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Method for purification of cytidinediphosphoric choline

A purification method and phosphorylcholine technology are applied in the field of purification of cytidine diphosphocholine, which can solve problems such as not

Active Publication Date: 2009-06-03
KYOWA HAKKO BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Known purification methods of CDP-choline prepared by chemical synthesis methods include: the method of using anion-exchange resin (Patent Document 1), the purification method of combining strongly acidic ion-exchange resin and weakly basic ion-exchange resin (Patent Document 1) 2), use two kinds of ion-exchange resins in the latter method, although phosphorylcholine, cytidine-5'-monophosphate (hereinafter referred to as CMP) contained in the CDP-choline solution can be separated, it is not possible to Method for effectively separating uracil or uridine-5'-triphosphate (hereinafter abbreviated as UTP)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Purification of CDP-choline using Strongly Acidic Cation Exchange Resins

[0105] 50 g of CDP-choline (manufactured by Wako Pure Chemical Industries), 2 g of uracil (manufactured by Nacalai Tesque), and 1 g of UTP (manufactured by Nacalai Tesque) were dissolved in water to prepare 5 L of a CDP-choline solution. The solution was adjusted to pH 3.0 with sulfuric acid, and passed through a column filled with 10 L of strongly acidic cation exchange resin Diamond ionPK208 (type H) with a crosslinking degree of 4%. Then, water was passed through, and components whose respective concentrations of uracil and UTP were lower than 0.1% (w / w) relative to CDP-choline were obtained by high-performance liquid chromatography analysis. The CDP-choline fraction was concentrated to 100 mL, and 350 mL of ethanol was slowly added. The precipitated crystals were collected by filtration, washed with 100% ethanol solution, and then dried under reduced pressure at 20° C. for three days. As a ...

Embodiment 2

[0107] Purification of CDP-choline from cultures of microorganisms capable of producing CDP-choline

[0108] Escherichia coli MM294 / pCKG55 strain (FERM BP-3717) having enzyme activity of PyrG, CCT, and CKI was inoculated into L medium [containing 10 g / L tryptone (manufactured by Difco), 5 g / L yeast extract (manufactured by Difco), 5 g / L NaCl, liquid medium adjusted to pH 7.2] in a test tube, shake culture at 300 rpm at 25° C. for 24 hours. 20 mL of the culture solution was inoculated into a 2L Erlenmeyer flask with baffles filled with 400 mL of L medium containing 50 μg / mL ampicillin, and cultured at 25° C. with rotary shaking at 190 rpm for 16 hours. 125 mL of this culture solution was inoculated into 2.5 L of 5 g / L glucose (separately sterilized), 5 g / L peptone (manufactured by Jidong Pharmaceutical Co., Ltd.), 6 g / L Na 2 HPO 4 、3g / L KH 2 PO 4 , 5g / L NaCl, 1g / LNH 4 Cl, 250mg / L MgSO 4 ·7H 2 O (separately sterilized) and 4 μg / L vitamin B1 (separately sterilized) liquid ...

Embodiment 3

[0113] CDP-choline sodium salt is purified from a culture of a microorganism having the ability to produce CDP-choline.

[0114] With sulfuric acid, the four reaction solutions obtained in the same manner as in Example 2 were adjusted to pH 1.0, and the bacterial cells were separated by centrifugation (7000rpm, 10 minutes), and water was added to the resulting supernatant to make 6L (6.0g / L CDP -choline, 0.5g / L uracil, 1.0g / L UTP). The cytidine diphosphocholine solution was overfilled with a 10 L column of strong acidic cation exchange resin Diamond ion SK104 (type H) with a crosslinking degree of 4%. Water was continuously fed, and components with respective concentrations lower than 0.1% (w / w) relative to CDP-choline uracil and UTP were obtained through high-performance liquid chromatography analysis. The CDP-choline fraction was adjusted to pH 7.5 with sodium hydroxide, decolorized using activated carbon, and then concentrated to 100 mL. 400 mL of ethanol was slowly added...

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Abstract

A method for purification of cytidinediphosphoric choline comprises the steps of contacting a cytidinediphosphoric choline solution having a pH of 0.5 to 5.0 (inclusive) and containing a nucleic acid analogue with a H-type strongly acidic cation exchange resin, and eluting cytidinediphosphoric choline adsorbed on the resin with water or an aqueous solution having an ion concentration of 0.1 mol / L or lower to thereby isolate and purify cytidinediphosphoric choline.

Description

technical field [0001] The invention relates to a method for purifying cytidine diphosphate choline which can be used as raw material of medicine and nutritional food. Background technique [0002] The preparation method of cytidine diphosphate choline (hereinafter abbreviated as CDP-choline) is known to be a chemical synthesis method (Patent Document 1, Patent Document 2), a method using a microbial culture or an enzyme (Patent Document 3, Patent Document 4 ). Known purification methods of CDP-choline prepared by chemical synthesis methods include: the method of using anion-exchange resin (Patent Document 1), the purification method of combining strongly acidic ion-exchange resin and weakly basic ion-exchange resin (Patent Document 1) 2), use two kinds of ion-exchange resins in the latter method, although phosphorylcholine, cytidine-5'-monophosphate (hereinafter referred to as CMP) contained in the CDP-choline solution can be separated, it is not possible to A method for ...

Claims

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Application Information

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IPC IPC(8): C07H19/10C12P19/32
CPCC07H19/10C12P19/32
Inventor 村田英城目代强盐见道夫
Owner KYOWA HAKKO BIO CO LTD
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