TWI591181B - Method and composition for producing sodium cyclic phosphatidic acid - Google Patents

Description

Method and composition for producing cyclic sodium phosphatidate

The present invention relates to a method for producing a cyclic sodium phosphatidate and a composition comprising the cyclic phosphatidic acid obtained by the above production method.

Cyclic phosphatidic acid (hereinafter, abbreviated as cPA) is known to have physiological activity such as inhibition of metastasis and infiltration of cancer cells (Non-Patent Document 1), and is expected to be a pharmaceutical or functional food containing an antitumor agent. Use, in addition, due to the hyaluronic acid synthesis promoting effect in the body, is added to cosmetics.

Conventionally, as a method for producing such a cyclic phosphatidic acid, a method of chemical synthesis (Patent Documents 1 and 2) or a method of reacting an enzyme which acts on phospholipase D and a lyso-form phospholipid is known. (Patent Documents 3 and 4). Since the cyclic phosphatidic acid-based lipid is insoluble in water, it needs to be a water-soluble salt such as a sodium salt, and is converted into sodium by treating the chemically synthesized cyclic phosphatidic acid with a strong base such as sodium hydride or sodium hydroxide. A salt method to prepare a cyclic sodium phosphatidate.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. Hei 6-228169

[Patent Document 2] Japanese Patent Laid-Open No. Hei 7-258278

[Patent Document 3] Japanese Laid-Open Patent Publication No. 2001-178489

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2008-222643

[Non-patent literature]

[Non-Patent Document 1] Biochemica et Biophysica Acta 15288 (2002), p. 1-7

Since the cyclic phosphatidic acid is unstable, the method using the strong base described in the above scientific synthesis method is not preferable, and it is difficult to obtain the cyclic phosphatidic acid in high yield and high purity in the chemical synthesis method, and it is desired to be mild and simple. The method of preparing a cyclic sodium phosphatidate in high yield and high purity.

The inventors of the present invention have conducted an effort to investigate a method for preparing a cyclic sodium phosphatidate, and found that phospholipase D and a lysolipid phospholipid are excluded by a system containing an organic solvent and/or water (except for hydride) After adding sodium chloride to the reaction product obtained by the action, the solvent is removed from the reactant, and the cyclic sodium phosphate can be prepared in a simple manner, with high yield and high purity under extremely mild conditions. The present invention is based on this finding.

That is, according to the present invention, the following invention is provided.

(1) A method for producing a cyclic sodium phosphatidate, which comprises adding a reaction solution obtained by causing phospholipase D and a lysolipid phospholipid (except for a hydride) in a system containing an organic solvent and/or water; After the sodium salt, the solvent is removed from the reactants.

(2) A method for producing a cyclic sodium phosphatidate according to (1), wherein the sodium salt is sodium chloride.

(3) The method for producing a cyclic sodium phosphatidate according to (1) or (2), wherein the phospholipase D and the lysolipid phospholipid are reacted in a system containing an organic solvent and/or water. The sodium salt is added in the presence of a chelating agent.

(4) A method for producing a cyclic sodium phosphatidate according to (3), wherein the chelating agent is EDTA.

(5) The method for producing a cyclic phosphatidic acid according to any one of (1) to (4), wherein the lysophospholipid is a phospholipid A2 and a phospholipid derived from soybean or a phospholipid derived from egg yolk or from Hemolysinated phospholipids derived from the phospholipids of corn.

(6) The method for producing a cyclic phosphatidic acid according to any one of (1) to (5), wherein the lysolipid phospholipid is a phospholipid derived from phospholipase A2 and phospholipid derived from soybean or from egg yolk The reactant derived from the phospholipid of corn can be dissolved in a system containing an organic solvent and/or water without the action of the purified lysolipid phospholipid to cause phospholipase D to act.

(7) A composition containing a cyclic phosphatidic acid having a purity of 40% or more, which is obtained by the method for producing a cyclic phosphatidic acid according to any one of (1) to (6).

(8) A solution containing a cyclic sodium phosphatidate of 1 mg/mL or more, which is obtained by the method for producing a cyclic sodium phosphatidate according to any one of (1) to (6).

(9) The solution according to (8), wherein the solution containing the cyclic sodium phosphatidate is an aqueous solution.

According to the present invention, by the action of phospholipase D and lysophospholipid in a system containing an organic solvent and/or water, the cyclic sodium phosphate can be produced in a highly gentle and simple manner with high yield and high purity. According to the present invention, as long as the reaction product is treated with a sodium salt, the cyclic phosphatidic acid can be prepared without being subjected to the separation and purification of the cyclic phosphatidic acid, and therefore it is particularly useful as a cyclic phospholipid which is used as a raw material such as soybean or the like. A method of producing sodium. The cyclic sodium phosphatidate obtained by the production conditions of the present invention, particularly when a phospholipid derived from soybean or a phospholipid derived from egg yolk or a phospholipid derived from corn is used as a raw material, has high water solubility at room temperature and can be not particularly The aqueous solution was prepared by purification.

Hereinafter, the present invention will be described in further detail.

The present invention is characterized in that a solvent is removed from a reactant after adding sodium chloride to a reaction product obtained by reacting phospholipase D with a lysolipid phospholipid (except for a hydride) in a system containing an organic solvent and/or water. .

Regarding the preparation of the cyclic sodium phosphatidylcholine, it is disclosed that the lysophosphatidylcholine from hydrogenated soybean is dissolved in water, sodium acetate buffer and 2M sodium chloride are added, and reacted with phospholipase D, followed by sodium hydroxide aqueous solution. And extracting with chloroform/methanol, recovering the organic solvent layer, and distilling off the solvent to obtain a cyclic sodium phosphatidate (Patent Document 4), but only revealing the fatty acid composition of the obtained substance, and there is no description of the sodium content, etc. It is clearly revealed that it is a cyclic sodium phosphatidate.

The inventors of the present invention discovered the enzyme reaction of phospholipase D, even if it does not exist. Calcium ions are also sufficiently carried out, and sodium chloride blocks the enzyme reaction. Further, the lysate-type phospholipid is dissolved in a system containing an organic solvent and/or water, and an acetic acid buffer is added to adjust the pH to 5.5 to 6.5 to carry out an enzyme reaction of phospholipase D, whereby the reaction proceeds efficiently, and After the completion of the reaction, a cyclic sodium phosphinate was successfully produced in a good yield by adding an aqueous solution of a sodium salt. Further, the phospholipase D reaction is continuously carried out by using a phospholipid derived from soybean phospholipid or a phospholipid derived from egg yolk or a phospholipid derived from corn as a raw material without a single purification of phospholipase A2. At the time, a small amount of calcium ions remain in the reaction solution, and the calcium salt of the cyclic phosphatidic acid is purified. However, after the reaction is completed, a sodium phosphate having a high purity can be obtained by combining sodium ions and a chelating agent.

The sodium salt used in the present invention may be sodium chloride, sodium sulfate, sodium acetate or sodium nitrate, but is preferably sodium chloride or sodium sulfate; more preferably sodium chloride. Preferably, the concentration of sodium chloride used is from 0.5 M to 3 M, more preferably from 2 to 3 M.

The chelating agent used in the present invention is sodium edetate (EDTA), diethylenetriaminepentaacetic acid, glycol ether diaminetetraacetic acid, citric acid, tartaric acid, phytic acid, etc., but is preferably ethylenediamine. Sodium tetraacetate (EDTA), diethylenetriaminepentaacetic acid, glycol ether diaminetetraacetic acid; optimally EDTA.

The organic solvent used in the present invention may be chloroform, dichloromethane, toluene, diethyl ether, ethyl acetate, hexane or the like, but is preferably chloroform, dichloromethane or toluene; more preferably chloroform or toluene.

The lysolipid phospholipid used in the present invention is preferably lysophosphatidylcholine (LPC) when considering the matrix specificity of phospholipase D, and is preferably derived from The phospholipid of soybean or the phospholipid derived from egg yolk or the phospholipid derived from corn is the raw material, and the phospholipase A2 acts as the LPC. The lysolipid phospholipid used in the present invention is a hydrogenated lysolipid phospholipid, and an unhydrogenated lysolipid phospholipid is used.

The phospholipase D used in the present invention is not particularly limited as long as it acts on the above-mentioned lysophospholipid, and is particularly preferably a phospholipase D derived from Streptomyces sp. or Actinomadula sp.

The reaction of the lysate-type phospholipid with the phospholipase D is not particularly limited as long as it can exhibit the activity of the enzyme, but it is preferred to dissolve the lysolipid phospholipid in an organic solvent such as chloroform, and add an acetate buffer to have a pH of 5.0. Adjust to 7.0, add 10 to 100 units/ml of phospholipase D, warm to 25 ° C ~ 50 ° C, while stirring continuously, react for about 5 to 30 hours. After the completion of the reaction, 2 to 3 M of sodium chloride and 0.1 to 0.3 M of EDTA were added, and the mixture was continuously stirred. The organic solvent layer was collected by centrifuging the reaction product (3000 rpm, 5 minutes), and the solvent was distilled off to obtain a powdery solid of a cyclic sodium phosphate. After completion of the reaction, an organic solvent such as methanol may be added as needed to recover the organic solvent layer. When a higher purity cyclic sodium phosphate is desired, it may be purified by using a cerium oxide gel or an adsorption resin.

According to the present invention, a composition containing a cyclic phosphatidic acid having a purity of 40% or more obtained by the above-described method for producing a cyclic sodium phosphatidylate of the present invention is provided. The purity is preferably 45% or more, more preferably 50% or more. The purity stated in the present invention is based on a standard (97% purity product) as a control group, and the density of the thin layer chromatography is measured by a densitometer, and the purity obtained by the area ratio quantification is obtained. . Standard product (97% pure product) The high-purity cyclic sodium phosphatidate obtained in Example 3 was subjected to re-chromatography to obtain a fatty acid composition as described in Example 5.

Further, according to the present invention, a solution containing 1 mg/mL or more of cyclic sodium phosphatidate obtained by the above-described method for producing a cyclic sodium phosphatidylate of the present invention is provided. The concentration of the cyclic sodium phosphatidate is not particularly limited as long as it is 1 mg/mL or more, and may be 2 mg/mL or more, 3 mg/mL or more, or 5 mg/mL or more.

The invention is illustrated by the following examples, but the invention is not limited by the examples.

[Examples] Example 1; Method for producing cyclic phosphatidic acid sodium salt

10 g of soybean phospholipid (lecithin content: 70%) (unhydride) was dissolved in 100 mL of 1 M acetate buffer (pH 6.5) containing 0.3 M calcium chloride, and 6000 units of phospholipase from Streptomyces were added. A2 was stirred at 40 ° C for 18 hours to cause a reaction. After the reaction solution was adjusted to pH 2.5 to deactivate the enzyme, 100 mL of chloroform and 50 mL of methanol were added, and the mixture was stirred and mixed to extract a lipid component. The chloroform layer was collected and solidified by drying under reduced pressure on a rotary evaporator. 100 mL of acetone was added to the solid component to precipitate a phospholipid to remove free fatty acids. 5 g of the precipitate was dissolved in 40 mL of chloroform, 10 mL of 1 M acetic acid buffer (pH 5.5) was added, and 1500 units of phospholipase D from Actinomadula was further added thereto, and the mixture was stirred at 40 ° C for 18 hours while the reaction was carried out. 20 mL of 3 M sodium chloride and 20 mL of a 0.1 M EDTA solution were added to the reaction mixture, and the mixture was stirred at 40 ° C for 1 hour. Further adding After adding 20 mL of methanol, the mixture was thoroughly stirred, centrifuged at 3000 rpm for 5 minutes, and a chloroform layer was collected. This solution was dried and solidified under reduced pressure on a rotary evaporator to obtain 3.8 g of a cyclic phosphatidic acid salt. Since the cyclic phosphatidic acid Na 3.8 g was obtained from the lecithin content of 70% (7 g in 10 g), the yield was 54.3%. The purity analysis of the cyclic phosphatidic acid sodium salt was carried out using cerium oxide gel tablets and developing with chloroform:methanol:acetic acid:5% 5% sodium sulfite (100:40:12:5, V/V) in 5% acetic acid. Copper: 8% phosphoric acid: 2% sulfuric acid mixed solution was immersed for a short period of time, air-dried, and heated at 180 ° C for about 10 minutes, and then the resulting spot was subjected to a scanner (manufactured by Atto Co., Ltd.). That is, the standard (97% purity product) was used as a control group, and the point of the thin layer chromatography was measured by a densitometer, and the area ratio was quantified. The purity of the cyclic phosphatidic acid sodium salt in the product obtained in the above step was 54%.

Example 2; Method for producing cyclic phosphatidic acid sodium salt without using chelating agent

10 g of soybean phospholipid (lecithin content: 70%) (unhydride) was dissolved in 100 mL of 1 M acetic acid buffer (pH 6.5) containing 0.3 M calcium chloride, and 6000 units of phospholipase from Streptomyces were added. A2 was stirred at 40 ° C for 18 hours to cause a reaction. After the reaction solution was adjusted to pH 2.5 to deactivate the enzyme, 100 mL of chloroform and 50 mL of methanol were added, and the mixture was stirred and mixed to extract a lipid component. The chloroform layer was collected and solidified by drying under reduced pressure on a rotary evaporator. 100 mL of acetone was added to the solid component to precipitate a phospholipid to remove free fatty acids. 5 g of the precipitate was dissolved in 40 mL of chloroform, 10 mL of 1 M acetic acid buffer (pH 5.5) was added, and 1500 units of phospholipase D from Actinomadula was further added, and the mixture was stirred at 40 ° C for 18 hours. The reaction is carried out. 20 mL of 3 M sodium chloride was added to the reaction mixture, and the mixture was stirred at 40 ° C for 1 hour. Further, 20 mL of methanol was added, and the mixture was thoroughly stirred, and then centrifuged for 3000 rpm for 5 minutes to collect a chloroform layer. This solution was dried and solidified under reduced pressure on a rotary evaporator to obtain 3.7 g of a cyclic phosphatidic acid salt. Since the cyclic phosphatidic acid Na 3.7 g was obtained from the lecithin content of 70% (7 g in 10 g), the yield was 52.9%. The purity analysis of the cyclic phosphatidic acid sodium salt was carried out by the method described in Example 1. The purity of the cyclic phosphatidic acid sodium salt in the product obtained in this step was 53%.

Example 3; Method for producing high-purity cyclic phospholipid sodium salt

500 mg of the cyclic phosphatidic acid sodium salt obtained in Example 1 was dissolved in 5 mL of chloroform containing 10% methanol, loaded into a cerium oxide gel column, developed in the same solvent, and further developed with chloroform containing 20% methanol. , take 10 mL of the fraction. The separated fraction containing the cyclic phosphatidic acid sodium salt was confirmed by the TLC method shown in Example 1 and collected, and dried under reduced pressure on a rotary evaporator to obtain 320 mg of a powder of a cyclic phosphatidic acid salt. The purity of the cyclic phosphatidic acid sodium salt of this sample was 95%.

Example 4; Analysis of sodium content in ion chromatography

For the cyclic phosphatidic acid sodium salt obtained in Example 1 and Example 2, the sodium content was determined by ion chromatography. The column was IonPac CS14 (manufactured by Dionex, Japan), the mobile phase was 10 mM methanesulfonic acid aqueous solution, the flow rate was 1.0 mL/min, the column temperature was 30 ° C, and the injection amount was 10 μL. The detection was carried out by a conductivity detector. The standard solution used cationic mixed standard solution II (Li ⁺ 0.5 mg / L, Na ⁺ 2 mg / L, NH ₄ ⁺ 2 mg / L, K ⁺ 5 mg / L, Ca ²⁺ 5 mg / L, Mg ²⁺ 5 mg / L) . The column temperature was 30 ° C and the injection amount was 10 μL. As a result of the analysis, it was found that a sodium salt such as a cyclic phosphatidic acid was a sodium salt.

Example 5;

The fatty acid composition of the high-purity cyclic phosphatidic acid obtained in Example 3 was analyzed by gas chromatography. The sample was dissolved in methanolic hydrochloric acid to make the sample 20 mg/mL, and the mixture was heated at 65 ° C for 30 minutes. After returning to room temperature, an equal amount of water was added, followed by an equal amount of hexane, and the mixture was thoroughly stirred and mixed. The mixture was centrifuged at 3000 rpm for 5 minutes, and 2 μL of a hexane layer was poured into a capillary column to analyze constituent fatty acids. The analysis is as follows.

Example 6; state of aqueous solution of cyclic sodium phosphatidate

The 1 mg/mL aqueous solution of the cyclic phosphatidic acid sodium obtained in Example 1 and Example 2 was prepared, and the absorbance at 660 nm was measured. The absorbance of the sample obtained in Example 1 was 0.05, and the absorbance of the sample obtained in Example 2 was 0.15. The sample obtained in Example 1 was obtained by using a sample obtained by a method for producing a chelating agent, so that the calcium salt was not present and became transparent.

Comparative Example 1; state of aqueous solution of cyclic sodium phosphatidate

The sodium hyaluronate was obtained by the same operation as described in Example 1 except that the soybean phospholipid described in Example 1 was used instead of the hydrogenated soybean phospholipid. A 1 mg/mL aqueous solution of this cyclic sodium phosphatidate was prepared, and the absorbance at 660 nm was measured. The absorbance was 0.33.

Example 7: Method for producing cyclic phosphatidyl sodium salt

10 g of soybean phospholipid (lecithin content: 70%) (unhydride) was dissolved in 100 mL of 1 M acetic acid buffer (pH 6.5) containing 0.3 M calcium chloride, and 6000 units of phospholipase from Streptomyces were added. A2 was stirred at 40 ° C for 18 hours to cause a reaction. After the reaction solution was adjusted to pH 2.5 to inactivate the enzyme, the pH was adjusted to 5.5. Next, 1500 units of phospholipase D derived from the genus Actinomadula were added to the reaction solution, and the mixture was stirred at 40 ° C for 18 hours while the reaction was carried out. 100 mL of hexane was added to the reaction mixture, and the mixture was stirred and extracted at room temperature for 30 minutes. The hexane layer was collected by centrifugation, and 50 mL of an aqueous solution of 10 mM EDTA, 0.1 M acetic acid buffer pH 6.5, and 0.5 M sodium chloride was added thereto, and the mixture was stirred at room temperature for 30 minutes, and then collected by centrifugation. The hexane layer was concentrated on a rotary evaporator. 50 mL of acetone was added thereto, and the mixture was thoroughly stirred and centrifuged to remove the acetone layer. This operation was repeated twice to remove free fatty acids. The acetone insoluble matter was collected, and dried under reduced pressure to give dicated sodium phosphite 3.6 g.

Claims (8)

A method for producing a cyclic sodium phosphatidate, which comprises adding a sodium salt to a reaction product obtained by reacting phospholipase D with a lysolipid phospholipid (except for a hydride) in a system containing an organic solvent and/or water The solvent is removed from the reactant, and the lysophosphatidic phospholipid is a lytic phospholipid obtained by reacting phospholipase A2 with a phospholipid derived from soybean, and the phospholipase D is derived from phospholipase D of Actinomadula. A method for producing a cyclic sodium phosphatidate according to claim 1, wherein the sodium salt is sodium chloride. The method for producing a cyclic phosphatidic acid sodium according to the first or second aspect of the invention, wherein the phospholipase D and the lysolipid phospholipid are reacted in a system containing an organic solvent and/or water, The sodium salt is added in the presence of a mixture. A method for producing a cyclic sodium phosphatidate according to claim 3, wherein the chelating agent is EDTA. The method for producing a cyclic sodium phosphatidylcholine according to claim 1 or 2, wherein the reactant obtained by reacting phospholipase A2 with phospholipid derived from soybean is dissolved without separately separating the purified lysolipid phospholipid Phospholipase D is allowed to act in a system containing an organic solvent and/or water. A method for producing a cyclic phosphatidic acid sodium according to claim 1 or 2, which comprises a composition containing a cyclic phosphatidic acid having a purity of 40% or more. A method for producing a cyclic phosphatidic acid sodium according to claim 1 or 2, which comprises dissolving a sodium citrate containing 1 mg/mL or more. liquid. A method for producing a cyclic sodium phosphatidate according to claim 7, wherein the solution containing the cyclic sodium phosphatidate is an aqueous solution.