JP2000281688A - Production of mixed acid-type 1,2-diacyl-3- glycerophospholipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a high-purity mixed acid-type 1,2-diacyl-3-glycerophospholipid industrially easily by a single method through combining any carboxylic acids at 1-position and 2-position. SOLUTION: This method for producing a high-purity mixed acid-type 1,2- diacyl-3-glycerophospholipid of the general formula (R1 and R2 are each a 12-22C acyl group differing from each other; X is a residue resulting from excluding one hydroxyl group from a base containing hydroxyl groups) is such one that 1,2-diacyl-3 glycerylphosphatidylcholine is, as a raw material, partially deacylated with lipase and purified with a silica gel column followed by introducing different acyl groups into the deacylated positions with the corresponding acid anhydrides to synthesize a mixed acid-type 1,2-diacyl-3-glycerophospholipid, and repeating crystallizing purification at least 3 times for the synthesized product with a specific mixed solvent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a mixed acid type 1,2-
The present invention relates to a method for producing diacyl-3-glycerophospholipid.

[0002]

2. Description of the Related Art Phospholipids have hitherto been widely used as emulsifiers. In recent years, phospholipids have attracted attention as drug carriers, artificial blood, artificial cells, etc. as liposome base materials. Various uses in the fields of medicine, pharmacy, and engineering are considered as lipids having biological activity and drug action. In order to meet such various demands, various phospholipids having appropriate functions according to the respective applications have been demanded, and many methods for producing synthetic phospholipids have been reported so far. For example, a conventional method for producing a glycerophospholipid having two identical fatty acid residues bonded thereto is described in JP-A-51-91213.
Regarding a method for purifying lysophospholipids, see
The method for producing glycerophospholipids using a base exchange reaction with phospholipase D is disclosed in Japanese Patent Application Laid-Open No. 5-292981. However, in these known methods, since the two kinds of fatty acid residues are the same, the range of use thereof is limited, and in terms of functions, it is insufficient to satisfy the performance corresponding to demand. On the other hand, the mixed acid type 1,2-diacyl-3-glycerophospholipid in which two kinds of acyl groups are bonded is contained in a large amount in a living body, and the fatty acid composition differs depending on the site in the living body. Among them, the mixed acid type 1,2-diacyl-3-glycerophospholipid in which the 1-position is a saturated fatty acid and the 2-position is an unsaturated fatty acid is the most abundant in the living body, and has a high affinity to the living body. . For application to medical liposomes and diagnostic agents, there is a strong demand for high-purity mixed acid-type 1,2-diacyl-3-glycerophospholipids having a structure and composition similar to natural phospholipids with good biocompatibility. Therefore, a method for producing a high-purity mixed acid glycerophospholipid is disclosed in Japanese Patent Application Laid-Open No. 52-8 / 1982.
No. 9622, JP-A-63-54385, JP-A-63-54384, JP-A-63-157993.
Publications are known. However, the production techniques described in these publications have, on the chemical structure, a restriction that the fatty acid composition must be a polymerizable fatty acid and a restriction that the type of phospholipid must be phosphatidylcholine or phosphatidylinositol, and There is a disadvantage that it is not possible to produce a mixed acid type 1,2-diacyl-3-glycerophospholipid having a structure. In order to properly cope with various uses, mixed acid types 1 and 2 having different fatty acid residues at the 1- and 2-positions under such conditions without structural restrictions.
A method for producing -diacyl-3-glycerophospholipid with high purity is not known. Under such circumstances, mixed acid type 1,2- having various functions according to various uses
Providing a method for efficiently producing diacyl-3-glycerophospholipids is of great industrial significance.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been made from the above-mentioned viewpoints, and is intended to provide a mixed acid type 1,2-diacyl-3-glycerophospholipid with a high purity and a high purity of 1,2-diacyl-3-glycerophospholipid. It is an object of the present invention to provide a method which can be easily produced on an industrial scale by a single production method even if the compound is a combination of carboxylic acids.

[0004]

Means for Solving the Problems In view of the above problems, the present inventors have made intensive studies, and as a result, have found that the above problems can be solved by using a specific solvent mixture in acyl group exchange. . That is, the present invention includes the following items. (1)

Embedded image (Wherein, R ¹ and R ² are acyl groups having 12 to 22 carbon atoms, R ¹ and R ² are not the same, and X is a residue obtained by removing one hydroxyl group from a hydroxyl group-containing base) When producing the mixed acid type 1,2-diacyl-3-glycerophospholipid represented by the above formula [2] from 1,2-diacyl-3-glycerylphosphatidylcholine represented by the formula [1] as a starting material, the following ( a) the process,
A method for producing a mixed acid type 1,2-diacyl-3-glycerophospholipid, which is carried out in the order of the steps (b), (c), (d), (e) and (f). (A) Step After deacylation of 1,2-diacyl-3-glycerylphosphatidylcholine with lipase, crystallization is performed using a mixed organic solvent of hexane: acetone: methanol = 7-5: 4-0: 2-0 (volume ratio). Precipitation to obtain monoacyl-3-glycerylphosphatidylcholine. (B) Step The monoacyl-3-glycerylphosphatidylcholine obtained in the step (a) is reacted with a fatty acid anhydride in the presence of a tertiary amine to give a mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine reaction solution. The reaction solution is adsorbed on a silica gel column, and a mixed solvent of chloroform: methanol = 95-80: 20-5 (volume ratio) is passed through the column to elute and remove impurities from the column. : Water = 85 to 75: 20 to 18:
A step of flowing a mixed solvent of 3 to 1.5 (volume ratio) to separate mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine into the mixed solvent. (C) Step The mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine and the base are reacted with phospholipase D to give a mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine into which an arbitrary acyl group obtained in the step (b) has been introduced.
Obtaining a 2-diacyl-glycerophospholipid. (D) Step Mixed acid form 1,2-diacyl-3-glycerylphosphatidylcholine or mixed acid form 1,2- obtained in step (c)
The diacyl-glycerophospholipid is dissolved in chloroform: methanol: acetone = 13-0: 2-0: 9-7 (volume ratio) to a concentration of 5-50% by weight, and mixed acid type 1,2
-A step of crystallizing the diacyl-3-glycerophospholipid. Step (e) Mixed acid type 1,2-diacyl-3 obtained in step (d) above
-A glycerophospholipid crystal is prepared by adding chloroform: methanol: acetone = 13 to 9: 2 to 1:11 by weight 5 to 20 times.
To 8 (volume ratio) to crystallize the mixed acid type 1,2-diacyl-3-glycerophospholipid. (F) Step The mixed acid type 1,2-diacyl-3 obtained in the above step (e).
-A glycerophospholipid crystal is prepared by adding chloroform: methanol: acetone = 13 to 9: 2-1: 7 to 5 to 20 times by weight.
6 (volume ratio), and mixed acid type 1,2-diacyl-
A step of crystallizing 3-glycerophospholipid. (2) The mixed acid type 1,2-diacyl-3-glycerophospholipid is the glycerophospholipid represented by the formula [3] (1).
The method for producing a mixed acid type 1,2-diacyl-3-glycerophospholipid according to the above item.

Embedded image (Wherein one of R ³ and R ⁴ has 12 to 22 carbon atoms)
And the other is an unsaturated acyl group having 12 to 22 carbon atoms, and X represents a residue obtained by removing one hydroxyl group from a base having a hydroxyl group. )

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the step (a) of the present invention, 1,2-diacyl-3-glycerylphosphatidylcholine is partially hydrolyzed with an enzyme lipase to synthesize monoacyl-3-glycerylphosphatidylcholine. This is a step in which monoacyl-3-glycerylphosphatidylcholine is selectively obtained as crystals by precipitation. Of the present invention
The 1,2-diacyl-3-glycerylphosphatidylcholine used in the step (a) may be a commercially available synthetic product, but a low-purity 1,2-diacyl-3-glycerylphosphatidylcholine has a purity of 98% because a large amount of by-products is produced during the deacylation reaction. % Or more is particularly preferable, and also one in which two acyl groups are the same acyl group from the viewpoint of purity. The acyl group of 1,2-diacyl-3-glycerylphosphatidylcholine is a saturated or unsaturated acyl group having 12 to 22 carbon atoms, such as lauric acid, tricadic acid, myristic acid,
Linear saturated carboxylic acids such as pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachinic acid, heneicosanoic acid, and behenic acid, and 2-dodecenoic acid, 3-dodecenoic acid, 4-dodecenoic acid, and 5-dodecenoic acid , 11-dodesenic acid, 2-tridesenic acid, cis-9-c
is-9-tridecenoic acid, 12-tridecenoic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, 6-pentadecenoic acid, cis-9-pentadecenoic acid,
14-pentadecenoic acid, 2-hexadecenoic acid, trans-
3-hexadecenoic acid, cis-7-hexadecenoic acid, cis-
9-palmitoleic acid, trans-9-hexadecenoic acid,
2-heptadecenoic acid, cis-7-heptadecenoic acid, cis-
8-heptadecenoic acid, cis-9-heptadecenoic acid, trans
-2-octadecenoic acid, cis-2-octadecenoic acid, tra
ns-3-octadecenoic acid, cis-3-octadecenoic acid, t
rans-4-octadecenoic acid, petroselinic acid, petroelaidic acid, cis-7-octadecenoic acid, trans-7-octadecenoic acid, cis-8-octadecenoic acid, trans-8-
Octadecenoic acid, oleic acid, elaidic acid, cis-1
1-octadecenoic acid, basenic acid, cis-9-nonadecenoic acid, gondolic acid, trans-gondoic acid, erucic acid, brassic acid, trans-8, trans-10-octadecadienoic acid, linoleic acid, linoelaidic acid, cis -9, t
rans-11-octadecadienoic acid, trans-10, cis-
12-octadecadienoic acid, cis-9, cis-11-octadecadienoic acid, cis-10, cis-12-octadecadienoic acid, trans-10, trans-12-octadecadienoic acid, trans-9, trans-11-octadecadienoic acid, α
-Eleostearic acid, β-eleostearic acid, cis
-9, cis-11, trans-13-octadecatrienoic acid, linolenic acid, linolenic elaidic acid, pseudoeleostearic acid, α-parinaric acid, β-parinaric acid, 2,2-dimethyl-cis-9, Linear unsaturated carboxylic acids such as cis-12-octadecadienoic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid can be used.

[0006] The lipase used in the step (a) of the present invention is a catalyst for deacylation.
Position the lipase specifically acting can phospholipase A ₁ or Ruimidori enzyme, phospholipase A ₂ or Ruimidori enzyme particularly preferably used. Phospholipase A ₁ acting specifically at position ₁ includes various bacteria,
For example, Escherichia coli, Mycobacterium frei, Bacillus megaterium, Bacillus subtilis,
Or phospholipase A obtained from various organs of animals, for example, bovine pancreas, rat thymus, kidney, spleen, lung, etc.
₁ etc. can be used. Of these phospholipase A ₁ for bovine pancreas it has been used industrially origin can be particularly preferably used. As the phospholipase A ₂ to act specifically at the 2-position, snake venom, for example Kurotarasu-Adomanteusu, Naja Naja venom such Torimeresurasu-Furabobirideisu, bee venom, for example toxins, such as bees, lizard poisons, for example Heroderuma & Poisons such as horidam, scorpion poisons, such as Loirus quinques
Can be used poisons such Toriatasu or animal various organs, for example, porcine pancreatic phospholipase A ₂ and the like obtained from such rat liver. Among them, phospholipase A ₂ to the porcine pancreas has been used industrially and origin can be particularly preferably used. The amount of the lipase used in the production method of the present invention can be 100 to 300 units, preferably 150 to 250 units, per 1 g of 1,2-diacyl-3-glycerylphosphatidylcholine. If it is less than 100 units, the reaction rate will be low, and if it exceeds 300 units, there will be no improvement in the reaction rate commensurate with the amount used, resulting in poor economics. The temperature of the deacylation reaction by partial hydrolysis in the step (a) is preferably in the range of 0 to 80 ° C, more preferably 10 to 60 ° C, and still more preferably 30 to 50 ° C. Outside this reaction temperature range, the deacylation reaction rate is significantly reduced. As the organic solvent used in the deacylation reaction in the step (a), any solvent can be used without particular limitation as long as it dissolves phosphatidylcholine, such as chloroform, dichloromethane, halogenated solvents such as carbon tetrachloride, hexane, pentane and the like. Hydrocarbon solvents, aromatic solvents such as benzene and xylene, and ether solvents such as dimethyl ether, diethyl ether and methyl ethyl ether. These may be used alone or in combination of two or more. Can be.

[0007] The amount of the organic solvent used is 5 to 100 times the weight of phosphatidylcholine. If it is less than 5 times, the viscosity of the solution becomes high, and if it exceeds 100 times, the industrial production efficiency deteriorates. Water used in the deacylation reaction of the step (a) is
Distilled water that does not contain metal ions that inhibit the enzymatic reaction is preferable, and a buffer is added to adjust the pH to 6.5 to 9.0.
It is more preferable to use the above buffer solution. For example, hydrochloric acid-tris (hydroxymethyl) aminomethane buffer, phosphate buffer, pyrophosphate-sodium hydroxide buffer, boric acid-
Sodium hydroxide buffer, boric acid-sodium carbonate buffer can be used. The amount of water used is 0.1 to 2 times the weight of the phosphatidylcholine organic solvent solution. After completion of the reaction, the layers are separated to obtain a lower organic solvent layer. The organic solvent layer is concentrated using an evaporator or the like. In the crystallization in the step (a), an organic solvent of hexane: acetone: methanol = 7 to 5: 4 to 0: 2 to 0 (volume ratio) is used. The solution is dissolved at a temperature of 35 to 45 ° C., the phospholipid concentration is adjusted to 5 to 50% by weight, preferably 20 to 40% by weight, and the temperature of this solution is 0 to 30 ° C., preferably 3 to 30% by weight.
The temperature is lowered to −10 ° C. to crystallize the phospholipid, which is filtered to obtain a phospholipid powder. If the crystallization conditions are outside the range of the solvent composition and the temperature conditions, the purity or yield of the target phospholipid decreases. By crystallization, unreacted 1,2-diacyl-3-glycerylphosphatidylcholine and fatty acids as impurities formed after the reaction can be removed. In the step (b) of the present invention, an arbitrary acyl group is introduced into the monoacyl-3-glycerylphosphatidylcholine obtained in the step (a) to obtain a mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine, The mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine is eluted from the reaction solution using silica gel column chromatography, and the eluate can be concentrated by lyophilization or the like to be crystallized. The acyl group introduced in the acylation reaction in the step (b) of the present invention is a natural or synthetic linear acyl group of a saturated or unsaturated carboxylic acid having 12 to 22 carbon atoms. Groups can be introduced. As the acyl group raw material introduced in the step (b) of the present invention, a corresponding carboxylic acid anhydride is used.

The carboxylic acid corresponding to the acyl group introduced in the step (b) of the present invention includes, for example, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid,
Straight-chain saturated carboxylic acids such as margaric acid, stearic acid, nonadecanoic acid, arachinic acid, heneicosanoic acid, and behenic acid; 2-tridesenic acid, cis-9-tridesenic acid, 12-tridesenic acid,
4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, 6-pentadecenoic acid, cis-9-pentadecenoic acid, 14-pentadecenoic acid, 2-hexadecenoic acid, tr
ans-3-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-palmitoleic acid, trans-9-hexadecenoic acid, 2-heptadecenoic acid, cis-7-heptadecenoic acid, c
is-8-heptadecenoic acid, cis-9-heptadecenoic acid, t
rans-2-octadecenoic acid, cis-2-octadecenoic acid, trans-3-octadecenoic acid, cis-3-octadecenoic acid, trans-4-octadecenoic acid, petroselinic acid,
Petroselaidic acid, cis-7-octadecenoic acid, tra
ns-7-octadecenoic acid, cis-8-octadecenoic acid, t
rans-8-octadecenoic acid, oleic acid, elaidic acid, cis-11-octadecenoic acid, basenic acid, cis-9
-Nonadecenoic acid, gondolic acid, trans-gondoic acid, erucic acid, brassic acid, trans-8, trans-10
-Octadecadienoic acid, linoleic acid, linoelaidic acid, cis-9-trans-11-octadecadienoic acid, tran
s-10, cis-12-octadecadienoic acid, cis-9, cis
-11-octadecadienoic acid, cis-10, cis-12
Octadecadienoic acid, trans-10, trans-12-octadecadienoic acid, trans-9, trans-11-octadecadienoic acid, α-eleostearic acid, β-eleostearic acid, cis-9, cis -11, trans-13-octadecatrienoic acid, linolenic acid, linolenic elaidic acid, pseudoeleostearic acid, α-parinaric acid, β-parinaric acid, 2,2-dimethyl-cis-9, cis-12 One or more acyl groups such as linear unsaturated carboxylic acids such as octadecadienoic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid can be mixed and introduced. As the acyl group, preferably myristic acid,
Palmitic, stearic, arachinic, behenic and palmitoleic, oleic, elaidic, erucic, linoleic, linolenic, eicosapentaenoic, docosahexaenoic, arachidonic acids can be used. Among them, acyl groups of linear unsaturated carboxylic acids such as oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid are particularly preferable.

Further, as the fatty acid anhydride used in the step (b), the anhydride of the above carboxylic acid can be used without any particular limitation. A commercially available product can be used, and an anhydride can be appropriately produced from the above fatty acid using a condensing agent such as dicyclohexylcarbodiimide and used. When two or more kinds of fatty acid anhydrides are used in the step (b), a mixture of two or more kinds of reactants is obtained. In step (b), the amount of the fatty acid anhydride to be added is monoacyl-
1 to 20 for 3-glyceryl phosphatidylcholine
It is good to be equivalent, preferably 1 to 5 equivalents. If the amount is less than 1 equivalent, the acylation reaction does not proceed sufficiently, and the purity and yield are reduced. Even when the amount exceeds 20 equivalents, the reaction rate and the reaction rate are hardly improved in proportion to the amount added, which is not desirable from the viewpoint of economy. The acylation reaction of the step (b) of the present invention comprises:
Organic solvents can be used. The organic solvent that can be used for this reaction may be any solvent that dissolves phosphatidylcholine, such as chloroform, dichloromethane, halogenated solvents such as carbon tetrachloride, hexane, and the like.
Hydrocarbon solvents such as pentane, benzene, aromatic solvents such as xylene, dimethyl ether, diethyl ether,
Examples thereof include ether solvents such as methyl ethyl ether and the like, and they may be used alone or in combination of two or more. The amount of the organic solvent used can be 5 to 100 times the weight ratio of phosphatidylcholine, and less than 5 times the viscosity of the solution increases,
If the amount exceeds 100 times, the production efficiency is deteriorated. The present invention (b)
A tertiary amine is used as a basic catalyst used in the acylation reaction in the step. For example, dimethylaminopyridine,
Pyridine derivatives such as dimethyl-4-amino-2-methylpyridine and 4-pyrrolidinopyrrolidine, and tertiary amines such as triethylamine and tributylamine can be used.
The use of pyrrolidinopyrrolidine is particularly preferred. The amount of the tertiary amine catalyst to be added may be about 0.01 to 20 equivalents, preferably 0.1 to 2 equivalents, to the raw material. If it is less than 0.01 equivalent, the reaction rate will be low, and if it exceeds 20 equivalents, the reaction rate will not be high and the economy will be poor.
The acylation reaction is carried out at a temperature of 0 to 80 ° C, preferably 10 to 50 ° C.
It can be performed in the range of ° C. If the temperature is lower than 0 ° C., the reaction rate is decreased. After the completion of the acylation reaction, as a post-treatment, solid substances such as a catalyst are removed by filtration to obtain a mixed acid-type 1,2-diacyl-3-glycerylphosphatidylcholine reaction solution.

In the step (b) of the present invention, any silica gel column that adsorbs the reaction solution can be used without particular limitation as long as it can separate phospholipids. it can. In step (b), the separation and purification of the adsorbed substance on the silica gel column is performed using mixed acid type 1,
Separation and purification can be performed using a solvent that selectively elutes 2-diacyl-3-glycerylphosphatidylcholine. In this case, two-step elution is performed using two kinds of solvents. In the separation and purification using a silica gel column in the step (b) of the present invention, an elution operation can be performed in two steps using the following two types of mixed solvents. (1) chloroform: methanol = 95-80: 20-
5 (volume ratio) of the mixed solvent is used in a ratio of 400 to 600 ml per 1 g of the monoacyl-3-glycerylphosphatidylcholine used in the acylation reaction in the step (b). By this elution operation, mainly free fatty acids and basic catalysts are eluted. (2) chloroform: methanol: water = 85-75: 2
A mixed solvent of 0 to 18: 3 to 1.5 (volume ratio) is used in a ratio of 400 to 600 ml per 1 g of monoacyl-3-glycerylphosphatidylcholine. By this elution operation, the desired mixed acid type 1,2-diacyl-3
Glyceryl phosphatidylcholine is eluted. In the purification of mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine by the above method, unreacted acids, by-products and catalysts can be efficiently removed by silica gel column chromatography, and a highly pure mixed acid can be obtained by a simple process. Type 1,2-diacyl-3-glycerylphosphatidylcholine can be obtained. Step (c) of the present invention is a step of converting mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine into mixed acid type 1,2-diacyl-3-glycerophospholipid by phospholipase D in the presence of a base. The organic solvent used in the reaction system of the step (c) of the present invention may be any solvent capable of dissolving phosphatidylcholine, such as chloroform, dichloromethane, a halogen-based solvent such as carbon tetrachloride, hexane, a hydrocarbon-based solvent such as pentane, Aromatic solvents such as benzene and xylene,
Ether solvents such as dimethyl ether, diethyl ether and methyl ethyl ether can be used alone or in combination of two or more. The amount of the organic solvent used is 5 to 100 times the weight of phosphatidylcholine. If the amount is less than 5 times, the viscosity of the solution becomes high, and if it exceeds 100 times, the production efficiency becomes poor.

The phosphorivase D used in the step (c) is derived from the genus Streptomyces, and is obtained, for example, from a phosphorivase D-producing bacterium such as Streptomyces chromophoscus, Streptomyces hatchjoensis, or Streptomyces prunicola. Phospholipase D can be used. The amount of phospholipase D used is
The amount is from 5 to 90 units, preferably from 10 to 80 units, per 1 g of the mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine. If it is less than 5 units, the reaction rate is low, and if it exceeds 90 units, the improvement in the reaction rate is small, and the economic efficiency is deteriorated. Examples of the base used in the step (c) include aliphatic alcohols such as methanol, ethanol and propanol; nitrogen-containing alcohols such as ethanolamine, serine and 1-amino-2-propanol; ethylene glycol, propylene glycol and glycerol. Examples thereof include polyols, and generally commercially available products may be used.
The amount of the base to be used is 5 to 100 mol, preferably 20 to 80 mol, per 1 mol of phosphatidylcholine. If the amount is less than 5 mol, the conversion may be low, and if it exceeds 100 mol, the yield may be reduced. The base exchange reaction in the step (c) can be performed at a temperature of about 20 to 60 ° C, preferably 35 to 45 ° C. If the temperature is lower than 20 ° C., the reaction rate decreases. The water used in the step (c) is preferably distilled water which does not contain metal ions which inhibit the enzyme reaction, and more preferably a buffer having a pH of 5.0 to 7.0 by adding a buffer. For example, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, citric acid-sodium citrate buffer, 3,3-dimethylglutaric acid-sodium hydroxide buffer, succinic acid-sodium hydroxide buffer, phosphate buffer There are liquids. The amount of water used is 0.1 to 2 times the weight ratio of the mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine organic solvent solution.

In the step (d) of the present invention, the organic solvent solution obtained in the step (c) is concentrated to dryness, and chloroform: methanol: acetone = 13 to 0: 2 to 0: 9 to 7 (volume ratio). After adjusting the phospholipid concentration of the solution to 5 to 50% by weight, preferably 20 to 40% by weight, the temperature of the solution is 0 to 30 ° C, preferably 3 to 1%.
The temperature is lowered to 0 ° C., and the phospholipid is crystallized and filtered to obtain a phospholipid powder. Outside the range, the purity or yield of the target phospholipid is reduced. In the step (e) of the present invention, the phospholipid crystals obtained in the step (d) are chloroform: methanol: acetone at a weight ratio of 5 to 20 times: 13 to 9: 2 to 1: 1 to 8 (volume mixing). Ratio) in a mixed solvent at 35 to 45 ° C., and after adjusting the phospholipid concentration of the solution to 5 to 50% by weight, preferably 20 to 40% by weight, the temperature is 0 to 30 ° C., preferably 3 to 10 ° C. Is a step of crystallizing the phospholipid and filtering to obtain a phospholipid powder. Outside the range, the purity or yield of the target phospholipid is reduced. Step (f) of the present invention comprises:
The crystals of the phospholipid obtained in the step (e) are chloroform: methanol: acetone = 13 to 9: 2 to 5 to 20 times by weight.
After dissolving in a mixed solvent of 1: 7 to 6 (volume ratio) at 35 to 45 ° C and adjusting the phospholipid concentration of the solution to 5 to 50% by weight, preferably 20 to 40% by weight, the temperature is 0 to 30 ° C. Preferably, a step of crystallizing the phospholipid at 3 to 10 ° C. and filtering to obtain a phospholipid powder. Outside the range, the purity or yield of the target phospholipid is reduced.

[0013]

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to the following examples. The composition analysis and the purity measurement of the phospholipid were performed by thin-layer chromatography (hereinafter, referred to as TLC). TLC
Using Kieselgel 60 (Merck & Co. Inc.), 2 microliters of a 5% by weight sample was spotted, and chloroform: methanol: distilled water: 28% aqueous ammonia = 65: 25: 4: 0.3 (volume) Ratio),
The solution was prepared by spraying a solution of 10 g of copper sulfate (anhydrous) and 8 ml of phosphoric acid (85%) in 100 ml of distilled water and then heating. In the method for producing a fatty acid anhydride used in Examples, the fatty acid was dissolved in chloroform so as to have a molar concentration of 1, and dicyclohexylcarbodiimide was added at 40 ° C. so as to have a molar concentration of 0.7, and white dicyclohexyl was added. When urea was precipitated, dicyclohexylurea was removed by filtration.

Example 1 Dipalmitoyl phosphatidylcholine (120 g) was dissolved in chloroform (1800 ml), and hydrochloric acid-100 mmol tris (hydroxymethyl) aminomethane buffer (pH = 8.0) (880 ml), 100 mM calcium chloride aqueous solution (1320 ml) and recitase (24000) After adding the unit and reacting at 40 ° C. for 24 hours, the reaction solution was transferred to a separating funnel and allowed to stand for 1 hour. 2
After confirming that the layers were separated, the lower layer was separated into a flask, the solvent was distilled off with an evaporator, 5000 ml of hexane was added, the mixture was cooled to 4 ° C, kept at the same temperature for 1 hour, filtered, and precipitated. 62.4 g of the product were obtained. The composition of the precipitate obtained was 95.5% by weight of monopalmitoyl phosphatidylcholine and 4.5% by weight of free fatty acid. After adding 10 g of the precipitate, 177.4 g of oleic anhydride and 4.9 g of dimethylaminopyridine and reacting at 40 ° C. for 24 hours, the reaction solution was applied to a silica gel column (silica gel: Daisogel IR-60 (manufactured by Daiso Corporation)). The mixture was adsorbed onto a column (volume: 500 ml), and a mixed solvent of chloroform: methanol = 80: 20 (volume ratio) was flowed five times the column volume, followed by mixing of chloroform: methanol: water = 80: 20: 3 (volume ratio). The eluate eluted was collected by flowing the solvent in an amount 5 times the column volume. The solvent was distilled off from the obtained eluate using an evaporator, dispersed in water for injection, and lyophilized to obtain 14 g of a dried product. The composition of the obtained dried product is 1-palmitoyl-2-oleoyl-3-phosphatidylcholine 97% by weight, monopalmitoylphosphatidylcholine 0.5% by weight, free fatty acid 2.5% by weight.
Met. 10 g of the dried product was dissolved in 80 ml of chloroform, and ammonium acetate / acetic acid buffer (pH
= 6.25) Glycerin 27.25 in 33 ml
g, 200 units of phospholipase D dissolved and added
After reacting at 18 ° C. for 18 hours, the reaction solution is transferred to a separating funnel and left for 1 hour to separate the lower layer. The solvent was distilled off from the separated lower layer using an evaporator, and methanol: acetone = 1.
100 ml of a 5/85 (volume ratio) mixed solution was added, the mixture was cooled to 4 ° C., kept at the same temperature for 1 hour, and then filtered to remove crude 1-palmitoyl-2-oleoyl-3-phosphatidylglycerol ammonium salt. Obtained. This was dissolved in 200 ml of a mixed solution of chloroform: methanol: acetone = 13/1/9 (volume ratio), precipitated by cooling, and collected by filtration. The obtained crystals were dissolved again in 100 ml of a mixed solution of chloroform: methanol: acetone = 13/1/6 (volume ratio), cooled to 4 ° C.
After keeping at the same temperature for 1 hour, the mixture was filtered and dried,
g was obtained. The composition of the obtained crystals was 99.5% by weight of purified 1-palmitoyl-2-oleoyl-3-phosphatidylglycerol ammonium salt, 1-palmitoyl-2
Oleoyl-3-phosphatidylcholine 0.1% by weight, lyso-form 0.2% by weight, and free fatty acid 0.2% by weight.

[0015] Example 2 distearoylphosphatidylcholine 130g was dissolved in chloroform 1800 ml, except that the enzyme phospholipase A ₁ solution 26000 units Example 1
Hexane: acetone: methanol =
6: 2: 1 (volume ratio) 2700 ml and add 4
After cooling to ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 67.6 g of a precipitate. The composition of the obtained precipitate was 95% by weight of monostearoyl phosphatidylcholine and 5% by weight of free fatty acid. 10 g of the precipitate, 177.4 g of linoleic anhydride and 4.9 g of dimethylaminopyridine were added and reacted in the same manner as in Example 1. After adsorbing on a silica gel column, chloroform: methanol = 9: 1 (volume ratio), chloroform: methanol: water = 80: 18:
The eluate was collected at 2 (volume ratio) in the same manner as in Example 1, and lyophilized to obtain 14 g of a dried product. The composition of the obtained dried product was 1-linoleoyl-2-stearoyl-3-phosphatidylcholine 96.5% by weight, monostearoylphosphatidylcholine 0.5% by weight, and free fatty acid 3%. 10 g of the dried product was dissolved in 270 ml of chloroform, 5.8 g of ethanolamine was dissolved in 100 ml of distilled water, and the pH was adjusted to 6.0 with acetic acid.
800 units of phospholipase D was added, and the mixture was added to a chloroform solution of 1-linoleoyl-2-stearoyl-phosphatidylcholine, followed by stirring at 40 ° C for 6 hours. The reaction solution was transferred to a separatory funnel, allowed to stand for 1 hour, the lower layer was separated into a flask, concentrated, added with 210 ml of acetone, cooled to 4 ° C, kept at the same temperature for 1 hour, and filtered. Thus, crude 1-linoleoyl-2-stearoyl-3-phosphatidylethanolamine was obtained. The obtained crystal was treated in the same manner as in Example 1 to obtain 6.65 g. The composition of the obtained crystal was 99.5% by weight of purified 1-linoleoyl-2-stearoyl-3-phosphatidylethanolamine, 0.1% by weight of 1-linoleoyl-2-stearoyl-phosphatidylcholine, 0.3% by weight of lyso-form. Was 0.2% by weight of free fatty acids.

Example 3 Dipalmitoylphosphatidylcholine (120 g) was dissolved in chloroform (1800 ml), and the reaction was carried out in the same manner as in Example 1 except that the enzyme was changed to 24,000 units of phospholipase A _2. Hexane: acetone: methanol = 6: 3:
1 (volume ratio) was added to 3000 ml, cooled to 4 ° C., kept at the same temperature for 1 hour, and filtered to obtain a precipitate 6
2.4 g were obtained. The composition of the resulting precipitate was monopalmitoyl phosphatidylcholine 95% by weight, free fatty acid 5
% By weight. Linoleic anhydride 2.
After adding 86 g and reacting in the same manner as in Example 2 and adsorbing on a silica gel column, chloroform: methanol = 9.
5: 5 (volume ratio), chloroform: methanol: water = 8
The eluate was collected at 0: 18: 1.5 (volume ratio) in the same manner as in Example 1 and lyophilized to obtain 14 g of a dried product. The composition of the obtained dried product was 96.5% by weight of 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine, 0.5% by weight of monopalmitoylphosphatidylcholine, and 3% of free fatty acid. 10 g of the dried product was dissolved in 80 ml of chloroform, and sodium acetate / acetic acid buffer (pH =
6.25) 27.25 g of glycerin in 33 ml,
800 units of phospholipase D was dissolved, added to a chloroform solution of 1-linoleoyl-2-stearoyl-3-phosphatidylcholine, and reacted at 40 ° C. for 18 hours.
The reaction solution was transferred to a separating funnel, allowed to stand for 1 hour, the lower layer was separated and concentrated, and 100 ml of a mixed solution of methanol: acetone = 15/85 (volume ratio) was added to the obtained concentrate. After cooling to 4 ° C. and keeping at the same temperature for 1 hour, the mixture was filtered to obtain 1-palmitoyl-2-linoleoyl oil.
3-Phosphatidylglycerol sodium salt was obtained.
The obtained crystals were treated in the same manner as in Example 1 to obtain 7.14 g. The composition of the obtained crystals was purified 1-palmitoyl-
99.5% by weight of 2-linoleoyl-3-phosphatidylglycerol sodium salt, 0.2% by weight of 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine,
The lyso form was 0.1% by weight and the free fatty acid was 0.2% by weight.

Example 4 120 g of dipalmitoylphosphatidylcholine was reacted in the same manner as in Example 1, 3,000 ml of hexane: acetone = 7: 4 (volume ratio) was added, the mixture was cooled to 4 ° C., and kept at the same temperature for 1 hour. After filtration, 62.3 g of a precipitate was obtained. The composition of the obtained precipitate was 95.5% by weight of monopalmitoyl phosphatidylcholine and 4.5% by weight of free fatty acid.
Met. 10 g of precipitate, 177.4 of linoleic anhydride
g, 4.9 g of dimethylaminopyridine was added.
And adsorbed on a silica gel column, chloroform: methanol = 8: 2 (volume ratio), chloroform: methanol: water = 75: 20: 3 (volume ratio), eluate in the same manner as in Example 1. , Freeze-dried and dried 1
4 g were obtained. The composition of the obtained dried product is 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine 9
6.5% by weight, 0.5% by weight of monopalmitoylphosphatidylcholine and 3% of free fatty acids. 10 g dry matter
Was reacted in the same manner as in Example 2, and methanol: acetone =
100 ml of a 15/85 (volume ratio) mixed solution was added, cooled to 4 ° C., kept at the same temperature for 1 hour, and then filtered to obtain 1-palmitoyl-2-linoleoyl-3-phosphatidylethanolamine. . The obtained crystals were treated in the same manner as in Example 1 to obtain 6.66 g. The composition of the obtained crystals was 99.3% by weight of purified 1-palmitoyl-2-linoleoyl-3-phosphatidylethanolamine, 0.2% by weight of 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine, and 0.2% of the lyso form. % Of free fatty acids and 0.3% by weight of free fatty acids.

Example 5 130 g of distearoylphosphatidylcholine was reacted in the same manner as in Example 2 to give hexane: acetone: methanol =
5: 4: 2 (volume ratio) 2700 ml was added and 4
After cooling to ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 67.6 g of a precipitate. The composition of the obtained precipitate was 95% by weight of monostearoyl phosphatidylcholine and 5% by weight of free fatty acid. 177.4 g of linoleic anhydride and 4.9 g of dimethylaminopyridine were added to 10 g of the precipitate, reacted at 40 ° C. for 24 hours, adsorbed on a silica gel column, and chloroform: methanol = 9.
0:10 (volume ratio), chloroform: methanol: water =
The eluate was collected at 75: 20: 1.5 (volume ratio) in the same manner as in Example 1 and lyophilized to obtain 14 g of a dried product. The composition of the obtained dried product was 1-linoleoyl-2-stearoyl-3-phosphatidylcholine 96.5% by weight, monostearoylphosphatidylcholine 0.5% by weight, and free fatty acid 3%. 10 g of the dried product was dissolved in 80 ml of chloroform, and 33 ml of sodium acetate / acetate buffer (pH = 6.25) was added to 27.25 of serine.
g, 200 units of phospholipase D dissolved and added
After reacting at 18 ° C. for 18 hours, the reaction solution is transferred to a separating funnel and left for 1 hour to separate the lower layer. The separated lower layer was concentrated, and chloroform: methanol: acetone = 13/2.
The mixture was cooled to 4 ° C., kept at the same temperature for 1 hour, and filtered to obtain crude 1-linoleoyl-2-stearoyl-3-phosphatidylserine sodium salt. The obtained crystals are chloroform:
It was dissolved in 200 ml of a mixed solution of methanol / acetone = 13/1/11 (volume ratio), precipitated by cooling, and collected by filtration. This is again chloroform: methanol:
It was dissolved in 100 ml of a mixed solution of acetone = 9/2/6 (volume ratio), precipitated by cooling, filtered and dried to obtain 7.27 g. The composition of the obtained crystal was purified 1
-Linoleoyl-2-stearoyl-3-phosphatidylserine sodium salt 99.4% by weight, 1-linoleoyl-2-stearoyl-phosphatidylcholine 0.2% by weight, lyso-form 0.2% by weight, free fatty acid 0.2% by weight. there were.

Example 6 Dipalmitoyl phosphatidylcholine (120 g) was reacted in the same manner as in Example 3, 3,000 ml of hexane was added, the mixture was cooled to 4 ° C., kept at the same temperature for 1 hour, filtered, and 62.4 g of a precipitate was obtained. I got The composition of the precipitate obtained was 95.5% by weight of monopalmitoyl phosphatidylcholine and 4.5% by weight of free fatty acid. 10 g of precipitate
180 g of arachidonic anhydride and 4.9 g of dimethylaminopyridine were added, reacted at 40 ° C. for 24 hours, adsorbed on a silica gel column, and then chloroform: methanol = 95: 5 (volume ratio), chloroform: methanol:
The eluate was collected in the same manner as in Example 1 with water = 85: 18: 1.5 (volume ratio) and freeze-dried to obtain 14 g of a dried product. The composition of the obtained dried product was 95.5% by weight of 1-palmitoyl-2-arachidonoyl-3-phosphatidylcholine,
3.5% by weight of monopalmitoyl phosphatidylcholine,
The free fatty acid was 1% by weight. Example 2 using 10 g of dried product
After adding 100 ml of a mixed solution of chloroform: methanol: acetone = 6/1/7 (volume ratio), the mixture was cooled to 4 ° C. and kept at the same temperature for 1 hour.
Filter to give crude 1-palmitoyl-2-arachidonoyl-3
-Phosphatidylethanolamine was obtained. The obtained crystals were subjected to chloroform: methanol: acetone = 9/2/8.
The mixture was dissolved in 200 ml of the mixed solution (volume ratio), precipitated by cooling, and collected by filtration. This is again chloroform:
Dissolved in 100 ml of a mixed solution of methanol: acetone = 13/1/7 (volume ratio), precipitated by cooling,
The crystals were collected by filtration and dried to obtain 6.67 g. The composition of the obtained crystals was purified 1-palmitoyl-2-arachidonoyl-3.
99.4% by weight of phosphatidylethanolamine, 1
-Palmitoyl-2-arachidonoyl-3-phosphatidylcholine 0.2% by weight, lyso-form 0.2% by weight and free fatty acid 0.2% by weight.

Example 7 130 g of distearoyl phosphatidylcholine was reacted in the same manner as in Example 2 to obtain hexane: acetone: methanol =
6: 3: 2 (volume ratio) 2700 ml and add 4
After cooling to ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 67.6 g of a precipitate. The composition of the obtained precipitate was 95.5% by weight of monostearoyl phosphatidylcholine and 4.5% by weight of free fatty acid. 177.4 g of oleic anhydride and 4.9 g of dimethylaminopyridine were added to 10 g of the precipitate.
, And reacted in the same manner as in Example 1. The reaction solution was adsorbed on a silica gel column, and chloroform: methanol = 8.
0:20 (volume ratio), chloroform: methanol: water =
The eluate was collected at 80: 19: 1 (volume ratio) in the same manner as in Example 1 and freeze-dried to obtain 14 g of a dried product. The composition of the obtained dried product is 1-oleoyl-2-stearoyl-3
-97% by weight of phosphatidylcholine, 0.5% by weight of monostearoyl phosphatidylcholine and 2.5% by weight of free fatty acids. 10 g of the dried product was reacted in the same manner as in Example 3, 100 ml of acetone was added, and the mixture was cooled to 4 ° C.
After maintaining at the same temperature for 1 hour, the mixture was filtered to obtain crude 1-palmitoyl-2-oleoyl-3-phosphatidylglycerol sodium salt. The obtained crystals were dissolved in 200 ml of a mixed solution of chloroform: methanol: acetone = 11/1/9 (volume ratio), precipitated by cooling, and collected by filtration. This was dissolved again in 100 ml of a mixed solution of chloroform: methanol: acetone = 11/1/7 (volume ratio), precipitated by cooling, filtered, and dried.
7.14 g of crystals were obtained. The composition of the obtained crystals was 99.4% by weight of purified 1-palmitoyl-2-oleoyl-3-phosphatidylglycerol sodium salt, 1-oleoyl-2-stearoyl-phosphatidylcholine 0.1% by weight.
1% by weight, lyso-form 0.2% by weight, free fatty acid 0.3% by weight
Met.

Comparative Example 1 Dipalmitoylphosphatidylcholine (120 g) was reacted in the same manner as in Example 1, 5,000 ml of acetone was added, the mixture was cooled to 4 ° C., kept at the same temperature for 1 hour, and filtered to obtain 52.4 g of a precipitate. Obtained. The composition of the obtained precipitate is
Monopalmitoyl phosphatidylcholine was 89.5% by weight and free fatty acid was 10.5% by weight. 10 g of precipitate
And oleic anhydride were reacted in the same manner as in Example 1, the reaction solution was adsorbed on a silica gel column, and chloroform: methanol = 8: 2 (volume ratio), chloroform: methanol: water = 80: 20: 3 (volume ratio) The eluate was collected in the same manner as in Example 1 and freeze-dried to obtain 9 g of a dried product. The composition of the obtained dried product was 12.5% by weight of 1-palmitoyl-2-oleoyl-phosphatidylcholine, 2.5% by weight of monopalmitoylphosphatidylcholine, and 5% by weight of free fatty acid. 10 g of the dried product was reacted in the same manner as in Example 1, 100 ml of a mixed solution of methanol / acetone = 15/85 (volume ratio) was added, cooled to 4 ° C., kept at the same temperature for 1 hour, and filtered. The crude 1-palmitoyl-2-oleoyl-3-phosphatidylglycerol ammonium salt was obtained. This was mixed with a mixed solution 20 of chloroform: methanol: acetone = 13/1/9 (volume ratio).
It was dissolved in 0 ml, precipitated by cooling, and collected by filtration. The obtained crystals are dissolved again in 100 ml of a mixed solution of chloroform: methanol: acetone = 13/1/6 (volume ratio), cooled to 4 ° C., kept at the same temperature for 1 hour, filtered and dried. 6.09 g. The composition of the obtained crystals was as follows: purified 1-palmitoyl-2-oleoyl-3-phosphatidyliso-form glycerol ammonium salt 90.5% by weight, monopalmitoyl phosphatidylcholine 2% by weight, lyso-form 1% by weight, free fatty acid 6.5 % By weight. If the composition of the mixed solvent in the step (a) is out of the range of the present invention, the purity and the yield will be low.

Comparative Example 2 130 g of distearoyl phosphatidylcholine was reacted in the same manner as in Example 2 and hexane: acetone: methanol was used.
6: 2: 1 (volume ratio) 2700 ml and add 4
After cooling to ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 67.6 g of a precipitate. The composition of the obtained precipitate was 95% by weight of monostearoyl phosphatidylcholine and 5% by weight of free fatty acid. After reacting 10 g of the precipitate in the same manner as in Example 2, the reaction solution was adsorbed on a silica gel column,
The eluate was collected and lyophilized in the same manner as in Example 1 using chloroform: methanol = 80: 25 (volume ratio) and chloroform: methanol: water = 80: 20: 5 (volume ratio).
8 g of a dried product was obtained. The composition of the resulting dried product was 17.5% by weight of 1-linoleoyl-2-stearoyl-3-phosphatidylcholine, 7.5% by weight of monostearoylphosphatidylcholine, and 5% by weight of free fatty acid. 10 g of the dried product was reacted in the same manner as in Example 2, and acetone 2100 was used.
Milliliter was added to obtain crude 1-linoleoyl-2-stearoyl-3-phosphatidylethanolamine.
The obtained crystals were treated in the same manner as in Example 1 to obtain 5.49 g. The composition of the obtained crystal was purified 1-linoleoyl-
91.5% by weight of 2-stearoyl-3-phosphatidylethanolamine, 3.5% by weight of 1-linoleoyl-2-stearoyl-3-phosphatidylcholine, Lyso-form 2
% Of free fatty acids and 2% by weight of free fatty acids. If the composition of the mixed solvent in the step (b) is out of the range of the present invention, the purity and the yield will be low.

Comparative Example 3 Dipalmitoyl phosphatidylcholine (120 g) was reacted in the same manner as in Example 3, and hexane: acetone: methanol was used.
3: 2: 5 (volume ratio) Add 3000 ml and add 4
After cooling to ℃ ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 52.4 g of a precipitate. The composition of the obtained precipitate was 90% by weight of monopalmitoyl phosphatidylcholine and 10% by weight of free fatty acid. After reacting 10 g of the precipitate in the same manner as in Example 3, the reaction solution was adsorbed on a silica gel column, and chloroform: methanol = 75: 25 (volume ratio), chloroform: methanol: water = 80: 19: 1.
The eluate was collected in the same manner as in Example 1 (by volume ratio) and freeze-dried to obtain 8 g of a dried product. The composition of the obtained dried product is 1
-Palmitoyl-2-linoleoyl-3-phosphatidylcholine 92.5% by weight, monopalmitoylphosphatidylcholine 5.5% by weight and free fatty acids 2% by weight. 10 g of the dried product was reacted in the same manner as in Example 3, and a mixed solution of methanol: acetone = 15/85 (volume ratio) 100
Milliliter was added to obtain crude 1-palmitoyl-2-linoleoyl-3-phosphatidylglycerol sodium salt. The obtained crystals were treated in the same manner as in Example 1, and 5.
73 g were obtained. The composition of the obtained crystals was 11.5% by weight of 1-palmitoyl-2-linoleoyl-3-phosphatidylglycerol sodium salt, 1-palmitoyl-2
-Linoleoyl-3-phosphatidylcholine 3.5% by weight, lyso-form 3% by weight, free fatty acid 2% by weight.
If the composition of the mixed solvent in the steps (a) and (b) is out of the range of the present invention, the purity and the yield will be low.

Comparative Example 4 130 g of distearoylphosphatidylcholine was reacted in the same manner as in Example 2, and hexane: acetone: methanol =
6: 2: 1 (volume ratio) 2700 ml and add 4
After cooling to ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 67.6 g of a precipitate. The composition of the obtained precipitate was 95% by weight of monostearoyl phosphatidylcholine and 5% by weight of free fatty acid. After reacting 10 g of the precipitate in the same manner as in Example 2, the reaction solution was adsorbed on a silica gel column,
The eluate was collected in the same manner as in Example 1 with chloroform: methanol = 9: 1 (volume ratio), chloroform: methanol = 1: 1 (volume ratio), and chloroform: methanol = 3: 7 (volume ratio), and lyophilized. Then, 8 g of a dried product was obtained. The composition of the obtained dried product was 19.5% by weight of 1-linoleoyl-2-stearoyl-phosphatidylcholine, 6.5% by weight of monopalmitoylphosphatidylcholine, and 4% by weight of free fatty acid. 10 g of the dried product was reacted in the same manner as in Example 2, and 2100 ml of acetone was added thereto.
Linoleoyl-2-stearoyl-3-phosphatidylethanolamine was obtained. The obtained crystals were treated in the same manner as in Example 1 to obtain 5.49 g. The composition of the obtained crystals was 91.5% by weight of purified 1-linoleoyl-2-stearoyl-3-phosphatidylethanolamine, 5.5% by weight of 1-linoleoyl-2-stearoyl-3-phosphatidylcholine, 2% by weight of lyso-form. 2% by weight of free fatty acids. If the composition of the mixed solvent in the step (b) is out of the range of the present invention, the purity and the yield will be low.

Comparative Example 5 Dipalmitoyl phosphatidylcholine (120 g) was reacted in the same manner as in Example 3, and hexane: acetone: methanol was used.
3: 2: 5 (volume ratio) Add 3000 ml and add 4
After cooling to ℃ ° C. and maintaining at the same temperature for 1 hour, the mixture was filtered to obtain 52.4 g of a precipitate. The composition of the resulting precipitate was monopalmitoyl phosphatidylcholine 95% by weight and free fatty acid 5% by weight. After reacting 10 g of the precipitate in the same manner as in Example 5, the reaction solution was adsorbed on a silica gel column,
Chloroform, chloroform: methanol: water = 65:
The eluate was collected and lyophilized at 25: 2 (volume ratio) in the same manner as in Example 1 to obtain 8 g of a dried product. The composition of the obtained dried product was 10.5% by weight of 1-palmitoyl-2-oleoyl-3-phosphatidylcholine, 5.5% by weight of monopalmitoyl phosphatidylcholine, and 4% by weight of free fatty acid.
Met. 10 g of the dried product was reacted in the same manner as in Example 3, 100 ml of a mixed solution of methanol / acetone = 15/85 (volume ratio) was added, and crude 1-palmitoyl-2-
Oleoyl-3-phosphatidylserine sodium salt was obtained. The obtained crystal was treated in the same manner as in Example 1, and 5.7 was obtained.
3 g were obtained. The composition of the obtained crystals was 91% by weight of purified 1-palmitoyl-2-oleoyl-3-phosphatidylserine sodium salt, 5% by weight of 1-palmitoyl-2-oleoyl-3-phosphatidylcholine, 2% by weight of lyso-form, The free fatty acid was 2% by weight. If the composition of the mixed solvent in the steps (a) and (b) is out of the range of the present invention, the purity and the yield will be low.

Comparative Example 6 Dipalmitoylphosphatidylcholine (120 g) was dissolved in chloroform (1800 ml), and the reaction was carried out in the same manner as in Example 1 except that the enzyme was changed to 24,000 units of phospholipase A _1.
After cooling to the same temperature and maintaining at the same temperature for 1 hour, 62.4 g of a precipitate was obtained by filtration. The composition of the obtained precipitate was monopalmitoyl phosphatidylcholine 95% by weight and free fatty acid 5% by weight. 10 g of the precipitate, 168.5 g of myristic anhydride and 4.9 g of dimethylaminopyridine were added.
After reacting at 40 ° C. for 24 hours, the reaction solution was adsorbed on a silica gel column, and chloroform: methanol = 9: 1.
The eluate was collected in the same manner as in Example 1 (volume ratio), chloroform: methanol = 1: 1 (volume ratio), and chloroform: methanol = 3: 7 (volume ratio), and lyophilized to obtain 8 g of a dried product. Was. The composition of the obtained dried product is 1-myristoyl-2-palmitoyl-3-phosphatidylcholine 9
0.5% by weight, 5.5% by weight of monopalmitoyl phosphatidylcholine and 4% by weight of free fatty acids. Dried product 1
0 g was reacted in the same manner as in Example 3, and a mixed solution 1 of chloroform: methanol: acetone = 13/2/8 (volume ratio) was used.
2,000 ml, and the crude 1-myristoyl-2-
Palmitoyl-3-phosphatidylglycerol sodium salt was obtained. The obtained crystals were mixed with a mixed solution 200 of chloroform: methanol: acetone = 8/1/5 (volume ratio).
It was dissolved in 0 ml, precipitated by cooling, and collected by filtration. This is again chloroform: methanol: acetone =
Dissolved in 1000 ml of a mixed solution of 8/1/5 (volume ratio), precipitated by cooling, collected by filtration and dried.
g was obtained. The composition of the obtained crystal was 91.5% by weight of purified 1-myristoyl-2-palmitoyl-3-phosphatidylglycerol sodium salt, 1-myristoyl-2
-Palmitoyl-3-phosphatidylcholine 4.5% by weight, lyso-form 3% by weight, free fatty acid 2% by weight.
If the composition of the mixed solvent in the steps (b), (e) and (f) is out of the range of the present invention, the purity and the yield will be low.

Comparative Example 7 Distearoyl phosphatidylcholine (130 g) was reacted in the same manner as in Example 3, and hexane: acetone: methanol was used.
3: 2: 5 (volume ratio) 2700 ml was added and 4
After cooling to the same temperature and keeping at the same temperature for 1 hour, 57.1 g of a precipitate was obtained by filtration. The composition of the obtained precipitate was 95% by weight of monostearoyl phosphatidylcholine and 5% by weight of free fatty acid. After reacting 10 g of the precipitate with 177.4 g of oleic anhydride in the same manner as in Example 1, the reaction solution was adsorbed on a silica gel column, and chloroform: methanol = 80: 25 (volume ratio), chloroform: methanol: water = 80. : 20: 5 (volume ratio), the eluate was collected and lyophilized in the same manner as in Example 1 to obtain 8 g of a dried product. The composition of the obtained dried product was 19.5% by weight of 1-stearoyl-2-oleoyl-3-phosphatidylcholine, 6.5% by weight of monostearoylphosphatidylcholine, and 4% by weight of free fatty acid. 10 g of the dried product was reacted in the same manner as in Example 5, and chloroform: methanol: acetone = 13.
100 ml of a mixed solution of ２/2/8 (volume ratio) was added to obtain crude 1-stearoyl-2-oleoyl-3-phosphatidylglycerol sodium salt. The obtained crystals were subjected to chloroform: methanol: acetone = 13/1 /
5 (volume ratio) dissolved in 200 ml mixed solution,
It was precipitated by cooling and collected by filtration. This was again dissolved in 100 ml of a mixed solution of chloroform: methanol: acetone = 7/1/3 (volume ratio), precipitated by cooling, filtered and dried to obtain 5.13 g. The composition of the obtained crystal was purified 1-stearoyl-2-oleoyl-3.
Phosphatidylglycerol sodium salt: 90.5% by weight, 1-stearoyl-2-oleoyl-3-phosphatidylcholine 6.5% by weight, lyso-form 1% by weight, free fatty acid 2% by weight. If the composition of the mixed solvent in the steps (a), (b), (d), (e), and (f) is out of the range of the present invention, the purity and the yield will be low.

[0028]

Industrial Applicability According to the present invention, a wide variety of high-purity phospholipids having a desired acyl group can be obtained in the same industrially easy production process in high yield. Since the phospholipid obtained by the present invention has high purity, it is suitable for use in the fields of pharmaceuticals and cosmetics.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Ken Yabe 4-12-1 Oshonishimachi, Amagasaki-shi, Hyogo F-term (reference) 4B064 CA21 CB03 CB26 CC03 CD07 CD15 CE15 DA01 DA16 4H050 AA02 AC40 AC70 AD15 AD17 BA51 BB11 BB12 BB14 BB16 BB31 BD70

Claims (2)

[Claims] [Claim 1] (Wherein, R ¹ and R ² are acyl groups having 12 to 22 carbon atoms, R ¹ and R ² are not the same, and X is a residue obtained by removing one hydroxyl group from a hydroxyl group-containing base) When producing the mixed acid type 1,2-diacyl-3-glycerophospholipid represented by the above formula [2] from 1,2-diacyl-3-glycerylphosphatidylcholine represented by the formula [1] as a starting material, the following ( a) the process,
A method for producing a mixed acid type 1,2-diacyl-3-glycerophospholipid, which is carried out in the order of the steps (b), (c), (d), (e) and (f). (A) Step After deacylation of 1,2-diacyl-3-glycerylphosphatidylcholine with lipase, crystallization is performed using a mixed organic solvent of hexane: acetone: methanol = 7-5: 4-0: 2-0 (volume ratio). Precipitation to obtain monoacyl-3-glycerylphosphatidylcholine. (B) Step The monoacyl-3-glycerylphosphatidylcholine obtained in the step (a) is reacted with a fatty acid anhydride in the presence of a tertiary amine to give a mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine reaction solution. The reaction solution is adsorbed on a silica gel column, and a mixed solvent of chloroform: methanol = 95-80: 20-5 (volume ratio) is passed through the column to elute and remove impurities from the column. : Water = 85 to 75: 20 to 18:
A step of flowing a mixed solvent of 3 to 1.5 (volume ratio) to separate mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine into the mixed solvent. (C) Step The mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine and the base are reacted with phospholipase D to give a mixed acid type 1,2-diacyl-3-glycerylphosphatidylcholine into which an arbitrary acyl group obtained in the step (b) has been introduced.
Obtaining a 2-diacyl-glycerophospholipid. (D) Step Mixed acid form 1,2-diacyl-3-glycerylphosphatidylcholine or mixed acid form 1,2- obtained in step (c)
The diacyl-glycerophospholipid is dissolved in chloroform: methanol: acetone = 13-0: 2-0: 9-7 (volume ratio) to a concentration of 5-50% by weight, and mixed acid type 1,2
-A step of crystallizing the diacyl-3-glycerophospholipid. Step (e) Mixed acid type 1,2-diacyl-3 obtained in step (d) above
-A glycerophospholipid crystal is prepared by adding chloroform: methanol: acetone = 13 to 9: 2 to 1:11 by weight 5 to 20 times.
To 8 (volume ratio) to crystallize the mixed acid type 1,2-diacyl-3-glycerophospholipid. (F) Step The mixed acid type 1,2-diacyl-3 obtained in the above step (e).
-A glycerophospholipid crystal is prepared by adding chloroform: methanol: acetone = 13 to 9: 2-1: 7 to 5 to 20 times by weight.
6 (volume ratio), and mixed acid type 1,2-diacyl-
A step of crystallizing 3-glycerophospholipid. 2. The method for producing a mixed acid type 1,2-diacyl-3-glycerophospholipid according to claim 1, wherein the mixed acid type 1,2-diacyl-3-glycerophospholipid is a glycerophospholipid represented by the formula [3]. Method. Embedded image (Wherein one of R ³ and R ⁴ has 12 to 22 carbon atoms)
And the other is an unsaturated acyl group having 12 to 22 carbon atoms, and X represents a residue obtained by removing one hydroxyl group from a base having a hydroxyl group. )