CN114507263A - Desalting method of beta-nicotinamide mononucleotide - Google Patents
Desalting method of beta-nicotinamide mononucleotide Download PDFInfo
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- CN114507263A CN114507263A CN202011276956.6A CN202011276956A CN114507263A CN 114507263 A CN114507263 A CN 114507263A CN 202011276956 A CN202011276956 A CN 202011276956A CN 114507263 A CN114507263 A CN 114507263A
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- nicotinamide mononucleotide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/048—Pyridine radicals
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Abstract
The invention discloses a desalting method of beta-nicotinamide mononucleotide. The method comprises the steps of adjusting a reaction solution containing inorganic salt and beta-nicotinamide mononucleotide to a specific pH value, flowing through a separation column filled with specific ion exchange resin, eluting with water, and performing nanofiltration to obtain a desalted beta-nicotinamide mononucleotide aqueous solution. The beta-nicotinamide mononucleotide aqueous solution obtained by desalting can be directly subjected to sterile filtration and then freeze-dried to produce food-grade beta-nicotinamide mononucleotide freeze-dried powder.
Description
The technical field is as follows:
the invention belongs to the field of pharmaceutical chemistry, and particularly relates to a desalting method of beta-nicotinamide mononucleotide.
Background art:
the Nicotinamide Mononucleotide is also called beta-Nicotinamide Mononucleotide (beta-Nicotinamide mononuleotide, beta-NMN), and has a structure shown as a compound I.
Beta-nicotinamide mononucleotide plays an important role in the production of energy in human cells and is involved in intracellular NAD+(nicotinamide adenine dinucleotide, an important coenzyme for cellular energy conversion) is NAD+Is one of the key precursors of (a). The David Scinclair research team reported in the literature Science,2017,355,1312-1317 NAD+The increase in the bodies of the mice can delay the aging signs of tissues and muscles of the aged mice, and the research work makes human beings greatly step towards realizing the longevity dream. NAD (nicotinamide adenine dinucleotide)+The molecular weight is too large, and the oral absorption and utilization rate is low. But with the addition of NAD+Research on precursor small molecule substance beta-nicotinamide mononucleotide finds that in vivo NAD can be effectively improved by taking beta-nicotinamide mononucleotide+The concentration of the beta-nicotinamide mononucleotide can obviously improve the metabolism of a human body, so that the beta-nicotinamide mononucleotide becomes a medicine for preventing the aging. It also promotes the medical, food and cosmetic industries of the worldResearchers are constantly researching and developing beta-nicotinamide mononucleotide.
There are many reports on methods for producing β -nicotinamide mononucleotide, mainly including chemical synthesis, fermentation and enzymatic production. The enzyme method is particularly concerned by people because of low cost, safe production and environmental protection. However, in the biotransformation production process of beta-nicotinamide mononucleotide, a large amount of inorganic salts including potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium chloride and the like are used as buffer salts or biological reaction trace elements, so that the divalent and trivalent salts in the crude product are high in content and difficult to remove.
If the conventional ion exchange method is adopted, the product cannot be effectively separated because the product contains similar phosphate groups and cation structures.
If the membrane separation technology is adopted, due to the fact that the molecular weight of the product is small, the permeability of divalent and trivalent salts is low in the range of selectable nanofiltration membranes, and the desalting effect is poor.
Therefore, it is desirable to develop a method for desalting β -nicotinamide mononucleotide, which is simple and easy to handle.
The invention content is as follows:
the invention aims to provide a nicotinamide mononucleotide desalting method which is simple to operate and easy to industrialize, aiming at the defects of the prior art.
The invention provides a desalting method of beta-nicotinamide mononucleotide, which comprises the following steps: adjusting the pH value of a reaction solution containing inorganic salt and beta-nicotinamide mononucleotide to 2.5-3.0 by using hydrochloric acid, then flowing through a separation column filled with strong-base anion exchange resin, eluting by using water, adjusting the pH value of the obtained effluent to 7.5-8.0 by using a sodium hydroxide aqueous solution, flowing through a separation column filled with strong-acid cation exchange resin, eluting by using water, and performing nanofiltration on the obtained effluent to obtain a desalted beta-nicotinamide mononucleotide aqueous solution.
Further, the reaction solution containing inorganic salt and beta-nicotinamide mononucleotide is a reaction solution from biological preparation, wherein the reaction solution contains about 100g/L of inorganic salt and 30g/L of beta-nicotinamide mononucleotide.
Further, the concentration of the hydrochloric acid is 2-3 mol/L.
Still further, the strongly basic anion exchange resin is selected from 201 x 4, 201 x 7U, JK206, 202, D213, WDA-OH, etc., preferably strongly basic anion exchange resin D202.
Further, the concentration of the sodium hydroxide aqueous solution is 2-3 mol/L.
Further, the strong-acid cation exchange resin is selected from 001 x 1-16, D001, JK006, WDC-H, etc., and preferably the strong-acid cation exchange resin D001.
Further, the salt content in the desalted beta-nicotinamide mononucleotide aqueous solution is less than 0.1g/L, and the content of beta-nicotinamide mononucleotide is 80-100 g/L.
Further, the desalted beta-nicotinamide mononucleotide aqueous solution obtained after nanofiltration can be directly subjected to aseptic filtration and then freeze-dried to produce food-grade beta-nicotinamide mononucleotide freeze-dried powder.
Further, after nanofiltration, desalted beta-nicotinamide mononucleotide aqueous solution is obtained and is crystallized and dried to obtain high-content beta-nicotinamide mononucleotide solid.
The invention has the beneficial effects that: the invention utilizes the amphiprotic characteristic of beta-nicotinamide mononucleotide molecules, converts divalent and trivalent ions in the beta-nicotinamide mononucleotide molecules into monovalent ions by adopting ion exchange resin under specific pH, and then carries out nanofiltration to obtain the beta-nicotinamide mononucleotide aqueous solution. The beta-nicotinamide mononucleotide aqueous solution obtained by desalting can be directly subjected to sterile filtration and then freeze-dried to produce food-grade beta-nicotinamide mononucleotide freeze-dried powder.
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Detailed Description
The technical content of the present invention is further described below with reference to specific examples for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
Example 1 preparation of beta-Nicotinamide mononucleotide reaction solution
Adding pH 8.5 aqueous buffer solution (concentration of aqueous buffer solution is 50 m)M sodium phosphate buffer) was added to the reaction vessel, and 40mM nicotinamide, 40mM ATP, 40mM D-5 ribose phosphate, and 10mM MgCl were added to the system2、10mM MnSO4Then 15% of K is used2CO3The pH was adjusted to 8.5. Then 40g/L of the immobilized whole cells were put into the raw material solution, stirred at a speed of 50rpm and a temperature of 37 ℃ by means of an automatic titrator apparatus using 15% K2CO3Controlling the pH value of the solution at 8.5, and reacting for 3 hours to obtain a beta-nicotinamide mononucleotide reaction solution.
Example 2 desalting of beta-Nicotinamide mononucleotide reaction solution
Taking 500mL of beta-nicotinamide mononucleotide reaction solution (30g/L) prepared in example 1, adjusting the pH value to be 2.5 by using 10% hydrochloric acid, keeping the flow rate at 15 ℃ for 4m/h, slowly passing through a 200mL of D202 resin bed layer (the length-diameter ratio is 12:1), collecting 700mL of beta-nicotinamide mononucleotide effluent, adjusting the pH value to be 7.5 by using 10% sodium hydroxide aqueous solution, keeping the flow rate at 6m/h, slowly passing through a 200mL of D001 resin bed layer (the length-diameter ratio is 9:1), collecting 900mL of beta-nicotinamide mononucleotide effluent, concentrating the beta-nicotinamide mononucleotide effluent to 150g/L by using a nanofiltration membrane, and freeze-drying to obtain 14g of beta-nicotinamide mononucleotide freeze-dried powder, wherein the yield is 93.33%, the purity is 99.7% and the content is 99.2%.
Example 3 preparation of beta-Nicotinamide mononucleotide reaction solution
Adding aqueous buffer (aqueous buffer is 50mM dipotassium hydrogen phosphate buffer) with pH 8.5 into reaction vessel, adding 40mM nicotinamide, 40mM ATP, 40mM D-5 ribose phosphate, and 10mM MgCl into the system2、10mM MnSO4Then 15% of K is used2CO3The pH was adjusted to 8.5. Then 40g/L of the immobilized whole cells were put into the raw material solution, stirred at a speed of 50rpm and a temperature of 37 ℃ by means of an automatic titrator apparatus using 15% K2CO3Controlling the pH value of the solution at 8.5, and reacting for 3 hours to obtain the beta-nicotinamide mononucleotide reaction solution.
Example 4 desalting of beta-Nicotinamide mononucleotide reaction solution
Taking 500mL of beta-nicotinamide mononucleotide reaction solution (30g/L) prepared in example 3, adjusting the pH value to 3.0 by using 10% hydrochloric acid, keeping the flow rate at 15 ℃ for 4m/h, slowly passing through a 200mL D213 resin bed layer, collecting 700mL of beta-nicotinamide mononucleotide effluent, adjusting the pH value to 8.0 by using 10% sodium hydroxide aqueous solution, keeping the flow rate at 6m/h, slowly passing through a 200mL JK006 resin bed layer, collecting 900mL of beta-nicotinamide mononucleotide effluent, concentrating to 150g/L by using a nanofiltration membrane, adding 1000g of ethanol, separating out a large amount of solid, stirring for 1 hour, performing suction filtration, and performing vacuum drying at 30 ℃ for 20 hours to obtain 13.8g of white solid, namely high-content beta-nicotinamide mononucleotide, wherein the yield is 92.00%, the purity is 99.9%, and the content is 99.4%.
Claims (6)
1. A desalting method of beta-nicotinamide mononucleotide is characterized in that hydrochloric acid is used for adjusting the pH value of reaction liquid containing inorganic salt and beta-nicotinamide mononucleotide to 2.5-3.0, then the reaction liquid flows through a separation column filled with strong-base anion exchange resin, water is used for elution, the pH value of obtained effluent liquid is adjusted to 7.5-8.0 by sodium hydroxide water solution, then the effluent liquid flows through the separation column filled with strong-acid cation exchange resin, water is used for elution, and the obtained effluent liquid is subjected to nanofiltration to obtain desalted beta-nicotinamide mononucleotide water solution.
2. The method of claim 1, wherein the reaction solution comprising the inorganic salt and β -nicotinamide mononucleotide is a reaction solution from a biological process for preparing β -nicotinamide mononucleotide, wherein the inorganic salt comprises about 100g/L and the β -nicotinamide mononucleotide comprises 30 g/L.
3. The method according to claim 1, wherein the hydrochloric acid concentration is 2 to 3 mol/L.
4. The method of claim 1, wherein the aqueous sodium hydroxide solution has a concentration of 2 to 3 mol/L.
5. The method of claim 1, wherein the desalted aqueous solution of β -nicotinamide mononucleotide has a salt content of less than 0.1g/L and a β -nicotinamide mononucleotide content of 80-100 g/L.
6. The method of claim 1, wherein the desalted aqueous solution of β -nicotinamide mononucleotide can be directly subjected to sterile filtration and freeze-dried to produce food grade β -nicotinamide mononucleotide freeze-dried powder.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011276956.6A CN114507263A (en) | 2020-11-16 | 2020-11-16 | Desalting method of beta-nicotinamide mononucleotide |
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| CN202011276956.6A CN114507263A (en) | 2020-11-16 | 2020-11-16 | Desalting method of beta-nicotinamide mononucleotide |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4904587A (en) * | 1987-09-10 | 1990-02-27 | Takeda Chemical Industries, Ltd. | Production of D-ribose |
| TWI231746B (en) * | 2001-10-29 | 2005-05-01 | Great Wall Entpr Co Ltd | Desalinizing method for nutrition supplement product containing animal protein and processing manner for regenerating ion exchange resin thereof |
| CN111377983A (en) * | 2020-03-26 | 2020-07-07 | 音芙医药科技(上海)有限公司 | Preparation method of β -nicotinamide mononucleotide |
| CN111424064A (en) * | 2020-04-20 | 2020-07-17 | 比瑞博生物科技(北京)有限公司 | High-purity NMN preparation process based on enzyme method |
-
2020
- 2020-11-16 CN CN202011276956.6A patent/CN114507263A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4904587A (en) * | 1987-09-10 | 1990-02-27 | Takeda Chemical Industries, Ltd. | Production of D-ribose |
| TWI231746B (en) * | 2001-10-29 | 2005-05-01 | Great Wall Entpr Co Ltd | Desalinizing method for nutrition supplement product containing animal protein and processing manner for regenerating ion exchange resin thereof |
| CN111377983A (en) * | 2020-03-26 | 2020-07-07 | 音芙医药科技(上海)有限公司 | Preparation method of β -nicotinamide mononucleotide |
| CN111424064A (en) * | 2020-04-20 | 2020-07-17 | 比瑞博生物科技(北京)有限公司 | High-purity NMN preparation process based on enzyme method |
Non-Patent Citations (1)
| Title |
|---|
| 上海市合成树脂研究所: "《塑料工业》", 石油化学工业出版社, pages: 586 - 588 * |
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