ℃ to minus 62 ℃ for 22 to 26 hours; the centrifugation is preferably 3500 r/min-4500 r/min for 4 min-6 min.
The method comprises the steps of dissolving, dialyzing, desalting and freeze-drying crude succinyl pullulan chelate iron to obtain pure succinyl pullulan chelate iron.
In the invention, the dissolution is preferably a solution with the dissolution concentration of 0.01 g/mL-0.02 g/mL of succinyl pullulan chelate iron crude product by deionized water; the dialysis is preferably deionized water dialysis for 48-60 hours; the freeze-drying is preferably performed by pre-freezing at-58 ℃ to-62 ℃ for 22-26 hours, and then freeze-drying at-58 ℃ to-62 ℃ for 22-26 hours under the vacuum degree of 0.1 Pa. The polysaccharide chelated iron preparation is extremely easy to re-dissolve by a freeze-drying process.
The invention also provides the succinylated pullulan chelate iron preparation prepared by the preparation method of the succinylated pullulan chelate iron preparation in the scheme.
The infrared proves that the succinylated pullulan chelated iron preparation obtained by the invention has successful synthesis of the ST-PU chelated iron preparation, is extremely easy to redissolve, and has the effect of improving IDA mouse anemia. Meanwhile, the preparation method has the advantages of wide sources of main raw materials, low cost, mild synthesis conditions, simple production route, simple process flow, strong operability and easier control of production quality, and has price advantage and clinical use value compared with the existing similar products.
The invention also provides an application of the succinylated pullulan chelate iron preparation in the aspect of iron supplementing medicines.
The invention has no special limitation on the dosage form of the medicine, and the succinylated pullulan chelated iron preparation is adopted to prepare the medicine acceptable dosage form. The preparation method of the medicine is not particularly limited, and the preparation method of the corresponding dosage form is adopted. The content of the succinylated pullulan chelated iron preparation in the medicine is not particularly limited, and the content of the conventional active substances in the medicine is adopted.
For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples. Materials, reagents and the like used in the examples and test examples of the present invention can be obtained commercially unless otherwise specified; the methods used in the examples and test examples of the present invention are conventional methods unless otherwise specified.
Example 1
(1) The method comprises the steps of mixing pullulan polysaccharide, 4-dimethylaminopyridine and succinic anhydride according to a mass ratio of 20:3:12 is dissolved in dimethyl sulfoxide, the dosage of the dimethyl sulfoxide is 1.4mL of dimethyl sulfoxide is added per gram of pullulan, and succinyl pullulan is obtained by stirring and reacting while heating;
the degree of substitution of succinyl groups in succinyl pullulan was determined by titration analysis (same below):
accurately weighing 0.15g of succinyl pullulan in a 50mL beaker, and respectively weighing 1mL of ethanol, 10mL of deionized water and 0.2mol/L of NH 4 Adding 4mL of Cl buffer solution into a beaker, stirring for dissolution, adjusting the pH to 6.0-7.0, and precisely adding 0.05mol/L of CuSO 4 10.0mL of standard solution, shaking up, standing for 15min, transferring toIn a 50mL volumetric flask, the volume was fixed, shaken well, and filtered through a Buchner funnel. The subsequent filtrate is measured with a precision of 10mL, and the pH is adjusted to 7.5-8.0. PAN was used as indicator. And (3) titrating with EDTA standard solution until the solution is green, namely the titration end point. The results of the titration were corrected with a blank test. Calculating the substitution degree of the sample according to the formula:
Figure BDA0004148271180000071
Figure BDA0004148271180000072
wherein, B: sodium succinate content, wherein 123 is the relative molecular mass of sodium succinate; w: weighing the sample, g; CEDTA: EDTA standard solution concentration, mol/L; v (V) 0 : the volume of EDTA is consumed for blank, and the volume of EDTA is mL; v (V) 1 : the volume of EDTA, mL, was consumed as a sample.
The degree of substitution of succinyl in the obtained succinyl pullulan was detected to be 71%.
(2) Weighing 2g of succinyl pullulan (ST-PU), 1g of sodium citrate, placing into a three-necked flask, adding 100mL of distilled water for dissolution, heating in a water bath at 70 ℃ and continuously stirring, and concentrating FeCl with the concentration of 2mol/L 3 Slowly dripping the solution into the solution, simultaneously dripping NaOH solution with the concentration of 2mol/L, controlling the dripping speed and the dripping amount of the solution and the NaOH solution, regulating the pH value of the reaction solution to 8, and dripping FeCl in the reaction 3 The solution starts to form precipitate and is immediately dissolved, when the reddish brown precipitate in the solution is not dissolved any more, the reaction is saturated, water bath heating is continued for 2 hours, and a reaction solution containing succinyl pullulan chelated iron is obtained;
(3) Centrifuging the reaction solution at 4000r/min for 5min to remove precipitate, concentrating the supernatant by a rotary evaporator to 1/4 of the original supernatant volume, adding 3 times of absolute ethyl alcohol for alcohol precipitation, standing for 24h, carrying out suction filtration, dissolving the obtained precipitate according to the proportion of adding 1mL of deionized water into each 0.1g of precipitate, dialyzing with deionized water for 24h, and freeze-drying the obtained dialysate: the obtained dialysate was pre-treated at-60 DEG CFreezing for 24 hr, and lyophilizing at-60deg.C under vacuum degree of 0.1Pa for 24 hr to obtain succinyl pullulan chelated iron (ST-PU-Fe) 3+ ) Crude products;
(4) In ST-PU-Fe 3+ Adding deionized water into the crude product to prepare a solution with the concentration of 0.01g/mL, adding the solution into a dialysis bag, dialyzing with deionized water for 48h, pre-freezing the obtained dialysate at-60 ℃ for 24h, and freeze-drying the obtained pre-frozen product at-60 ℃ under the vacuum degree of 0.1Pa for 24h to obtain granular ST-PU-Fe 3+ Pure product.
Example 2
(1) The pullulan polysaccharide, 4-dimethylaminopyridine and succinic anhydride are mixed according to the mass ratio of 22:4:14 is dissolved in dimethyl sulfoxide, wherein the dosage of the dimethyl sulfoxide is 1.6mL of dimethyl sulfoxide is added per gram of pullulan, and succinyl pullulan is obtained by stirring and reacting while heating; the substitution degree of succinyl in the obtained succinyl pullulan is 78% by titration analysis;
(2) Weighing 2g of succinyl pullulan (ST-PU), 2g of sodium citrate, putting into a three-necked flask, adding 100mL of distilled water for dissolution, heating in a water bath at 60 ℃ and continuously stirring, and concentrating FeCl with the concentration of 2.5mol/L 3 Slowly dripping the solution into the solution, simultaneously dripping NaOH solution with the concentration of 2.5mol/L, controlling the dripping speed and the dripping amount of the solution and the NaOH solution, regulating the pH value of the reaction solution to 7, and dripping FeCl in the reaction 3 The solution starts to form precipitate and is immediately dissolved, when the reddish brown precipitate in the solution is not dissolved any more, the reaction is saturated, water bath heating is continued for 1h, and a reaction solution containing succinyl pullulan chelated iron is obtained;
(3) Centrifuging the reaction solution 4500r/min for 4min to remove precipitate, concentrating the supernatant by a rotary evaporator to 1/3 of the original supernatant volume, adding 3 times of absolute ethyl alcohol for alcohol precipitation, standing for 26h, performing suction filtration, dissolving the obtained precipitate according to the proportion of adding 2mL of deionized water into each 0.1g of precipitate, dialyzing with deionized water for 24h, and freeze-drying the obtained dialysate: pre-freezing the obtained dialysate at-58deg.C for 26 hr, lyophilizing the obtained pre-frozen product at-62deg.C under vacuum degree of 0.1Pa for 22 hr to obtain succinyl pullulan chelateIron (ST-PU-Fe) 3+ ) Crude products;
(4) In ST-PU-Fe 3+ Adding deionized water into the crude product to prepare a solution with the concentration of 0.02g/mL, adding the solution into a dialysis bag, dialyzing with deionized water for 48h, pre-freezing the obtained dialysate at-60 ℃ for 24h, and freeze-drying the obtained pre-frozen product at-60 ℃ under the vacuum degree of 0.1Pa for 24h to obtain granular ST-PU-Fe 3+ Pure product.
Example 3
(1) The pullulan polysaccharide, 4-dimethylaminopyridine and succinic anhydride are mixed according to the mass ratio of 18:2:10 is dissolved in dimethyl sulfoxide, the dosage of the dimethyl sulfoxide is 1.5mL of dimethyl sulfoxide is added per gram of pullulan, and succinyl pullulan is obtained by stirring and reacting while heating; the substitution degree of succinyl in the obtained succinyl pullulan is 75% by titration analysis;
(2) Weighing 1g of succinyl pullulan (ST-PU), 2g of sodium citrate, placing into a three-necked flask, adding 100mL of distilled water for dissolution, heating in a water bath at 80 ℃ and continuously stirring, and concentrating FeCl with the concentration of 1.5mol/L 3 Slowly dripping the solution into the solution, simultaneously dripping NaOH solution with the concentration of 1.5mol/L, controlling the dripping speed and the dripping amount of the solution and the NaOH solution, regulating the pH value of the reaction solution to 9, and dripping FeCl in the reaction 3 The solution starts to form precipitate and is immediately dissolved, when the reddish brown precipitate in the solution is not dissolved any more, the reaction is saturated, water bath heating is continued for 3 hours, and a reaction solution containing succinyl pullulan chelated iron is obtained;
(3) Centrifuging the reaction solution at 3500r/min for 6min to remove precipitate, concentrating the supernatant by a rotary evaporator to 1/4 of the original supernatant volume, adding 3 times of absolute ethyl alcohol for alcohol precipitation, standing for 22h, carrying out suction filtration, dissolving the obtained precipitate according to the proportion of adding 1.5mL of deionized water into each 0.1g of precipitate, dialyzing with deionized water for 48h, and freeze-drying the obtained dialysate: pre-freezing the obtained dialysate at-60deg.C for 24 hr, lyophilizing the obtained pre-frozen product at-60deg.C under vacuum of 0.1Pa for 24 hr to obtain succinyl pullulan chelated iron (ST-PU-Fe) 3+ ) Crude products;
(4) In ST-PU-Fe 3+ Adding the crude product into the mixture to removeIonic water is prepared into a solution with the concentration of 0.015g/mL, the solution is added into a dialysis bag, deionized water is used for dialysis for 60 hours, the obtained dialysate is pre-frozen for 22 hours at the temperature of minus 62 ℃, and the obtained pre-frozen product is frozen and dried for 26 hours under the vacuum degree condition of minus 62 ℃ and 0.1Pa, thus obtaining granular ST-PU-Fe 3+ Pure product.
Test example 1 Spectrum detection
(1) And (3) infrared detection: firstly, using base Pullulan (PU) as a reference to estimate whether succinyl pullulan (ST-PU) is synthesized, and then using succinyl pullulan (ST-PU) as a reference to estimate whether succinyl pullulan chelate iron (ST-PU-Fe) is synthesized 3+ ). By KBr tabletting method, 40-4000 cm -1 Wave number scanning. As a result of the examination shown in FIG. 1, it can be seen from FIG. 1 that the sample prepared in example 1 was succinylated pullulan at 3418cm -1 the-OH stretching vibration at the position is weakened and at 1734cm -1 New ester group peak appears at the position, ST-PU and FeCl 3 ST-PU-Fe generated by the coordination reaction 3+ Has characteristic absorption peak of polysaccharide, compared with ST-PU-OH characteristic absorption peak of 3418cm -1 Move to 3386cm -1 At 1636cm -1 New ST-PU-Fe appears 3+ Matching with absorption peak. In addition, ST-PU-Fe 3+ Inorganic salt donor FeCl of complex infrared spectrogram and iron ion 3 The infrared spectrograms of the (B) are obviously different, which proves that the ST-PU-Fe is successfully synthesized 3+ A complex.
(2) Taking samples prepared in examples 1-3, and performing ultraviolet detection by taking succinyl pullulan (ST-PU) as a control:
and respectively dissolving a certain amount of ST-PU and ST-PU-Fe (III) complex in distilled water, scanning the ultraviolet spectrum with the wavelength of 200 nm-600 nm on an ultraviolet spectrophotometer, and analyzing the difference of ultraviolet scanning curves between the ST-PU and ST-PU-Fe (III) complex.
The detection results are shown in FIG. 2. As can be seen from FIG. 2, the ST-PU exhibits a strong absorption peak at 280nm, which is caused by n-pi transition of electrons generated by the-COOH P-pi conjugation in the ST-PU. When ST-PU and Fe (III) form a complex, the absorption peak of ST-PU at 280nm disappears, which shows that the P-pi conjugation of carboxymethyl in the complex changes, so that the ST-PU-Fe (III) complex shows a gently decreasing curve between 200 and 500 nm. The Uv spectrum shows that coordination occurs between Fe (III) and ST-PU, and electron transfer occurs between Fe (III) and ST-PU in ST-PU-Fe (III), so that Uv absorption of ST-PU-Fe (III) is changed.
Test example 2ST-PU-Fe (III) test for improving iron deficiency anemia in mice
Healthy three-week-old male ICR mice without specific pathogens (Specific Pathogen Free, SPF) are 18-22 g in weight, layered according to weight, and randomly divided into a normal control group and an anemia model group. The normal control group was fed with a normal diet with an iron content of 200 mg iron per kg diet, and the anaemia model group reduced the iron content of the basal diet to 7 mg iron per kg diet, and the combined bleeding method was fed with a low iron feed, resulting in anaemia in mice. After successful molding, the control group was subjected to gastric lavage with normal saline as a control, and the anemia model group was randomly divided into a model control group, a ferrous sulfate group, a commercial iron dextran group and a ST-PU-Fe (III) (this is a sample obtained in example 1, the same applies below) dose group, and the treatment effect was verified by gastric lavage 28d, and the specific dosing amounts are shown in Table 1, and the components were fed in cages during the experiment, fed with standard feed, and drunk freely.
Table 1 mice dosing amounts
Figure BDA0004148271180000101
After 28d of divided gastric lavage, the blood routine measurement results are shown in fig. 3, and the HGB content of each iron supplementing group is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and the RBC content is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and the HCT content is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, wherein HGB content of ST-PU-Fe (III) group is 133.21g/L, RBC content is increased to 8.5510 12 The HCT content is increased to 0.42L/L, the contents of Hemoglobin (HGB), red blood cell number (RBC) and Hematocrit (HCT) are all improved, the levels are all obviously increased, and the anemia symptoms are all clearAnd the composition is obvious in recovery, and has good curative effect compared with an IDA model group.
The results of serum biochemical indexes are shown in fig. 4-6, and as can be seen from fig. 4-6, the Serum Iron (SI) concentration of mice in the IDA model group is 34.62 mu mol/L, the total iron binding force (TIBC) level of the mice is 136.20 mu mol/L, the Transferrin (TRF) content of the mice is 23.66mg/dL, the SI concentration of normal control group is 50.00 mu mol/L, the TIBC level is 82.31 mu mol/L, the TRF content is 18.61mg/dL, and compared with the normal control group, the SI content of the mice in the IDA model group is obviously reduced, and the TIBC level and the TRF content are obviously increased. After 4 weeks of administration, three index parameters of each iron-supplementing group of mice are improved compared with those of IDA group of mice, SI concentration is obviously increased, and TIBC level and TRF content are obviously reduced. Under the same dosage, the SI content of each iron supplementing group is ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, the TIBC content is ST-PU-Fe (III) group < commercial dextran iron group < ferrous sulfate group < model control group, and the TRF content is ST-PU-Fe (III) group < commercial dextran iron group < ferrous sulfate group < model control group. Wherein, the SI concentration of the ST-PU-Fe (III) group is 50.51 mu mol/L, the TIBC level is 85.73 mu mol/L, the TRF content is 18.80mg/dL, and the result shows that the ST-PU-Fe (III) group has higher SI concentration and lower TIBC level and TRF content than the commercial dextran iron group and ferrous sulfate group.
The results of the measurement of the iron content of the tissues are shown in fig. 7, and as can be seen from fig. 7, the iron content of each iron supplementing group heart is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and the liver iron content is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and the content of spleen iron is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and the kidney iron content is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group. The results show that the heart iron content of the mice in the ST-PU-Fe (III) group is increased to 6.70 mu mol/gprot, the iron content is higher than that of the mice in the normal control group, the amplification is 2.29%, the liver iron content is increased to 15.34 mu mol/gprot, the iron content is higher than that of the mice in the normal control group, the amplification is 13.88%, the spleen iron content is increased to 9.05 mu mol/gprot, the iron content is higher than that of the mice in the normal control group, the amplification is 20.99%, the kidney iron content is increased to 5.63 mu mol/gprot, the iron content is higher than that of the mice in the normal control group, and the amplification is 17.54%.
After four weeks of iron supplementation, the organ indexes of mice in each group are shown in fig. 8, and as can be seen from fig. 8, the liver coefficients of mice in the IDA group are obviously improved, and the heart index, spleen index and kidney index with higher IDA are obviously reduced. The liver index of each iron supplement group is as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and the heart index, spleen index and kidney index are all: the results of the ST-PU-Fe (III) group < commercial iron dextran group < ferrous sulfate group < model control group show that the liver index of the mice in the ST-PU-Fe (III) group is increased to 4.14, the heart index is 0.54, the spleen index is 0.31, the kidney index is 1.37, the hypertrophy of the heart, spleen and kidney of the mice in the IDA group is obviously relieved, compared with other iron supplementing treatment groups, the effect of improving is best, and the mice are closest to the mice in the normal control group.
As shown in FIGS. 9 to 11, the results of the liver oxidative stress reaction of the mice are shown in FIGS. 9 to 11, the CAT content of the IDA mice is 31.36nmol/mgprot, the CAT content of the normal control mice is 194.65nmol/mgprot, the SOD content of the IDA mice is 14.79nmol/mgprot, the SOD content of the normal control mice is 27.24nmol/mgprot, the MDA content of the IDA mice is 15.39nmol/mgprot, the MDA content of the normal control mice is 9.28nmol/mgprot, the Catalase (CAT) value and the superoxide dismutase (SOD) value of the IDA mice are reduced, and the Methane Dicarboxylic Aldolase (MDA) value of the IDA mice is increased as compared with the normal mice. After treatment with iron supplement, CAT content of each iron supplement group was as follows: ST-PU-Fe (III) group > commercial dextran iron group > ferrous sulfate group > model control group, and SOD content is as follows: the content of MDA is ST-PU-Fe (III) group, commercial dextran iron group, ferrous sulfate group and model control group. The results showed that the CAT and SOD values were increased and the MDA value was decreased for each iron-supplemented group of mice compared to the IDA group, wherein the CAT value was 162.55nmol/mgprot, the SOD value was 26.79nmol/mgprot and the MDA value was 8.16nmol/mgprot for the ST-PU-Fe (III) group of mice. The CAT value and the SOD value of the mice in the ST-PU-Fe (III) group are increased most and are closest to those of the normal control group, and the MDA value is reduced most obviously and is lower than that of the normal control group. It can be seen that ST-PU-Fe (III) is optimal in promoting SOD and CAT activity and inhibiting MDA production.
In conclusion, the polysaccharide chelated iron preparation is successfully obtained, is extremely easy to re-dissolve and has the effect of improving IDA mouse anemia. Meanwhile, the preparation method has the advantages of wide sources of main raw materials, low cost, mild synthesis conditions, simple production route, simple process flow, strong operability and easier control of production quality, and has price advantage and clinical use value compared with the existing similar products.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The preparation method of the succinylated pullulan chelated iron preparation is characterized by comprising the following steps of:
s1, dissolving pullulan, 4-dimethylaminopyridine and succinic anhydride in dimethyl sulfoxide, and stirring for reaction while heating to obtain succinyl pullulan;
s2, preparing sodium citrate and succinyl pullulan obtained in the step S1 into succinyl pullulan aqueous solution, stirring and heating, dripping an iron ion solution and an alkaline regulator solution at the same time, stopping dripping after the reaction reaches saturation, and continuing to heat in a water bath to obtain a reaction solution containing succinyl pullulan chelated iron;
s3, centrifuging the reaction solution obtained in the step S2, taking a supernatant, concentrating the supernatant, precipitating with alcohol, standing, performing suction filtration, dissolving the obtained precipitate in water, dialyzing, and lyophilizing to obtain a crude succinyl pullulan chelate iron product;
s4, dissolving the crude succinyl pullulan chelate iron, dialyzing for desalting, and freeze-drying to obtain a pure succinyl pullulan chelate iron product.
2. The preparation method of the succinylated pullulan chelate iron preparation according to claim 1, wherein the weight ratio of the pullulan to the 4-dimethylaminopyridine to the succinic anhydride in the S1 is (18-22): (2-4): (10-14).
3. The preparation method of the succinylated pullulan chelated iron preparation according to claim 1, wherein the molecular weight of succinyl pullulan in S1 is 100-200 kDa, and the substitution degree of succinyl is 71-78%.
4. The preparation method of the succinylated pullulan chelate iron preparation according to claim 1, wherein the weight ratio of sodium citrate to succinyl pullulan in S2 is (1-2): (1-2); the concentration of succinyl pullulan in the succinyl pullulan aqueous solution is 0.01 g/mL-0.02 g/mL.
5. The method for preparing the succinylated pullulan chelate iron preparation according to claim 1, wherein the concentration of the iron ion solution in the S2 is 1.5 mol/L-2.5 mol/L, and the concentration of the alkaline regulator solution is 1.5 mol/L-2.5 mol/L; the adding amount and the adding speed of the iron ion solution and the alkaline regulator solution are controlled to maintain the pH value of the reaction system at 7-9, and precipitate is formed in the reaction system and is immediately dissolved; the reaction in S2 is saturated until the reddish brown precipitate in the solution is no longer dissolved.
6. The method for preparing the succinylated pullulan chelate iron preparation as defined in claim 1, wherein the stirring and heating in S2 is to 60-80 ℃ while stirring.
7. The method for preparing the succinylated pullulan chelated iron preparation according to claim 1, wherein the dialysis is deionized water dialysis for 24-60 hours; the freeze-drying is carried out at the temperature of minus 58 ℃ to minus 62 ℃ for 22 hours to 26 hours, and then freeze-drying is carried out at the vacuum degree of 0.1Pa and the temperature of minus 58 ℃ to minus 62 ℃ for 22 hours to 26 hours.
8. The method for preparing the succinylated pullulan chelate iron preparation according to claim 1, characterized in that the centrifugation in the step S3 is 3500 r/min-4500 r/min for 4 min-6 min; the alcohol precipitation in the step S3 is absolute alcohol precipitation with the volume of 3 times of the volume of the solution obtained after the addition and the concentration; and (3) standing at room temperature for 22-26 h.
9. A succinylated pullulan chelate iron preparation prepared by the preparation method of the succinylated pullulan chelate iron preparation according to any one of claims 1 to 8.
10. Use of the succinylated pullulan chelate iron preparation of claim 9 in iron supplementing medicine.
CN202310310554.0A 2023-03-28 2023-03-28 Succinylated pullulan chelated iron preparation as well as preparation method and application thereof Pending CN116421622A (en)

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