WO2020056714A1 - Method for preparing high-purity riboflavin sodium phosphate - Google Patents

Method for preparing high-purity riboflavin sodium phosphate Download PDF

Info

Publication number
WO2020056714A1
WO2020056714A1 PCT/CN2018/106904 CN2018106904W WO2020056714A1 WO 2020056714 A1 WO2020056714 A1 WO 2020056714A1 CN 2018106904 W CN2018106904 W CN 2018106904W WO 2020056714 A1 WO2020056714 A1 WO 2020056714A1
Authority
WO
WIPO (PCT)
Prior art keywords
solution
sodium phosphate
riboflavin sodium
preparing high
purity
Prior art date
Application number
PCT/CN2018/106904
Other languages
French (fr)
Chinese (zh)
Inventor
郭涛
张琦
张冬民
程双
Original Assignee
邦泰生物工程(深圳)有限公司
邦泰合盛生物科技（深圳）有限公司
江西安泽麦生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 邦泰生物工程(深圳)有限公司, 邦泰合盛生物科技（深圳）有限公司, 江西安泽麦生物科技有限公司 filed Critical 邦泰生物工程(深圳)有限公司
Priority to CN201880037970.5A priority Critical patent/CN111094309A/en
Priority to PCT/CN2018/106904 priority patent/WO2020056714A1/en
Publication of WO2020056714A1 publication Critical patent/WO2020056714A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C07D475/12—Heterocyclic compounds containing pteridine ring systems containing pteridine ring systems condensed with carbocyclic rings or ring systems
    • C07D475/14—Benz [g] pteridines, e.g. riboflavin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings

Definitions

  • the present invention relates to the technical field of preparation of medical monomers, and in particular, to a method for preparing high-purity riboflavin sodium phosphate by using a separation and purification technology.
  • Riboflavin Sodium Phosphate is a phosphate form of riboflavin (vitamin B2), also known as vitamin B2 sodium phosphate, flavin mononucleotide sodium, riboflavin-5'- (Dihydrogen phosphate) Monosodium salt, etc., whose CAS number is 130-40-5, and its structural formula is shown in the figure below.
  • Riboflavin sodium phosphate is currently commonly used as a riboflavin supplement for the treatment of cheilitis, cheilitis, glossitis, conjunctivitis, and scrotal inflammation due to riboflavin deficiency.
  • Common dosage forms are injections and oral tablets.
  • the domestic method for preparing riboflavin sodium phosphate is mainly a chemical synthesis method, which generally uses high-purity riboflavin as a starting material, phosphorus trichloride as a phosphorylation reagent, and tetrahydrofuran, acetonitrile, and Chemical reactions in the environment of organic solvents such as pyridine generate flavin single nucleotides, which are further converted to riboflavin sodium phosphate.
  • organic solvents such as pyridine
  • flavin single nucleotides which are further converted to riboflavin sodium phosphate.
  • biocatalytic methods have gradually emerged. It uses biological enzymes to catalyze the conversion of substrates into flavin single nucleotides. Further conversion into riboflavin sodium phosphate has the advantages of high product-specific conversion rate, energy saving and environmental protection.
  • both Chinese invention patent documents CN106674225 A and CN103396452B disclose a method for preparing high-purity riboflavin sodium phosphate through a crystallization process, but a large number of toxic and harmful organic solvents need to be used in the preparation process.
  • acetic acid is required in CN106674225A Methyl ester, acetone, cyclohexanol and methanol, while CN103396452B requires the use of acetonitrile, methanol and chloroform, which does not meet the environmental protection concept of sustainable development.
  • the object of the present invention is to provide a new method for preparing high-purity riboflavin sodium phosphate with low energy consumption, environmental protection, and low cost.
  • the present invention provides the following method for preparing high-purity riboflavin sodium phosphate, including the following steps: 1) pH of a crude solution of riboflavin sodium phosphate and / or flavin single nucleotide Adjust the value to 5.0-7.0; 2) Control the solution temperature to 20 ⁇ 40 ° C, and add 0.5 to 1.5 times the volume of the crude solution to a 9 5% (ml / ml) ethanol aqueous solution; 3) Reduce the temperature to 3 ⁇ 5 ° C, wait for the solid to precipitate; 4) Filter and remove the filtrate. The filter cake is dried to obtain pure riboflavin sodium phosphate.
  • Flavin Mononucleotide (English abbreviation FMN), also known as riboflavin-5-phosphate, is a prosthetic group of flavin protein, and plays an important role in the electron transfer of biological processes such as respiration, that is, As a prosthetic group of the flavinase group, it participates in the transfer of electrons from the substrate to the electron acceptor in a binding state with an apo-enzyme, and plays an important role in basic metabolism. Its CAS number is 146-17-8 Its structural formula is shown in the figure below.
  • the crude solution of riboflavin sodium phosphate and / or flavin single nucleotide in step 1) refers to a solution containing a large amount of impurities in addition to sodium riboflavin phosphate and / or flavin single nucleotide.
  • the solution includes: a solution obtained by dissolving riboflavin sodium phosphate and / or flavin mononucleotide purchased from the market with insufficient purity in water, and a crude product solution obtained by biocatalytic preparation of flavin mononucleotide.
  • the concentration of the crude solution of the riboflavin sodium phosphate and / or the flavin mononucleotide is 7.5 g / L or more based on the sodium riboflavin phosphate, so as to ensure that sufficient solids can be precipitated at a later stage, thereby ensuring that High enough yield.
  • Biocatalysis refers to the process of transforming substances using biological enzymes or biological organisms (cells, organelles, tissues, etc.) as catalysts (referred to as enzyme catalysis and biological fermentation, respectively). This reaction process is also called biological Transformation (biotransformation).
  • the biological organisms commonly used in biocatalysis are mainly microorganisms, and the essence is to use enzymes in the cells of the microorganisms to catalyze and promote the process of biological transformation.
  • the biocatalytic method has mild working conditions (basically completed in normal temperature, neutral, water and other environments)
  • the so-called crude product solution obtained by preparing a flavin mononucleotide by a biocatalytic method in the present invention specifically refers to an enzyme reaction solution after converting a substrate to a flavin mononucleotide by using a biological enzyme to catalyze a substrate, or a biological organism containing a biological enzyme through A biological fermentation broth containing flavin single nucleotides after removing biological organisms after fermentation culture and induction of expression.
  • the so-called biological enzyme refers to an enzyme that can specifically catalyze the conversion of a substrate into a flavin single nucleotide
  • the so-called substrate refers to a precursor material that can be converted into a flavin single nucleotide.
  • the substrate can be riboflavin And ATP can also be precursors that can be converted to riboflavin and / or ATP, and the corresponding biological enzyme is riboflavin kinase (EC 2.7.1.26), or riboflavin kinase (EC 2.7.1.26) Used in combination with one or more other enzymes.
  • the substrate can also be riboflavin and D-glucose-1-phosphate, or a precursor substance that can be converted into riboflavin and / or D-glucose-1-phosphate, and the corresponding biological enzyme is riboflavin.
  • Phosphotransferase EC2.7.1.42
  • a combination of riboflavin phosphotransferase EC2.7.1.42
  • step 1) of the method of the present invention the reason why the pH value is adjusted to 5.0 to 7.0 is that within this pH value range, riboflavin phosphate monosodium salt can be formed.
  • the adjustment object is a crude solution of flavin single nucleotide, sodium hydroxide is added for adjustment; when the adjustment object is a crude solution of riboflavin sodium phosphate, sodium hydroxide or hydrochloric acid is added for adjustment according to the pH value of the solution .
  • the pH of the solution is preferably adjusted to 5.5 to 6.5, and more preferably, the pH of the solution is adjusted to 6.0 to 6.3.
  • step 2) of the method of the present invention 95% ethanol is added as a poor solvent, and the polarity of the aqueous solution is reduced to reduce the solubility of sodium riboflavin phosphate in the solution, thereby reaching a supersaturated state and precipitating.
  • adding a 95% (ml / ml) ethanol aqueous solution with a concentration of 0.5 to 0.9 times the volume of the solution can save costs on the one hand and improve the uniformity of products between different batches on the other.
  • step 3 of the method of the present invention a riboflavin sodium phosphate product with high yield and content can be obtained when the temperature is lowered to 3 ⁇ 5 ° C. If the temperature is higher than this range, the yield is lower and the temperature is lower. In this range, the content decreases.
  • the inventor's research found that the precipitation of solids is greatly affected by temperature. If the cooling rate is too fast, it will cause the solids to burst out within a short period of time, resulting in different particle sizes, affecting the morphology of the product, and even causing solids.
  • the agglomerates are precipitated, so that a large amount of impurities are embedded, so that the product content is not high.
  • the cooling process in step 3) is completed within 1-2 hours.
  • the temperature reduction process of step 3) is performed according to the following steps: First, the temperature is lowered to 12 ⁇ 15 ° C, and after the solid precipitation rate in the solution reaches more than 40%, the temperature is lowered to 3 ⁇ 5 ° C.
  • a concentration of 0.3% to 0.5 times the volume of the crude solution is added to a concentration of 95% (ml / ml) of ethanol.
  • Filtration in step 4) of the method of the present invention refers to using a physical method to separate solids and liquids in the solution.
  • common filtration methods are applicable to the method of the present invention, including normal pressure filtration, reduced pressure filtration, centrifugal filtration, and the like.
  • step 4) of the method of the present invention preferably, the obtained filter cake is washed with a 75% (ml / ml) ethanol aqueous solution before the drying treatment.
  • the obtained filter cake is beaten with a 50% ethanol aqueous solution, and then the filter cake is collected by filtration.
  • the so-called beating refers to adding a certain kind of solvent with a lower solubility to the target substance and a higher solubility to other related substances to the solid substance, and dissolving other unnecessary related substances in the solvent by stirring, and the target substance and the solvent are suspended. Cloudy process.
  • the drying method is vacuum drying at a temperature of 30 ° C. for 5 to 10 hours.
  • the filter cake obtained in step 4) or the pure sodium riboflavin phosphate is dissolved in purified water, and steps 1) to 4 of the method of the present invention are repeated.
  • the crude solution of riboflavin sodium phosphate and / or flavin mononucleotide in step 1) of the method of the present invention is a crude product solution obtained by preparing a flavin mononucleotide by a biocatalytic method
  • the solution often contains a residual portion Biological enzymes, in order to prevent degradation of the target product by residual enzymes during the separation and purification of sodium riboflavin phosphate, it is preferable to perform the following crude solution of riboflavin sodium phosphate and / or flavin single nucleotide before step 1)
  • Pretreatment a) The pH value of the crude solution of riboflavin sodium phosphate and / or flavin single nucleotide is adjusted to 11.0 -12.0; b) The temperature of the solution is raised to 40 ⁇ 1 ° C, and at 40 ⁇ 1 ° C for 2 h; C) remove insolubles.
  • step c) of the pre-processing by adjusting the pH value of the solution to 11.0 to 12.0, and supplemented with heat treatment at a temperature of 40 ⁇ 1 ° C., the biological enzymes remaining in the solution can be inactivated.
  • sodium hydroxide is used to adjust the pH value, so that the flavin single nucleotide can be converted into sodium riboflavin phosphate; in particular, the preferred range of pH value is 11.0 ⁇ 11.5, and the stability of the target product is the best in this range.
  • step c) of the pre-processing the method of removing insoluble matter is preferably: centrifuging the solution after the heating in step b) to collect the supernatant.
  • the filtering operation in step 4) is often followed. It will be more difficult to easily cause clogging of the filtration membrane.
  • the above-mentioned pre-treatment further includes a step of pre-treating the solution obtained by removing insoluble matter obtained in step c) to remove the crude product solution. Residual impurities in the residue;
  • the so-called pretreatment refers to purifying the solution after removing the insoluble matter with an ion exchange resin, or sequentially subjecting the solution after removing the insoluble matter to microfiltration and ultrafiltration.
  • Microfiltration also known as microporous filtration, uses a microporous filter membrane as a filter medium and, under the pressure of 0.1 to 0.3 MPa, traps particles and bacteria between 0.1 and 1 micron, but allows macromolecular organics and inorganics. Salt passed.
  • the microfiltration membrane used in the method of the present invention is preferably a hollow fiber membrane having a pore diameter of 0.1 pm.
  • Ultrafiltration is also called ultrafiltration, and its principle is the same as that of microfiltration. The only difference is that the pore size of the ultrafiltration membrane used in ultrafiltration is smaller than that of the microfiltration membrane, and is generally below 0.01 microns.
  • the ion exchange resin is an anion exchange resin.
  • the anion exchange resin is a weakly basic anion exchange resin or a macroporous adsorption resin.
  • a 0.1 to 0.4 mol / L sodium chloride aqueous solution is used as the eluent of the ion exchange resin; in order to improve the yield of the product, more preferably, the concentration of the sodium chloride aqueous solution is 0.3 to 0.35 mol / L.
  • the method for regenerating the ion exchange resin includes the following steps: A. The resin is rinsed with a mixed solution of a 2mol / L sodium chloride solution and a 0.5mol / L hydrochloric acid solution until no impurities remain in the effluent; B. Rinse with purified water until the pH value of the eluent is 5.00 ⁇ 7.00; C. Rinse with 0.5mol / L sodium hydroxide solution until the eluent becomes strongly alkaline; D. Rinse with purified water until washing The dehydration pH is 9.00 ⁇ 10.00.
  • the above pretreated solution is concentrated to remove a large amount of water
  • the concentration treatment in the method of the present invention refers to a process in which the concentration of a solution is increased by reducing the solvent using a physical method, and includes a vacuum distillation method, an ultrafiltration method, a dialysis method, an adsorption method, and a freeze-drying method.
  • the concentration treatment method of the method of the present invention is nanofiltration concentration.
  • Nanofiltration is a pressure-driven membrane separation process between reverse osmosis and ultrafiltration. Nanofiltration membranes are used as filter media. The pore size of nanofiltration membranes ranges from several About nanometers, it allows solvent molecules or some relatively low molecular weight solutes or low-priced ions to penetrate, so as to achieve the effect of separation and concentration.
  • the molecular weight cut-off of the nanofiltration membrane used in the method of the present invention is 300 ⁇ 50 Daltons.
  • the pre-treated solution is concentrated to a concentration of riboflavin sodium phosphate in the solution above 7.5 g / L.
  • the method for preparing high-purity riboflavin sodium phosphate provided by the present invention has the following advantages:
  • a riboflavin sodium phosphate product with a purity of more than 99% can be prepared.
  • the purification target of Example 1 is a crude flavin single nucleotide product purchased from the market, and the purification targets of Examples 2 to 5 are Bangtai Biological Engineering (Shenzhen) Co., Ltd. using biological enzymes. Catalytic method (with riboflavin and ATP as substrates and catalysis with riboflavin kinase) to prepare the crude product solution of flavin single nucleotide.
  • the purity of the flavin single nucleotide crude product to be purified was 30.20%, and the content was 40.14%.
  • the purification process is as follows: take 30.01 g of the crude flavin single nucleotide product to be purified, dissolve it in 1.0 L of purified water, and after fully dissolving, adjust the pH of the solution to 6.0 with sodium hydroxide; after heating to 30 ° C Add 0.5L of 95% (ml / ml) ethanol aqueous solution and mix well; slowly cool to 13 ° C and wait for the solid to precipitate ; On-line detection by HPLC, after the solid precipitation rate in the solution reaches more than 40%, slowly cool down to 4 ° C, and add 0.4L of 95% (ml / ml) ethanol solution, mix well and continue to wait Precipitation of solids; online detection by HPLC, after the solids extraction rate in the solution reaches more than 90%, filter, collect the filter cake, and wash the filter cake with a 75% (ml / ml) ethanol solution, and finally wash the The filter cake was dried under vacuum at 30 ° C for 5
  • the crude product solution to be purified had a pH of 6.60, a volume of 27 L, and a flavin mononucleotide content in the solution of 186.02 g.
  • the purification process was as follows: the pH of the crude product solution was adjusted to 11.50 with sodium hydroxide, heated to 40 ° C. and heated for 2 h, and then centrifuged, and the supernatant was pumped into an anion resin column of type LXQ510 through a peristaltic pump, 0.3 mol / L sodium chloride aqueous solution 120L elution impurities, 0.35mol / L sodium chloride aqueous solution 120L elution product, the product eluate was 122L, the product content was 175.04g, the yield was 94.10%; the product eluate was concentrated by nanofiltration, The nanofiltration membrane allows small molecules with a molecular weight below 300 to pass through.
  • the volume capacity of the nanofiltration membrane is 6L, and the volume of the eluent is finally concentrated to 19.5L.
  • the product content of the concentrated solution is 172.74g in terms of flavin single nucleotides, and the mass concentration is 8.85g / L; adjust the pH value of the concentrated solution to 6.25 with 6m ol / L hydrochloric acid solution, transfer to a 50L glass reactor, and slowly add 25.6L (1.3BV) 95% (ml / ml) ethanol aqueous solution, temperature Control at 25 ⁇ 30 ° C. After the addition, slowly reduce the temperature to 13 ° C. Perform on-line detection by HPLC.
  • the crude product solution to be purified has a pH of 6.87, a volume of 38 L, and a flavin single nucleotide content in the solution of 255.29 g.
  • the purification process is: adjusting the pH value of the crude product solution to 12.00 with sodium hydroxide, heating to 40 ° C. for 2 h, centrifuging, and taking the supernatant for use; regenerating 90 L of anionic resin (LXQ510), the processing method is
  • Purified water was rinsed to pH 9.00 ⁇ 1 0.00; the above-mentioned spare supernatant was pumped into a regenerated anion resin column through a peristaltic pump, and 0.3 mol / L sodium chloride aqueous solution was used to elute impurities at 180 L, 0.3 mol / L chlorine
  • the product was eluted with 180L of sodium chloride aqueous solution to obtain 250L of product eluent, with a product content of 243.31g and a yield of 95.31%.
  • the product eluate was concentrated by nanofiltration, and the nanofiltration membrane allowed small molecules with a molecular weight below 300 to pass through.
  • the volume capacity is 6L, and the volume of the eluent is finally concentrated to 16 L.
  • the content of the product in the concentrated solution is 242.42 g in terms of flavin single nucleotides, and the mass concentration is 15.15 g / L.
  • the pH of the concentrated solution is 6 m ol / L hydrochloric acid solution.
  • the value was adjusted to 5.86, transferred to a 50L glass reactor, and slowly added 11.6L (0.73BV) 95% (ml / ml) ethanol aqueous solution, the temperature was controlled at 25 ⁇ 30 ° C, after the addition was completed, slowly cooled to 13 ° C , On-line detection by HPLC, until the solid precipitation rate in the solution reaches 40% After that, continue to cool down to 4 ° C, and add 6.4L (0.4BV) 95% (ml / ml) ethanol aqueous solution, mix well, maintain 4 ° C and stir for 4h, filter, and filter cake with a small amount of 75% (ml / ml) ) Wash with ethanol solution, 30.
  • the crude product solution to be purified had a pH of 6.77, a volume of 35 L, and a flavin mononucleotide content in the solution of 265.81 g.
  • the purification process is: adjusting the pH of the crude product solution with sodium hydroxide to 11.00, heating to 40 ° C and heating for 2h after centrifugation, taking the supernatant for use; the supernatant with a pore size of O. lum of microfiltration Membrane microfiltration. Ultrafiltration is performed by ultrafiltration using ultrafiltration membrane. Ultrafiltration is directly pumped into a nanofiltration machine for nanofiltration and concentration. The nanofiltration membrane allows small molecules with a molecular weight below 300 to pass. The volume capacity of the nanofiltration membrane is 12L.
  • the volume of the eluent was finally concentrated to 18L, and the product content in the concentrate was 255.74g based on flavin single nucleotides, the mass concentration was 14.21g / L, and the yield was 96.21%; the pH of the concentrated solution was adjusted with 6mol / L hydrochloric acid For 6.10, transfer to 50L glass reaction vessel, wash the barrel wall with 1L purified water, slowly add 11.4L (0.6BV) 95% (ml / ml) ethanol aqueous solution, and control the temperature at 25 ⁇ 35 ° C, after adding, slowly reduce the temperature to 13 ° C, and perform on-line detection by HPLC.
  • the pH of the crude product solution to be purified was 6.65, the volume was 41 L, and the flavin mononucleotide content in the solution was 341.94 g.
  • the purification process is: adjust the pH of the crude product solution with sodium hydroxide to 11.75, heated to 40 ° C and heated for 2h after centrifugation, take the supernatant for backup; the supernatant with a membrane pore size of O. lum of the micro
  • the membrane is filtered. After the ultrafiltration, the filtrate is directly transferred to a nanofiltration machine for nanofiltration and concentration.
  • the ultrafiltration membrane mainly retains peptides and miscellaneous proteins with a molecular weight of more than 1,000.
  • the nanofiltration membrane allows small molecules with a molecular weight of less than 300 to pass through.
  • the volume capacity is 12L.
  • the volume of the eluent is finally concentrated to 21L.
  • the product content of the concentrated solution is 338.24g based on flavin single nucleotides and the mass concentration is 16.11g / L.
  • the pH value of the concentrated solution is adjusted with a 6mol / L hydrochloric acid solution. 5.10, transfer to a 50L glass reactor, and slowly add 11.00L (0.6BV) 95% (ml / ml) ethanol aqueous solution, the temperature is controlled at 25 ⁇ 30 ° C, after adding, slowly cool down to 10 ° C, pass HPLC was used for on-line detection.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A method for preparing high-purity riboflavin sodium phosphate, comprising: adjusting the pH value to 5.0-7.0; controlling the solution temperature at 20-40℃, and adding an ethanol aqueous solution with a concentration of 95％ (ml/ml); reducing the temperature to 3-5℃ and waiting for a solid to precipitate; filtering the solid, and drying a filter cake to obtain a pure product, the riboflavin sodium phosphate. The method is suitable for the large-scale isolation and purification of a riboflavin sodium phosphate product with a purity of at least 99% from a biological enzyme reaction liquid or biological fermentation broth.

Description

高纯度核黄素磷酸钠的制备方法 技术领域  Method for preparing high-purity riboflavin sodium phosphate TECHNICAL FIELD
[0001] 本发明涉及医药单体制备的技术领域， 具体涉及采用分离纯化技术制备高纯度 核黄素磷酸钠的方法。  [0001] The present invention relates to the technical field of preparation of medical monomers, and in particular, to a method for preparing high-purity riboflavin sodium phosphate by using a separation and purification technology.
背景技术  Background technique
[0002] 核黄素磷酸钠 （Riboflavin Sodium Phosphate， 英文缩写 RSP） 是核黄素（维生 素 B2）的磷酸盐形式， 又名维生素 B2磷酸钠、 黄素单核苷酸钠、 核黄素 -5'- （二 氢磷酸酯） 单钠盐等， 其 CAS号为 130-40-5， 其结构式如下图所示。  [0002] Riboflavin Sodium Phosphate (RSP) is a phosphate form of riboflavin (vitamin B2), also known as vitamin B2 sodium phosphate, flavin mononucleotide sodium, riboflavin-5'- (Dihydrogen phosphate) Monosodium salt, etc., whose CAS number is 130-40-5, and its structural formula is shown in the figure below.
Figure imgf000002_0001
Figure imgf000002_0001
[0004] 核黄素磷酸钠目前普遍用作核黄素补充剂， 用于治疗因核黄素缺乏弓 I起的口角 炎、 唇炎、 舌炎、 眼结膜炎及阴囊炎等疾病， 临床上常见剂型为注射剂和口服 片剂。  [0004] Riboflavin sodium phosphate is currently commonly used as a riboflavin supplement for the treatment of cheilitis, cheilitis, glossitis, conjunctivitis, and scrotal inflammation due to riboflavin deficiency. Common dosage forms are injections and oral tablets.
[0005] 目前， 国内制备核黄素磷酸钠的方法主要为化学合成法， 其一般是以高纯度的 核黄素为起始原料， 以三氯氧磷为磷酸化试剂， 在四氢呋喃、 乙腈和吡啶等有 机溶剂的环境中发生化学反应生成黄素单核苷酸， 黄素单核苷酸再经进一步转 化生成核黄素磷酸钠。 但是化学合成法对环境的污染较为严重， 随着国家对环 境保护的逐渐重视， 取而代之的生物催化法逐渐兴起， 其是利用生物酶催化底 物转化成黄素单核苷酸， 黄素单核苷酸再经进一步转化生成核黄素磷酸钠， 该 方法具有产品定向转化率高、 节能环保的优点。  [0005] At present, the domestic method for preparing riboflavin sodium phosphate is mainly a chemical synthesis method, which generally uses high-purity riboflavin as a starting material, phosphorus trichloride as a phosphorylation reagent, and tetrahydrofuran, acetonitrile, and Chemical reactions in the environment of organic solvents such as pyridine generate flavin single nucleotides, which are further converted to riboflavin sodium phosphate. However, the environmental pollution caused by chemical synthesis is more serious. As the country attaches more and more importance to environmental protection, biocatalytic methods have gradually emerged. It uses biological enzymes to catalyze the conversion of substrates into flavin single nucleotides. Further conversion into riboflavin sodium phosphate has the advantages of high product-specific conversion rate, energy saving and environmental protection.
[0006] 无论是化学合成法还是生物催化法， 其制备得到的核黄素磷酸钠产品的纯度往 往不够高， 尚需进行进一步的分离纯化处理。 如中国发明专利文献 CN106674225 A和 CN103396452B中均公开了一种通过结晶工艺制备高纯度核黄素磷酸钠的方 法， 但是其制备过程中需要使用大量有毒有害的有机溶剂， 如， CN106674225A 中需要使用乙酸甲酯、 丙酮、 环己醇和甲醇， 而 CN103396452B中需要使用乙腈 、 甲醇和氯仿， 不符合可持续发展的环保理念。 [0006] Whether it is a chemical synthesis method or a biocatalytic method, the purity of the riboflavin sodium phosphate product obtained by the method is It is not high enough, and further separation and purification treatment is needed. For example, both Chinese invention patent documents CN106674225 A and CN103396452B disclose a method for preparing high-purity riboflavin sodium phosphate through a crystallization process, but a large number of toxic and harmful organic solvents need to be used in the preparation process. For example, acetic acid is required in CN106674225A Methyl ester, acetone, cyclohexanol and methanol, while CN103396452B requires the use of acetonitrile, methanol and chloroform, which does not meet the environmental protection concept of sustainable development.
发明概述  Summary of invention
技术问题  technical problem
问题的解决方案  Problem solution
技术解决方案  Technical solutions
[0007] 本发明的目的在于提供一种节能环保、 成本低廉的制备高纯度核黄素磷酸钠的 新方法。  [0007] The object of the present invention is to provide a new method for preparing high-purity riboflavin sodium phosphate with low energy consumption, environmental protection, and low cost.
[0008] 为实现上述目的， 本发明提供了如下一种高纯度核黄素磷酸钠的制备方法， 包 括如下步骤： 1） 将核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液的 pH值调节至 5. 0-7.0； 2） 控制溶液温度为 20~40°C， 并加入占粗品溶液体积 0.5~1.5倍的浓度为 9 5% （ml/ml） 的乙醇水溶液； 3） 降温至 3~5°C， 等待固体析出； 4） 过滤， 除去 滤液， 滤饼经干燥处理后即得核黄素磷酸钠纯品。  [0008] To achieve the above object, the present invention provides the following method for preparing high-purity riboflavin sodium phosphate, including the following steps: 1) pH of a crude solution of riboflavin sodium phosphate and / or flavin single nucleotide Adjust the value to 5.0-7.0; 2) Control the solution temperature to 20 ~ 40 ° C, and add 0.5 to 1.5 times the volume of the crude solution to a 9 5% (ml / ml) ethanol aqueous solution; 3) Reduce the temperature to 3 ~ 5 ° C, wait for the solid to precipitate; 4) Filter and remove the filtrate. The filter cake is dried to obtain pure riboflavin sodium phosphate.
[0009] 黄素单核苷酸 （Flavin Mononucleotide， 英文缩写 FMN） ， 亦称核黄素 -5 -磷酸 ， 是黄素蛋白的辅基， 对呼吸等生物氧化过程的电子传递起着重要的作用， 即 作为黄素酶组的辅基， 以与酶蛋白 （apo— enzyme） 的结合状态， 参与电子从底 物向电子接受体传递， 在基础代谢中起重要作用， 其 CAS号为 146-17-8 , 其结构 式如下图所示。  [0009] Flavin Mononucleotide (English abbreviation FMN), also known as riboflavin-5-phosphate, is a prosthetic group of flavin protein, and plays an important role in the electron transfer of biological processes such as respiration, that is, As a prosthetic group of the flavinase group, it participates in the transfer of electrons from the substrate to the electron acceptor in a binding state with an apo-enzyme, and plays an important role in basic metabolism. Its CAS number is 146-17-8 Its structural formula is shown in the figure below.
[] [0010] 本发明方法中， 步骤 1） 的核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液是指除 了含有核黄素磷酸钠和 /或黄素单核苷酸之外还含有大量杂质的溶液， 包括： 将 从市场中购买的纯度不够高的核黄素磷酸钠和 /或黄素单核苷酸溶解于水后得到 的溶液， 生物催化法制备黄素单核苷酸得到的粗产物溶液。 [] [0010] In the method of the present invention, the crude solution of riboflavin sodium phosphate and / or flavin single nucleotide in step 1) refers to a solution containing a large amount of impurities in addition to sodium riboflavin phosphate and / or flavin single nucleotide. The solution includes: a solution obtained by dissolving riboflavin sodium phosphate and / or flavin mononucleotide purchased from the market with insufficient purity in water, and a crude product solution obtained by biocatalytic preparation of flavin mononucleotide.
[0011] 优选地， 控制核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液的浓度以核黄素磷 酸钠计为 7.5g/L以上， 以确保后期能够析出足够多的固体， 从而保证足够高的收 率。  [0011] Preferably, the concentration of the crude solution of the riboflavin sodium phosphate and / or the flavin mononucleotide is 7.5 g / L or more based on the sodium riboflavin phosphate, so as to ensure that sufficient solids can be precipitated at a later stage, thereby ensuring that High enough yield.
[0012] 生物催化 （biocatalysis） 是指利用生物酶或者生物有机体 （细胞、 细胞器、 组 织等） 作为催化剂进行物质转化的过程 （分别称为酶催化和生物发酵） ， 这种 反应过程又称为生物转化 （biotransformation） 。 生物催化中常用的生物有机体 主要是微生物， 其本质是利用微生物细胞内的酶进行催化， 促进生物转化的进 程。 生物催化法具有作用条件温和 （基本上在常温、 中性、 水等环境中完成） [0012] Biocatalysis (biocatalysis) refers to the process of transforming substances using biological enzymes or biological organisms (cells, organelles, tissues, etc.) as catalysts (referred to as enzyme catalysis and biological fermentation, respectively). This reaction process is also called biological Transformation (biotransformation). The biological organisms commonly used in biocatalysis are mainly microorganisms, and the essence is to use enzymes in the cells of the microorganisms to catalyze and promote the process of biological transformation. The biocatalytic method has mild working conditions (basically completed in normal temperature, neutral, water and other environments)
; 独特、 高效的底物选择性 （因为催化过程中的酶具有专一性的特点， 即一种 酶只能催化一种特定的底物发生反应， 但是一种底物则可能被多种酶催化） ； 对于手性活性药物成分的合成具有独特性的优点。 ; Unique and efficient substrate selectivity (because the enzymes in the catalytic process have specific characteristics, that is, one enzyme can only catalyze a specific substrate to react, but one substrate may be affected by multiple enzymes Catalysis); It has unique advantages for the synthesis of chiral active pharmaceutical ingredients.
[0013] 本发明所谓生物催化法制备黄素单核苷酸得到的粗产物溶液具体是指利用生物 酶催化底物转化成黄素单核苷酸后的酶反应液， 或者是利用含有生物酶的生物 有机体经发酵培养和诱导表达后去除生物有机体后的含有黄素单核苷酸的生物 发酵液。 其中所谓生物酶是指可以特异性催化底物转化成黄素单核苷酸的酶， 所谓底物是指可以转化成黄素单核苷酸的前体物质。 譬如， 底物可以是核黄素 和 ATP， 也可以是能够转化成核黄素和 /或 ATP的前体物质， 与之对应的生物酶 则是核黄素激酶 (EC 2.7.1.26) ， 或者是核黄素激酶 (EC 2.7.1.26) 和一种或者 多种其他酶的联合使用。 又譬如， 底物还可以是核黄素和 D-葡萄糖 -1-磷酸， 或 者是能够转化成核黄素和 /或 D-葡萄糖 -1-磷酸的前体物质， 与之对应的生物酶则 是核黄素磷酸转移酶 (EC2.7.1.42) ， 或者是核黄素磷酸转移酶 (EC2.7.1.42) 和一种或者多种其他酶的联合使用。 [0013] The so-called crude product solution obtained by preparing a flavin mononucleotide by a biocatalytic method in the present invention specifically refers to an enzyme reaction solution after converting a substrate to a flavin mononucleotide by using a biological enzyme to catalyze a substrate, or a biological organism containing a biological enzyme through A biological fermentation broth containing flavin single nucleotides after removing biological organisms after fermentation culture and induction of expression. The so-called biological enzyme refers to an enzyme that can specifically catalyze the conversion of a substrate into a flavin single nucleotide, and the so-called substrate refers to a precursor material that can be converted into a flavin single nucleotide. For example, the substrate can be riboflavin And ATP can also be precursors that can be converted to riboflavin and / or ATP, and the corresponding biological enzyme is riboflavin kinase (EC 2.7.1.26), or riboflavin kinase (EC 2.7.1.26) Used in combination with one or more other enzymes. For another example, the substrate can also be riboflavin and D-glucose-1-phosphate, or a precursor substance that can be converted into riboflavin and / or D-glucose-1-phosphate, and the corresponding biological enzyme is riboflavin. Phosphotransferase (EC2.7.1.42), or a combination of riboflavin phosphotransferase (EC2.7.1.42) and one or more other enzymes.
[0014] 本发明方法的步骤 1) 中， 将 pH值调节为 5.0~7.0的原因在于， 在该 pH值范围内 ， 可定向形成核黄素磷酸一钠盐。 当调节对象为黄素单核苷酸的粗品溶液时， 则加入氢氧化钠进行调节； 当调节对象为核黄素磷酸钠的粗品溶液时， 则根据 溶液 pH值的不同加入氢氧化钠或者盐酸进行调节。 为了提高目标产物的收率、 获得外观形态更好的核黄素磷酸钠产品， 优选地， 将溶液的 pH值调节为 5.5~6.5 ， 更优选地， 将溶液的 pH值调节为 6.0~6.3。  [0014] In step 1) of the method of the present invention, the reason why the pH value is adjusted to 5.0 to 7.0 is that within this pH value range, riboflavin phosphate monosodium salt can be formed. When the adjustment object is a crude solution of flavin single nucleotide, sodium hydroxide is added for adjustment; when the adjustment object is a crude solution of riboflavin sodium phosphate, sodium hydroxide or hydrochloric acid is added for adjustment according to the pH value of the solution . In order to increase the yield of the target product and obtain a riboflavin sodium phosphate product with a better appearance, the pH of the solution is preferably adjusted to 5.5 to 6.5, and more preferably, the pH of the solution is adjusted to 6.0 to 6.3.
[0015] 本发明方法的步骤 2) 中， 加入 95%乙醇作为不良溶剂， 同时降低水溶液的极 性， 以使核黄素磷酸钠在溶液中的溶解度降低， 从而达到过饱和状态而沉淀析 出。 优选地， 加入占溶液体积 0.5~0.9倍的浓度为 95% (ml/ml) 的乙醇水溶液， 一方面可节约成本， 另一方面可提高不同批次间产品的均一性。  [0015] In step 2) of the method of the present invention, 95% ethanol is added as a poor solvent, and the polarity of the aqueous solution is reduced to reduce the solubility of sodium riboflavin phosphate in the solution, thereby reaching a supersaturated state and precipitating. Preferably, adding a 95% (ml / ml) ethanol aqueous solution with a concentration of 0.5 to 0.9 times the volume of the solution can save costs on the one hand and improve the uniformity of products between different batches on the other.
[0016] 本发明方法的步骤 3) 中， 降温至 3~5°C时可获得收率和含量均较高的核黄素磷 酸钠产品， 温度高于此范围则收率较低， 温度低于此范围则含量降低。  [0016] In step 3) of the method of the present invention, a riboflavin sodium phosphate product with high yield and content can be obtained when the temperature is lowered to 3 ~ 5 ° C. If the temperature is higher than this range, the yield is lower and the temperature is lower. In this range, the content decreases.
[0017] 发明人研究发现， 固体的析出受温度的影响比较大， 如若降温速度过快， 则会 导致固体短时间内爆发性析出， 从而颗粒大小不一， 影响产品的形态， 甚至会 造成固体聚团析出， 从而包埋大量杂质， 以致产品含量不高。 为了获得含量更 高、 形态更好的产品， 优选地， 步骤 3) 的降温过程控制在 l~2h内完成。  [0017] The inventor's research found that the precipitation of solids is greatly affected by temperature. If the cooling rate is too fast, it will cause the solids to burst out within a short period of time, resulting in different particle sizes, affecting the morphology of the product, and even causing solids. The agglomerates are precipitated, so that a large amount of impurities are embedded, so that the product content is not high. In order to obtain a product with a higher content and a better form, preferably, the cooling process in step 3) is completed within 1-2 hours.
[0018] 更优选地， 步骤 3) 的降温过程按如下步骤操作： 先降温至 12~15°C， 待溶液中 的固体析出率达 40%以上后， 再降温至 3~5°C。  [0018] More preferably, the temperature reduction process of step 3) is performed according to the following steps: First, the temperature is lowered to 12 ~ 15 ° C, and after the solid precipitation rate in the solution reaches more than 40%, the temperature is lowered to 3 ~ 5 ° C.
[0019] 为了进一步提高步骤 3) 中析出固体的量， 优选地， 待溶液中的固体析出率达 4 0%以上后， 再加入占粗品溶液体积 0.3~0.5倍的浓度为 95% (ml/ml) 的乙醇水溶 液。  [0019] In order to further increase the amount of solids precipitated in step 3), preferably, after the solids precipitation rate in the solution reaches 40% or more, a concentration of 0.3% to 0.5 times the volume of the crude solution is added to a concentration of 95% (ml / ml) of ethanol.
[0020] 本发明方法步骤 4) 中的过滤是指采用物理方法将溶液中的固体和液体进行分 离的过程， 常见的过滤方法均适用于本发明方法， 包括常压过滤、 减压过滤、 离心过滤等。 [0020] Filtration in step 4) of the method of the present invention refers to using a physical method to separate solids and liquids in the solution. In the separation process, common filtration methods are applicable to the method of the present invention, including normal pressure filtration, reduced pressure filtration, centrifugal filtration, and the like.
[0021] 本发明方法的步骤 4） 中， 优选地， 在干燥处理前， 用浓度为 75% （ml/ml） 的 乙醇水溶液对所得滤饼进行洗涤。  [0021] In step 4) of the method of the present invention, preferably, the obtained filter cake is washed with a 75% (ml / ml) ethanol aqueous solution before the drying treatment.
[0022] 更优选地， 待用浓度为 75% （ml/ml） 的乙醇水溶液洗涤后， 再用浓度为 95% （ ml/ml） 的乙醇水溶液洗涤， 可最大程度确保附着在产品表面的杂质被洗涤干净  [0022] More preferably, after washing with a 75% (ml / ml) ethanol aqueous solution, and then washing with a 95% (ml / ml) ethanol aqueous solution, the impurities attached to the surface of the product can be ensured to the greatest extent. Washed up
[0023] 为进一步降低杂质残留、 提高产品含量， 优选地， 在洗涤滤饼之前， 先用 50% 乙醇水溶液对所得滤饼进行打浆， 然后过滤收集滤饼。 所谓打浆， 是指向固体 物质中加入某种对目标物质溶解度较低而对其他有关物质有较高溶解度的溶剂 ， 通过搅拌使其他不需要的有关物质溶解在溶剂中， 而目标物质与溶剂呈悬浊 状态的过程。 [0023] In order to further reduce the residue of impurities and increase the product content, preferably, before washing the filter cake, the obtained filter cake is beaten with a 50% ethanol aqueous solution, and then the filter cake is collected by filtration. The so-called beating refers to adding a certain kind of solvent with a lower solubility to the target substance and a higher solubility to other related substances to the solid substance, and dissolving other unnecessary related substances in the solvent by stirring, and the target substance and the solvent are suspended. Cloudy process.
[0024] 优选地， 本发明方法的步骤 4） 中， 干燥处理的方式为于 30°C的温度下真空干 燥 5~ 10小时。  [0024] Preferably, in step 4) of the method of the present invention, the drying method is vacuum drying at a temperature of 30 ° C. for 5 to 10 hours.
[0025] 为了进一步提高核黄素磷酸钠纯品的纯度， 优选地， 将步骤 4） 所得的滤饼或 者核黄素磷酸钠纯品用纯化水溶解后重复本发明方法步骤 1） 至步骤 4） 的操作  [0025] In order to further improve the purity of the pure riboflavin sodium phosphate, preferably, the filter cake obtained in step 4) or the pure sodium riboflavin phosphate is dissolved in purified water, and steps 1) to 4 of the method of the present invention are repeated. ) Operation
[0026] 当本发明方法步骤 1） 的核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液为生物催 化法制备黄素单核苷酸得到的粗产物溶液时， 溶液中往往会含有残留的部分生 物酶， 为防止在对核黄素磷酸钠的分离纯化过程中残留酶使目标产物发生降解 ， 优选在步骤 1） 之前， 对核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液进行如下 前序处理： a） 将核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液的 pH值调节至 11.0 -12.0； b） 将溶液温度升温至 40±1°C， 并于 40±1°C温度下保持 2h; C） 除去不溶 物。 [0026] When the crude solution of riboflavin sodium phosphate and / or flavin mononucleotide in step 1) of the method of the present invention is a crude product solution obtained by preparing a flavin mononucleotide by a biocatalytic method, the solution often contains a residual portion Biological enzymes, in order to prevent degradation of the target product by residual enzymes during the separation and purification of sodium riboflavin phosphate, it is preferable to perform the following crude solution of riboflavin sodium phosphate and / or flavin single nucleotide before step 1) Pretreatment: a) The pH value of the crude solution of riboflavin sodium phosphate and / or flavin single nucleotide is adjusted to 11.0 -12.0; b) The temperature of the solution is raised to 40 ± 1 ° C, and at 40 ± 1 ° C for 2 h; C) remove insolubles.
[0027] 上述前序处理中， 通过将溶液的 pH值调节至 11.0~12.0, 再辅以 40±1°C温度下 加热处理， 可使溶液中残留的生物酶失活。 优选地， 使用氢氧化钠调节 pH值， 可使黄素单核苷酸转化为核黄素磷酸钠； 特别地， pH值的优选范围为 11.0~11.5 ， 此范围内目标产物的稳定性最好。 [0028] 上述前序处理的步骤 c） 中， 除去不溶物的方法优选为： 将步骤 b） 加热后的溶 液进行离心处理， 收集上清液。 [0027] In the foregoing pre-treatment, by adjusting the pH value of the solution to 11.0 to 12.0, and supplemented with heat treatment at a temperature of 40 ± 1 ° C., the biological enzymes remaining in the solution can be inactivated. Preferably, sodium hydroxide is used to adjust the pH value, so that the flavin single nucleotide can be converted into sodium riboflavin phosphate; in particular, the preferred range of pH value is 11.0 ~ 11.5, and the stability of the target product is the best in this range. [0028] In step c) of the pre-processing, the method of removing insoluble matter is preferably: centrifuging the solution after the heating in step b) to collect the supernatant.
[0029] 当本发明方法步骤 1） 的核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液为生物催 化法制备黄素单核苷酸得到的粗产物溶液时， 往往后续步骤 4） 的过滤操作会比 较困难， 容易造成过滤膜的堵塞， 为了使过滤操作更容易， 优选地， 上述前序 处理还包括将步骤 c） 获得的去除不溶物后的溶液进行预处理的步骤， 以除去粗 产物溶液中残留的杂质； 所谓预处理是指用离子交换树脂对去除不溶物后的溶 液进行纯化， 或者将去除不溶物后的溶液依次经微滤和超滤处理。  [0029] When the crude solution of riboflavin sodium phosphate and / or flavin mononucleotide in step 1) of the method of the present invention is a crude product solution obtained by preparing a flavin mononucleotide by a biocatalytic method, the filtering operation in step 4) is often followed. It will be more difficult to easily cause clogging of the filtration membrane. In order to make the filtration operation easier, preferably, the above-mentioned pre-treatment further includes a step of pre-treating the solution obtained by removing insoluble matter obtained in step c) to remove the crude product solution. Residual impurities in the residue; The so-called pretreatment refers to purifying the solution after removing the insoluble matter with an ion exchange resin, or sequentially subjecting the solution after removing the insoluble matter to microfiltration and ultrafiltration.
[0030] 微滤又称微孔过滤， 以微孔滤膜为过滤介质， 在 0.1〜 0.3MPa的压力推动下， 截留 0.1〜 1微米之间的颗粒和细菌， 但能允许大分子有机物和无机盐等通过。 本 发明方法中使用的微滤膜优选为 0.1pm孔径的中空纤维膜。  [0030] Microfiltration, also known as microporous filtration, uses a microporous filter membrane as a filter medium and, under the pressure of 0.1 to 0.3 MPa, traps particles and bacteria between 0.1 and 1 micron, but allows macromolecular organics and inorganics. Salt passed. The microfiltration membrane used in the method of the present invention is preferably a hollow fiber membrane having a pore diameter of 0.1 pm.
[0031] 超滤又称超过滤， 其原理与微滤相同， 区别仅在于， 超滤使用的超滤膜的孔径 小于微滤膜， 一般在 0.01微米以下。  [0031] Ultrafiltration is also called ultrafiltration, and its principle is the same as that of microfiltration. The only difference is that the pore size of the ultrafiltration membrane used in ultrafiltration is smaller than that of the microfiltration membrane, and is generally below 0.01 microns.
[0032] 优选地， 上述离子交换树脂为阴离子交换树脂。  [0032] Preferably, the ion exchange resin is an anion exchange resin.
[0033] 更优选地， 上述阴离子交换树脂为弱碱性阴离子交换树脂或大孔吸附树脂。  [0033] More preferably, the anion exchange resin is a weakly basic anion exchange resin or a macroporous adsorption resin.
[0034] 优选地， 用 0.1~0.4mol/L氯化钠水溶液作为离子交换树脂的洗脱液； 为提高产 品收率， 更优选地， 氯化钠水溶液的浓度为 0.3~0.35mol/L。  [0034] Preferably, a 0.1 to 0.4 mol / L sodium chloride aqueous solution is used as the eluent of the ion exchange resin; in order to improve the yield of the product, more preferably, the concentration of the sodium chloride aqueous solution is 0.3 to 0.35 mol / L.
[0035] 优选地， 对离子交换树脂进行再生处理的方法包括如下步骤： A、 用 2mol/L氯 化钠溶液与 0.5mol/L盐酸溶液的混合液淋洗树脂至流出液中无杂质残留； B、 用 纯化水淋洗至洗脱液的 pH值为 5.00~7.00; C、 用 0.5mol/L氢氧化钠溶液淋洗至洗 脱液呈强碱性； D、 用纯化水淋洗至洗脱液的 pH值为 9.00~10.00。  [0035] Preferably, the method for regenerating the ion exchange resin includes the following steps: A. The resin is rinsed with a mixed solution of a 2mol / L sodium chloride solution and a 0.5mol / L hydrochloric acid solution until no impurities remain in the effluent; B. Rinse with purified water until the pH value of the eluent is 5.00 ~ 7.00; C. Rinse with 0.5mol / L sodium hydroxide solution until the eluent becomes strongly alkaline; D. Rinse with purified water until washing The dehydration pH is 9.00 ~ 10.00.
[0036] 为了提高后续操作的效率， 减少溶剂的使用量， 并提高目的产物的收率， 优选 地， 在步骤 1） 之前， 将上述预处理后的溶液进行浓缩处理， 以除去大量的水分  [0036] In order to improve the efficiency of subsequent operations, reduce the amount of solvent used, and increase the yield of the target product, preferably, before step 1), the above pretreated solution is concentrated to remove a large amount of water
[0037] 本发明方法中的浓缩处理是指采用物理方法使溶剂减少而提高溶液的浓度的过 程， 包括减压蒸馏法、 超过滤法、 透析法、 吸附法、 冷冻干燥法等。 [0037] The concentration treatment in the method of the present invention refers to a process in which the concentration of a solution is increased by reducing the solvent using a physical method, and includes a vacuum distillation method, an ultrafiltration method, a dialysis method, an adsorption method, and a freeze-drying method.
[0038] 优选地， 本发明方法的浓缩处理方式为纳滤浓缩。 纳滤是一种介于反渗透和超 滤之间的压力驱动膜分离过程， 以纳滤膜为过滤介质， 纳滤膜的孔径范围在几 个纳米左右， 允许溶剂分子或某些相对分子质量较小的溶质或低价离子透过， 从而达到分离和浓缩的效果。 优选地， 本发明方法选用的纳滤膜的截留分子量 为 300±50道尔顿。 [0038] Preferably, the concentration treatment method of the method of the present invention is nanofiltration concentration. Nanofiltration is a pressure-driven membrane separation process between reverse osmosis and ultrafiltration. Nanofiltration membranes are used as filter media. The pore size of nanofiltration membranes ranges from several About nanometers, it allows solvent molecules or some relatively low molecular weight solutes or low-priced ions to penetrate, so as to achieve the effect of separation and concentration. Preferably, the molecular weight cut-off of the nanofiltration membrane used in the method of the present invention is 300 ± 50 Daltons.
[0039] 为获得较高的产品收率， 优选地， 将上述预处理后的溶液浓缩至溶液中核黄素 磷酸钠的浓度在 7.5g/L以上。  [0039] In order to obtain a higher product yield, preferably, the pre-treated solution is concentrated to a concentration of riboflavin sodium phosphate in the solution above 7.5 g / L.
发明的有益效果  The beneficial effects of the invention
有益效果  Beneficial effect
[0040] 与现有的纯化方法相比， 本发明提供的高纯度核黄素磷酸钠的制备方法具有如 下优点：  [0040] Compared with the existing purification methods, the method for preparing high-purity riboflavin sodium phosphate provided by the present invention has the following advantages:
[0041] 1、 全程无需使用有毒有害溶剂， 绿色无污染， 且对生产操作人员不会造成健 康损害；  [0041] 1. There is no need to use toxic and harmful solvents throughout the process, green and pollution-free, and will not cause health damage to production operators;
[0042] 2、 工艺简单易操作， 无特殊技能要求及安全防护要求；  [0042] 2, the process is simple and easy to operate, no special skills requirements and safety protection requirements;
[0043] 3、 工艺步骤较少， 效率和收率高， 成本低廉， 经济效益显著；  [0043] 3. There are fewer process steps, high efficiency and yield, low cost, and significant economic benefits;
[0044] 4、 可制备得到纯度在 99%以上的核黄素磷酸钠产品。  [0044] 4. A riboflavin sodium phosphate product with a purity of more than 99% can be prepared.
发明实施例  Invention Examples
本发明的实施方式  Embodiments of the invention
[0045] 下面结合具体实施例对本发明做进一步的详细说明， 以下实施例是对本发明的 解释， 本发明并不局限于以下实施例。  [0045] The present invention is further described in detail with reference to specific embodiments. The following embodiments are an explanation of the present invention, and the present invention is not limited to the following embodiments.
[0046] 以下实施例中所使用的原料及试剂， 除特别指明的以外， 均为从市场中购入。  [0046] Unless otherwise specified, the raw materials and reagents used in the following examples were purchased from the market.
[0047] 以下实施例中， 实施例 1的纯化对象为从市场中购入的黄素单核苷酸粗产品， 实施例 2至实施例 5的纯化对象为邦泰生物工程 （深圳） 有限公司采用生物酶催 化法 （以核黄素和 ATP为底物， 用核黄素激酶进行催化） 制备黄素单核苷酸得到 的粗产物溶液。 [0047] In the following examples, the purification target of Example 1 is a crude flavin single nucleotide product purchased from the market, and the purification targets of Examples 2 to 5 are Bangtai Biological Engineering (Shenzhen) Co., Ltd. using biological enzymes. Catalytic method (with riboflavin and ATP as substrates and catalysis with riboflavin kinase) to prepare the crude product solution of flavin single nucleotide.
[0048] 实施例 1  Example 1
[0049] 经检测， 待纯化的黄素单核苷酸粗产品的纯度为 30.20%， 含量为 40.14%。  [0049] After testing, the purity of the flavin single nucleotide crude product to be purified was 30.20%, and the content was 40.14%.
[0050] 纯化过程为： 取待纯化的黄素单核苷酸粗产品 30.01g， 溶解于 1.0L纯化水中， 待充分溶解后用氢氧化钠将溶液的 pH值调节至 6.0; 升温至 30°C后， 加入 0.5L浓 度为 95% （ml/ml） 的乙醇水溶液， 混合均匀； 缓慢降温至 13°C， 等待固体析出 ; 通过 HPLC进行在线检测， 待溶液中的固体析出率达 40%以上后， 再缓慢降温 至 4°C， 并加入 0.4L浓度为 95% (ml/ml) 的乙醇水溶液， 混合均匀， 继续等待固 体析出； 通过 HPLC进行在线检测， 待溶液中的固体析出率达 90%以上后， 过滤 ， 收集滤饼， 并用浓度为 75% (ml/ml) 的乙醇水溶液洗涤滤饼， 最后将洗涤后 的滤饼置于 30°C的温度下真空干燥 5小时后得橙黄色核黄素磷酸钠固体粉末 10.72 g， 干燥失重 4.48%， 钠离子含量 4.50%， 比旋光度为 +38.37°， 纯度为 99.33%; 含 量 (以核黄素为外标， 以干品计) 为 77.28% (药典要求范围为 74.0%-79.0%) ， 总收率以黄素单核苷酸计为 80.97%。 [0050] The purification process is as follows: take 30.01 g of the crude flavin single nucleotide product to be purified, dissolve it in 1.0 L of purified water, and after fully dissolving, adjust the pH of the solution to 6.0 with sodium hydroxide; after heating to 30 ° C Add 0.5L of 95% (ml / ml) ethanol aqueous solution and mix well; slowly cool to 13 ° C and wait for the solid to precipitate ; On-line detection by HPLC, after the solid precipitation rate in the solution reaches more than 40%, slowly cool down to 4 ° C, and add 0.4L of 95% (ml / ml) ethanol solution, mix well and continue to wait Precipitation of solids; online detection by HPLC, after the solids extraction rate in the solution reaches more than 90%, filter, collect the filter cake, and wash the filter cake with a 75% (ml / ml) ethanol solution, and finally wash the The filter cake was dried under vacuum at 30 ° C for 5 hours to obtain 10.72 g of orange-yellow riboflavin sodium phosphate solid powder, drying loss of 4.48%, sodium ion content of 4.50%, specific rotation of + 38.37 °, and purity of 99.33 %; The content (with riboflavin as the external standard and the dry product) was 77.28% (the pharmacopoeia requirements ranged from 74.0% to 79.0%), and the total yield was 80.97% based on the flavin single nucleotide.
[0051] 实施例 2  Example 2
[0052] 经检测， 待纯化的粗产物溶液的 pH值为 6.60， 体积为 27L， 溶液中黄素单核苷 酸的含量为 186.02g。  [0052] After testing, the crude product solution to be purified had a pH of 6.60, a volume of 27 L, and a flavin mononucleotide content in the solution of 186.02 g.
[0053] 纯化过程为： 用氢氧化钠调节粗产物溶液的 pH值为 11.50， 升温至 40°C加热 2h 后离心， 上清液通过蠕动泵泵入型号为 LXQ510的阴离子树脂柱中， 0.3mol/L氯 化钠水溶液 120L洗脱杂质， 0.35mol/L氯化钠水溶液 120L洗脱产品， 得产品洗脱 液 122L， 产品含量 175.04g， 收率 94.10% ; 将产品洗脱液纳滤浓缩， 纳滤膜允许 分子量低于 300的小分子通过， 纳滤膜体积容量为 6L， 洗脱液体积最终浓缩至 19. 5L， 浓缩液中产品含量以黄素单核苷酸计为 172.74g， 质量浓度为 8.85g/L; 用 6m ol/L盐酸溶液将浓缩液的 pH值调节为 6.25， 转入 50L玻璃反应釜中， 缓慢加入 25. 6L ( 1.3BV) 95% (ml/ml) 乙醇水溶液， 温度控制在 25~30°C， 加毕， 缓慢降温 至 13°C， 通过 HPLC进行在线检测， 待溶液中的固体析出率达 40%以上后， 继续 降温至 4°C， 维持 4°C搅拌 4h， 过滤， 滤饼用少量 75% (ml/ml) 乙醇水溶液洗涤 ， 30°C真空干燥 10h得橙黄色核黄素磷酸钠固体粉末 166.59g， 干燥失重 4.34%， 钠离子含量 4.72%， 比旋光度为 +38.68°， 纯度为 99.28%; 含量 (以核黄素为外标 ， 以干品计) 为 77.86% (药典要求范围为 74.0%-79.0%) ， 总收率以黄素单核苷 酸计为 81.44%。  [0053] The purification process was as follows: the pH of the crude product solution was adjusted to 11.50 with sodium hydroxide, heated to 40 ° C. and heated for 2 h, and then centrifuged, and the supernatant was pumped into an anion resin column of type LXQ510 through a peristaltic pump, 0.3 mol / L sodium chloride aqueous solution 120L elution impurities, 0.35mol / L sodium chloride aqueous solution 120L elution product, the product eluate was 122L, the product content was 175.04g, the yield was 94.10%; the product eluate was concentrated by nanofiltration, The nanofiltration membrane allows small molecules with a molecular weight below 300 to pass through. The volume capacity of the nanofiltration membrane is 6L, and the volume of the eluent is finally concentrated to 19.5L. The product content of the concentrated solution is 172.74g in terms of flavin single nucleotides, and the mass concentration is 8.85g / L; adjust the pH value of the concentrated solution to 6.25 with 6m ol / L hydrochloric acid solution, transfer to a 50L glass reactor, and slowly add 25.6L (1.3BV) 95% (ml / ml) ethanol aqueous solution, temperature Control at 25 ~ 30 ° C. After the addition, slowly reduce the temperature to 13 ° C. Perform on-line detection by HPLC. After the solid precipitation rate in the solution reaches more than 40%, continue to cool to 4 ° C and maintain 4 ° C for 4h. , Filter, filter cake with a small amount of 75% (ml / ml) Washed with an alcohol solution and dried under vacuum at 30 ° C for 10h to obtain 166.59g of orange-yellow riboflavin sodium phosphate solid powder, 4.34% loss on drying, sodium ion content of 4.72%, specific rotation of + 38.68 °, and purity of 99.28%; Riboflavin is an external standard, calculated on a dry basis, at 77.86% (the Pharmacopoeia requires a range of 74.0% -79.0%), and the total yield is 81.44% based on flavin single nucleotides.
[0054] 实施例 3  Example 3
[0055] 经检测， 待纯化的粗产物溶液的 pH值为 6.87， 体积为 38L， 溶液中黄素单核苷 酸的含量为 255.29g。 [0056] 纯化过程为： 用氢氧化钠调节粗产物溶液的 pH值为 12.00， 升温至 40°C加热 2h 后离心， 取上清液备用； 将阴离子树脂 (LXQ510) 90L再生处理， 处理方法为[0055] After testing, the crude product solution to be purified has a pH of 6.87, a volume of 38 L, and a flavin single nucleotide content in the solution of 255.29 g. [0056] The purification process is: adjusting the pH value of the crude product solution to 12.00 with sodium hydroxide, heating to 40 ° C. for 2 h, centrifuging, and taking the supernatant for use; regenerating 90 L of anionic resin (LXQ510), the processing method is
: 用 2mol/L : Use 2mol / L
氯化钠 +0.5mol/L盐酸水溶液 90L淋洗树脂， 纯化水淋洗至 pH为 5.00~7.00， 再用 0. 5mol/L氢氧化钠溶液 90L淋洗至流出液呈强碱性， 最后用纯化水淋洗至 PH9.00~ 1 0.00； 将前述备用的上清液通过蠕动泵泵入再生好的阴离子树脂柱中， 0.3mol/L 氯化钠水溶液 180L洗脱杂质， 0.3mol/L氯化钠水溶液 180L洗脱产品， 得产品洗脱 液 250L， 产品含量 243.31g， 收率 95.31% ; 将产品洗脱液纳滤浓缩， 纳滤膜允许 分子量低于 300的小分子通过， 纳滤膜体积容量为 6L， 洗脱液体积最终浓缩至 16 L， 浓缩液中产品含量以黄素单核苷酸计为 242.42g， 质量浓度为 15.15g/L; 用 6m ol/L盐酸溶液将浓缩液的 pH值调节为 5.86 , 转入 50L玻璃反应釜中， 缓慢加入 11. 6L (0.73BV) 95% (ml/ml) 乙醇水溶液， 温度控制在 25~30°C， 加毕， 缓慢降温 至 13°C， 通过 HPLC进行在线检测， 待溶液中的固体析出率达 40%以上后， 继续 降温至 4°C， 并加入 6.4L (0.4BV) 95% (ml/ml) 的乙醇水溶液， 混合均匀， 维 持 4°C搅拌 4h， 过滤， 滤饼用少量 75% (ml/ml) 乙醇水溶液洗涤， 30。(：真空干燥 10h得橙黄色核黄素磷酸钠固体粉末 233.86g， 干燥失重 4.40%， 钠离子含量 4.32% ， 比旋光度为 +38.20°， 纯度为 99.09%; 含量 (以核黄素为外标， 以干品计) 为 7 6.04% (药典要求范围为 74.0%-79.0%) ， 总收率以黄素单核苷酸计为 83.62%。  90L of sodium chloride + 0.5mol / L hydrochloric acid aqueous solution was used to rinse the resin, and the purified water was rinsed to a pH of 5.00 to 7.00, and then rinsed with 0.5L of 0.5mol / L sodium hydroxide solution until the effluent became strongly alkaline. Purified water was rinsed to pH 9.00 ~ 1 0.00; the above-mentioned spare supernatant was pumped into a regenerated anion resin column through a peristaltic pump, and 0.3 mol / L sodium chloride aqueous solution was used to elute impurities at 180 L, 0.3 mol / L chlorine The product was eluted with 180L of sodium chloride aqueous solution to obtain 250L of product eluent, with a product content of 243.31g and a yield of 95.31%. The product eluate was concentrated by nanofiltration, and the nanofiltration membrane allowed small molecules with a molecular weight below 300 to pass through. The volume capacity is 6L, and the volume of the eluent is finally concentrated to 16 L. The content of the product in the concentrated solution is 242.42 g in terms of flavin single nucleotides, and the mass concentration is 15.15 g / L. The pH of the concentrated solution is 6 m ol / L hydrochloric acid solution. The value was adjusted to 5.86, transferred to a 50L glass reactor, and slowly added 11.6L (0.73BV) 95% (ml / ml) ethanol aqueous solution, the temperature was controlled at 25 ~ 30 ° C, after the addition was completed, slowly cooled to 13 ° C , On-line detection by HPLC, until the solid precipitation rate in the solution reaches 40% After that, continue to cool down to 4 ° C, and add 6.4L (0.4BV) 95% (ml / ml) ethanol aqueous solution, mix well, maintain 4 ° C and stir for 4h, filter, and filter cake with a small amount of 75% (ml / ml) ) Wash with ethanol solution, 30. (: Vacuum drying for 10h to obtain 233.86g of orange-yellow riboflavin sodium phosphate solid powder, dry weight loss of 4.40%, sodium ion content of 4.32%, specific rotation of + 38.20 °, purity of 99.09%; content (with riboflavin as the outer (Standard, based on dry product) is 7 6.04% (pharmacopoeia requirement range is 74.0% -79.0%), and the total yield is 83.62% based on flavin single nucleotide.
[0057] 实施例 4 Example 4
[0058] 经检测， 待纯化的粗产物溶液的 pH值为 6.77， 体积为 35L， 溶液中黄素单核苷 酸含量为 265.81g。  [0058] After testing, the crude product solution to be purified had a pH of 6.77, a volume of 35 L, and a flavin mononucleotide content in the solution of 265.81 g.
[0059] 纯化过程为： 用氢氧化钠调节粗产物溶液的 pH值为 11.00， 升温至 40°C加热 2h 后离心， 取上清液备用； 将上清液用孔径为 O. lum的微滤膜微滤， 微滤液再用超 滤膜进行超滤， 超滤液直接泵入纳滤机进行纳滤浓缩， 纳滤膜允许分子量低于 3 00的小分子通过， 纳滤膜体积容量为 12L， 洗脱液体积最终浓缩至 18L， 浓缩液 中产品含量以黄素单核苷酸计为 255.74g， 质量浓度为 14.21g/L， 收率 96.21% ; 用 6mol/L盐酸将浓缩液的 pH值调节为 6.10， 转入 50L玻璃反应藎中， 用 1L纯化水 洗涤桶壁， 缓慢加入 11.4L (0.6BV) 95% (ml/ml) 乙醇水溶液， 温度控制在 25~ 35°C， 加毕， 缓慢降温至 13°C， 通过 HPLC进行在线检测， 待溶液中的固体析出 率达 40%以上后， 继续降温至 6°C， 维持 6°C搅拌 4h， 过滤， 滤饼先用少量 75% ( ml/ml) 乙醇水溶液洗涤， 再用少量浓度为 95% (ml/ml) 的乙醇水溶液洗涤， 30 °(：真空干燥 10h得橙黄色核黄素磷酸钠固体粉末 266.95g， 干燥失重 4.60%， 钠离 子含量 4.54%， 比旋光度为 +38.89°， 纯度为 99.43%; 含量 (以核黄素为外标， 以 干品计) 为 77.64% (药典要求范围为 74.0%-79.0%) ， 总收率以黄素单核苷酸计 为 91.25%。 Lum 的 微 filtration [0059] The purification process is: adjusting the pH of the crude product solution with sodium hydroxide to 11.00, heating to 40 ° C and heating for 2h after centrifugation, taking the supernatant for use; the supernatant with a pore size of O. lum of microfiltration Membrane microfiltration. Ultrafiltration is performed by ultrafiltration using ultrafiltration membrane. Ultrafiltration is directly pumped into a nanofiltration machine for nanofiltration and concentration. The nanofiltration membrane allows small molecules with a molecular weight below 300 to pass. The volume capacity of the nanofiltration membrane is 12L. The volume of the eluent was finally concentrated to 18L, and the product content in the concentrate was 255.74g based on flavin single nucleotides, the mass concentration was 14.21g / L, and the yield was 96.21%; the pH of the concentrated solution was adjusted with 6mol / L hydrochloric acid For 6.10, transfer to 50L glass reaction vessel, wash the barrel wall with 1L purified water, slowly add 11.4L (0.6BV) 95% (ml / ml) ethanol aqueous solution, and control the temperature at 25 ~ 35 ° C, after adding, slowly reduce the temperature to 13 ° C, and perform on-line detection by HPLC. After the solids precipitation rate in the solution reaches more than 40%, continue to cool to 6 ° C, maintain 6 ° C and stir for 4h, filter, filter The cake was first washed with a small amount of 75% (ml / ml) ethanol aqueous solution, and then with a small amount of 95% (ml / ml) ethanol aqueous solution, and then dried at 30 ° (: vacuum for 10h to obtain orange-yellow riboflavin sodium phosphate solid powder 266.95 g, dry weight loss 4.60%, sodium ion content 4.54%, specific rotation is + 38.89 °, purity is 99.43%; content (with riboflavin as external standard, based on dry product) is 77.64% (pharmacopoeia requirement range is 74.0 % -79.0%), and the total yield was 91.5% based on flavin single nucleotides.
[0060] 实施例 5  Example 5
[0061] 经检测， 待纯化的粗产物溶液的 pH值为 6.65， 体积为 41L， 溶液中黄素单核苷 酸含量为 341.94g。  [0061] After testing, the pH of the crude product solution to be purified was 6.65, the volume was 41 L, and the flavin mononucleotide content in the solution was 341.94 g.
[0062] 纯化过程为： 用氢氧化钠调节粗产物溶液的 pH值为 11.75， 升温至 40°C加热 2h 后离心， 取上清液备用； 将上清液用膜孔径为 O. lum的微滤膜过滤， 滤液经超滤 后直接转入纳滤机进行纳滤浓缩， 超滤膜主要截留分子量超过 1000的多肽及杂 蛋白， 纳滤膜允许分子量低于 300的小分子通过， 纳滤膜体积容量为 12L， 洗脱 液体积最终浓缩至 21L， 浓缩液中产品含量以黄素单核苷酸计为 338.24g， 质量浓 度为 16.11g/L; 用 6mol/L盐酸溶液将浓缩液的 pH值调节为 5.10， 转入 50L玻璃反 应釜中， 缓慢加入 11.00L (0.6BV) 95% (ml/ml) 乙醇水溶液， 温度控制在 25~3 0°C, 加毕， 缓慢降温至 10°C， 通过 HPLC进行在线检测， 待溶液中的固体析出率 达 40%以上后， 继续降温至 6°C， 并加入 6.4L (0.4BV) 95% (ml/ml) 的乙醇水 溶液， 混合均匀， 维持 6°C搅拌 4h， 过滤， 滤饼用 50%乙醇水溶液 2L打浆 2h， 温 度控制在 10~15°C， 过滤， 滤饼用少量 75% (ml/ml) 乙醇水溶液洗涤， 30°C真空 干燥 10h得橙黄色核黄素磷酸钠固体粉末 339.07g， 干燥失重 4.86%， 钠离子含量 4 .45% , 比旋光度为 +38.83°， 纯度为 99.34%; 含量 (以核黄素为外标， 以干品计 ) 为 77.30% (药典要求范围为 74.0%-79.0%) ， 总收率以黄素单核苷酸计为 89.93 Lum 的 微。 [0062] The purification process is: adjust the pH of the crude product solution with sodium hydroxide to 11.75, heated to 40 ° C and heated for 2h after centrifugation, take the supernatant for backup; the supernatant with a membrane pore size of O. lum of the micro The membrane is filtered. After the ultrafiltration, the filtrate is directly transferred to a nanofiltration machine for nanofiltration and concentration. The ultrafiltration membrane mainly retains peptides and miscellaneous proteins with a molecular weight of more than 1,000. The nanofiltration membrane allows small molecules with a molecular weight of less than 300 to pass through. The volume capacity is 12L. The volume of the eluent is finally concentrated to 21L. The product content of the concentrated solution is 338.24g based on flavin single nucleotides and the mass concentration is 16.11g / L. The pH value of the concentrated solution is adjusted with a 6mol / L hydrochloric acid solution. 5.10, transfer to a 50L glass reactor, and slowly add 11.00L (0.6BV) 95% (ml / ml) ethanol aqueous solution, the temperature is controlled at 25 ~ 30 ° C, after adding, slowly cool down to 10 ° C, pass HPLC was used for on-line detection. After the solid precipitation rate in the solution reached more than 40%, the temperature was further reduced to 6 ° C, and 6.4L (0.4BV) 95% (ml / ml) ethanol aqueous solution was added, mixed uniformly, and maintained at 6 ° Stir for 4h, filter, filter cake with 50% ethanol water 2L slurry for 2h, control the temperature at 10 ~ 15 ° C, filter, wash the filter cake with a small amount of 75% (ml / ml) ethanol aqueous solution, and vacuum dry at 30 ° C for 10h to obtain orange-yellow riboflavin sodium phosphate solid powder 339.07g, Loss on drying was 4.86%, sodium ion content was 4.45%, specific rotation was + 38.83 °, purity was 99.34%; content (with riboflavin as external standard, as dry product) was 77.30% (pharmacopoeia requirement range was 74.0 % -79.0%), the total yield is 89.93 based on flavin single nucleotide
% %

Claims

权利要求书 Claim
[权利要求 1] 高纯度核黄素磷酸钠的制备方法， 其特征在于包括如下步骤： i） 将 核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液的 pH值调节至 5.0 7.0; 2 ） 控制溶液温度为 20~40°C， 并加入占粗品溶液体积 0.5~1.5倍的浓度 为 95% （ml/ml） 的乙醇水溶液； 3） 降温至 3~5°C， 等待固体析出； 4 ） 过滤， 除去滤液， 滤饼经干燥处理后即得核黄素磷酸钠纯品。  [Claim 1] A method for preparing high-purity riboflavin sodium phosphate, comprising the steps of: i) adjusting the pH of a crude solution of sodium riboflavin phosphate and / or flavin single nucleotide to 5.0 7.0; 2 ) Control the solution temperature to 20 ~ 40 ° C, and add a 95% (ml / ml) ethanol solution with a concentration of 0.5 to 1.5 times the volume of the crude solution; 3) Reduce the temperature to 3 ~ 5 ° C and wait for the solid to precipitate; 4 ) Filtration to remove the filtrate. The filter cake is dried to obtain pure riboflavin sodium phosphate.
[权利要求 2] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 2] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that
: 所述核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液的浓度以核黄素 磷酸钠计为 7.5g/L以上。  : The concentration of the crude solution of the riboflavin sodium phosphate and / or the flavin single nucleotide is 7.5 g / L or more based on the riboflavin sodium phosphate.
[权利要求 3] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 3] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that
: 所述步骤 1） 中， 将溶液的 pH值调节为 5.5~6.5。  : In step 1), adjust the pH of the solution to 5.5 ~ 6.5.
[权利要求 4] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 4] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that
: 所述步骤 2） 中， 加入占溶液体积 0.5~0.9倍的浓度为 95% （ml/ml） 的乙醇水溶液。  : In step 2), add a 95% (ml / ml) ethanol aqueous solution with a concentration of 0.5 to 0.9 times the volume of the solution.
[权利要求 5] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于 ， 所述步骤 3） 的降温过程按如下步骤操作： 先降温至 12~15°C， 待溶 液中的固体析出率达 40%以上后， 再降温至 3~5°C。  [Claim 5] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that the temperature-lowering process of step 3) is performed as follows: first lower the temperature to 12-15 ° C, and wait until After the solid precipitation rate in the solution reaches more than 40%, the temperature is lowered to 3 ~ 5 ° C.
[权利要求 6] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 6] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that
: 所述步骤 3） 中， 待溶液中的固体析出率达 40%以上后， 再加入占 粗品溶液体积 0.3~0.5倍的浓度为 95% （ml/ml） 的乙醇水溶液。  In step 3), after the solids precipitation rate in the solution reaches more than 40%, an ethanol aqueous solution having a concentration of 95% (ml / ml) which is 0.3 to 0.5 times the volume of the crude solution is added.
[权利要求 7] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 7] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that
: 所述步骤 4） 中， 在干燥处理前， 用浓度为 75% （ml/ml） 的乙醇水 溶液对所述滤饼进行洗涤。  In step 4), before the drying treatment, the filter cake is washed with a 75% (ml / ml) ethanol water solution.
[权利要求 8] 根据权利要求 7所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 8] The method for preparing high-purity riboflavin sodium phosphate according to claim 7, characterized in that
: 在洗涤滤饼之前， 先用 50%乙醇水溶液对所述滤饼进行打浆， 然后 过滤收集滤饼。  : Before washing the filter cake, first beat the filter cake with a 50% ethanol aqueous solution, and then collect the filter cake by filtration.
[权利要求 9] 根据权利要求 1所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 9] The method for preparing high-purity riboflavin sodium phosphate according to claim 1, characterized in that
: 所述步骤 1） 中， 核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液为生 物催化法制备黄素单核苷酸得到的粗产物溶液。 : In step 1), a crude solution of riboflavin sodium phosphate and / or flavin single nucleotide is produced The crude product solution of flavin single nucleotide was prepared by the biocatalysis method.
[权利要求 10] 根据权利要求 9所述的高纯度核黄素磷酸钠的制备方法， 其特征在于 ， 在步骤 1） 之前， 对核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液进 行如下前序处理： a） 将核黄素磷酸钠和 /或黄素单核苷酸的粗品溶液 的 pH值调节至 11.0~12.0; b） 将溶液温度升温至 40±1°C， 并于 40±1°C 温度下保持 2h; c） 除去不溶物。  [Claim 10] The method for preparing high-purity riboflavin sodium phosphate according to claim 9, characterized in that before step 1), a crude solution of riboflavin sodium phosphate and / or flavin mononucleotide is performed. The pretreatment is as follows: a) The pH of the crude solution of riboflavin sodium phosphate and / or flavin single nucleotide is adjusted to 11.0 ~ 12.0; b) The temperature of the solution is raised to 40 ± 1 ° C, and the temperature is adjusted at 40 ± 1 ° C for 2h; c) remove insoluble matter.
[权利要求 11] 根据权利要求 10所述的高纯度核黄素磷酸钠的制备方法， 其特征在于 所述前序处理还包括将步骤 c） 获得的去除不溶物后的溶液进行预处 理的步骤， 所述预处理是指用离子交换树脂对去除不溶物后的溶液进 行纯化， 或者将去除不溶物后的溶液依次经微滤处理和超滤处理。  [Claim 11] The method for preparing high-purity riboflavin sodium phosphate according to claim 10, wherein the pre-treatment further comprises a step of pre-treating the solution obtained by removing insoluble matter obtained in step c). The pretreatment refers to using an ion exchange resin to purify the solution after removing the insoluble matter, or sequentially subjecting the solution after removing the insoluble matter to microfiltration treatment and ultrafiltration treatment.
[权利要求 12] 根据权利要求 11所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 12] The method for preparing high-purity riboflavin sodium phosphate according to claim 11, characterized in that
: 所述离子交换树脂为阴离子交换树脂。  : The ion exchange resin is an anion exchange resin.
[权利要求 13] 根据权利要求 11所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 13] The method for preparing high-purity riboflavin sodium phosphate according to claim 11, characterized in that
: 用 0. l~0.4mol/L氯化钠水溶液作为离子交换树脂的洗脱液。  : Use 0.1 ~ 0.4mol / L sodium chloride aqueous solution as eluent of ion exchange resin.
[权利要求 14] 根据权利要求 11所述的高纯度核黄素磷酸钠的制备方法， 其特征在于  [Claim 14] The method for preparing high-purity riboflavin sodium phosphate according to claim 11, characterized in that
: 在步骤 1） 之前， 将所述预处理后的溶液进行浓缩处理。  : Before step 1), the pretreated solution is concentrated.
[权利要求 15] 根据权利要求 11所述的高纯度核黄素磷酸钠的制备方法， 其特征在于 ， 通过如下方法对所述离子交换树脂进行再生处理： A、 用 2mol/L氯 化钠溶液与 0.5mol/L盐酸溶液的混合液淋洗树脂至流出液中无杂质残 留； B、 用纯化水淋洗至洗脱液的 pH值为 5.00~7.00; C、 用 0.5mol/L 氢氧化钠溶液淋洗至洗脱液呈强碱性； D、 用纯化水淋洗至洗脱液的 pH值为 9.00~10.00。  [Claim 15] The method for preparing high-purity riboflavin sodium phosphate according to claim 11, characterized in that the ion exchange resin is regenerated by the following method: A. Using a 2mol / L sodium chloride solution Rinse the resin with the 0.5mol / L hydrochloric acid solution until no impurities remain in the effluent; B. Rinse with purified water until the pH of the eluent is 5.00 ~ 7.00; C. Use 0.5mol / L sodium hydroxide Rinse the solution until the eluent is strongly alkaline; D. Rinse with purified water until the pH of the eluent is 9.00 ~ 10.00.
PCT/CN2018/106904 2018-09-21 2018-09-21 Method for preparing high-purity riboflavin sodium phosphate WO2020056714A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201880037970.5A CN111094309A (en) 2018-09-21 2018-09-21 Preparation method of high-purity riboflavin sodium phosphate
PCT/CN2018/106904 WO2020056714A1 (en) 2018-09-21 2018-09-21 Method for preparing high-purity riboflavin sodium phosphate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/106904 WO2020056714A1 (en) 2018-09-21 2018-09-21 Method for preparing high-purity riboflavin sodium phosphate

Publications (1)

Publication Number Publication Date
WO2020056714A1 true WO2020056714A1 (en) 2020-03-26

Family

ID=69888179

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/106904 WO2020056714A1 (en) 2018-09-21 2018-09-21 Method for preparing high-purity riboflavin sodium phosphate

Country Status (2)

Country Link
CN (1) CN111094309A (en)
WO (1) WO2020056714A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114702524A (en) * 2022-04-29 2022-07-05 湖北广济药业股份有限公司 Method for preparing high-purity riboflavin sodium phosphate

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5017700A (en) * 1988-10-19 1991-05-21 Basf Aktiengesellschaft Preparation of riboflavin-5'-phosphate (5'-FMN) and its sodium salt, and of riboflavin-4',5'-cyclophosphoric acid ester chloride as an intermediate
CN103435651A (en) * 2013-09-11 2013-12-11 天津天雷科技有限公司 Synthesis method and process of riboflavin sodium phosphate

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206233B (en) * 2011-04-06 2012-07-04 湖北广济药业股份有限公司 Industrial preparation method for riboflavine sodium phosphate
CN102617643B (en) * 2012-03-06 2013-04-10 黄金秀 Riboflavin sodium phosphate compound
CN104072540B (en) * 2014-05-22 2016-08-17 深圳朗欧医药集团有限公司 Riboflavin sodium phosphate compound and preparation method thereof
CN107286194A (en) * 2017-06-07 2017-10-24 山西集翔生物工程有限公司 A kind of riboflavin phosphate process for producing sodium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5017700A (en) * 1988-10-19 1991-05-21 Basf Aktiengesellschaft Preparation of riboflavin-5'-phosphate (5'-FMN) and its sodium salt, and of riboflavin-4',5'-cyclophosphoric acid ester chloride as an intermediate
CN103435651A (en) * 2013-09-11 2013-12-11 天津天雷科技有限公司 Synthesis method and process of riboflavin sodium phosphate

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHIEF EDITOR: ZHAO LINXIANG, CHEMICAL PHARMACEUTICAL TECHNOLOGY (M) THE FOURTH EDITION, BEIJING: CHINA MEDICAL SCIENCE PRESS, 31 August 2015 (2015-08-31), pages 102 - 104 *
WEI, ZHUAN ET AL.: "Preparation of Riboflavin 5'-Phosphate Sodium from Crude Riboflavin", CHINA FOOD ADDITIVES, 15 December 2011 (2011-12-15) *

Also Published As

Publication number Publication date
CN111094309A (en) 2020-05-01

Similar Documents

Publication Publication Date Title
CN105969819B (en) A kind of method of Production by Enzymes l-tyrosine
CN108285912B (en) Method for preparing and extracting pharmaceutical grade valine by fermentation
Boonmee et al. Improved lactic acid production by in situ removal of lactic acid during fermentation and a proposed scheme for its recovery
WO2021135886A1 (en) Transaminase mutant, immobilized transaminase and use in preparation of sitagliptin
CN109265498B (en) Method for preparing N-acetylneuraminic acid by integrated separation and purification of polysialic acid
CN107151685B (en) Method for producing chondroitin sulfate by fermentation method
WO2018107377A1 (en) Purification process of nadph
CN108285913B (en) Process for preparing and extracting L-glutamine
CN111087432A (en) Separation and extraction method of N-acetylneuraminic acid
CN107383135B (en) Method for separating and purifying beta-thymidine from fermentation liquor
CN103965064A (en) Method for extraction of L-alanine from L-alanine fermentation broth
WO2022257686A1 (en) Transaminase, immobilized transaminase and use for preparing sitagliptin
CN108484422B (en) Purification and crystallization process for extracting beta-alanine from enzyme conversion solution and system thereof
CN106220521B (en) A kind of method of full film extraction L isoleucines
CN105348122A (en) Method for purifying L-alanine final mother liquor
WO2020056714A1 (en) Method for preparing high-purity riboflavin sodium phosphate
CN112047828B (en) Method for preparing protocatechuic acid by biochemical method
CN116479068B (en) Method for preparing N1-methyl-pseudouridine monophosphate by using biological enzyme
CN113005161A (en) Preparation method of polysialic acid and polysialic acid product
CN108299220B (en) Method for extracting L-4-hydroxyisoleucine from fermentation catalytic liquid
CN110627634A (en) Method for separating and extracting lactic acid from daqu liquor by-product yellow water
CN103539688B (en) A kind of method of separation and Extraction Serine from Corynebacterium glutamicum fermented liquid
CN102533886A (en) Production process for synthetizing L-theanine through biological immobilized enzyme catalysis
CN111065644B (en) Method for preparing high-purity NAD
CN104178540B (en) Method for synthesizing ademetionine by biological catalysis method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18933852

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18933852

Country of ref document: EP

Kind code of ref document: A1