JPH04261190A - Purification of phosphatidic acids - Google Patents

Abstract

PURPOSE:To obtain the titled high-purity compound free from contaminants by converting (lyso)phosphatidic acid in a phospholipid mixture into a polyvalent metallic salt by a specific method. CONSTITUTION:A phospholipid mixture containing phosphatidic acid(PA) and/or lysophosphatidic acid(L-PA) is dispersed or dissolved in water or a nonpolar solvent (preferably n-hexane), reacted in the presence of a polyvalent metallic salt (preferably calcium chloride) and a polar solvent (preferably ethanol) under an alkali condition preferably at pH10-12 and the phospholipid mixture containing PA and/or L-PA is obtained to purify phosphatidic acids. Further after being dissolved in a polar solvent, preferably the mixture is blended with edible liquid fats and oils to selectively precipitate a polyvalent metallic salt of PA and/or L-PA.

Description

[Detailed description of the invention]

0001

[Industrial Field of Application] The present invention relates to a method for purifying phosphatidic acids (hereinafter abbreviated as PA) and/or lysophosphatidic acids (hereinafter abbreviated as L-PA) contained in phospholipids. Specifically, the present invention relates to a method for purifying highly pure PAs and/or L-PAs that do not contain impurities.

0002

[Prior art and problems to be solved by the invention] Currently,
Adsorption chromatography including column chromatography is generally used to purify phospholipids such as PA and L-PA. In particular, in the purification method using silicic acid column chromatography, each component is eluted and fractionated by changing the polarity of the developing solvent. P.A., L-P.
For the purification of A, a chloroform-methanol mixed solvent is used as a developing solvent, and by changing the mixing ratio, the polarity is changed and fractionation is performed. However, the contamination of contaminants is a problem, and to obtain a highly pure sample, it is necessary to pass it through the column several times, resulting in an increase in the amount of solvent used.
Problems such as a decrease in collection rate arise. Furthermore, a method has been used in which PA and L-PA separated on thin layer chromatography are detected using a non-decomposing reagent and the detected portion is extracted by scraping, but this method has problems with yield and is not suitable for industrial use. , it is considered not to be effective.

Therefore, the present invention provides highly pure PA, L-
The purpose of the present invention is to provide a method for efficiently purifying PAs.

[0004] In the present invention, PAs and/or L-P
Class A refers to metal salts of PA and/or L-PA and free PA and/or L-PA that do not contain metals.

[0005]

[Means for Solving the Problems] As a result of intensive research aimed at solving the above problems, the present inventors have completed the present invention. That is, the present invention provides a method for purifying phosphatidic acids and/or lysophosphatidic acids, which is characterized by including the following step (a). (a) A phospholipid mixture containing phosphatidic acid and/or lysophosphatidic acid is dispersed or dissolved in water or a non-polar solvent, and then reacted with an aqueous polyvalent metal salt solution in the presence of a polar solvent under alkaline conditions to obtain phosphatidic acid and/or lysophosphatidic acid. Or a step of producing a phospholipid mixture containing a polyvalent metal salt of lysophosphatidic acid.

[0006] The present invention also provides the above step (a) as well as
Furthermore, there is provided a method for purifying phosphatidic acids and/or lysophosphatidic acids, characterized by including the following step (b). (b) After dissolving the phospholipid mixture obtained in (a) in a non-polar solvent, a substance that precipitates the polyvalent metal salt of phosphatidic acid and/or lysophosphatidic acid is added, and phosphatidic acid and/or lysophosphatidine is added. A process of precipitating polyvalent metal salts of acids.

In step (a) of the present invention, first, a phospholipid mixture containing phosphatidic acid and/or lysophosphatidic acid is dispersed or dissolved in water or a nonpolar solvent. Examples of the nonpolar solvent used in this case include liquid organic compounds having a carbon number of 5 to 16 and a dielectric constant of 10 or less.
Examples include hydrocarbons having substituents such as hydrocarbons, halogenated hydrocarbons, and aromatic hydrocarbons. From the viewpoint of safety, hydrocarbons having 5 to 16 carbon atoms are preferred, and n-hexane is particularly preferred. Here, the relative dielectric constant is from page 1166 of the Chemical Handbook of the Chemical Society of Japan, Basic Edition, revised 2nd edition (1975).
This is based on the liquid organic compounds and relative dielectric constants of liquids described on pages 1168 and 1582 to 1583. Next, a mixed solution of a polyvalent metal salt aqueous solution and a polar solvent is added in an amount of 0.1 times or more, preferably 0.4 to 0.8 times the volume of the water or non-polar solvent. The volume ratio of the mixed solution is preferably 0.1 or more of polyvalent metal salt aqueous solution/polar solvent,
In particular, 0.5 to 2.0 is preferable. In addition, examples of the polar solvent used here include liquid organic compounds having a carbon number of 1 to 10 and a dielectric constant exceeding 10, and examples of liquid organic compounds having a dielectric constant exceeding 10 include monohydric alcohols having a carbon number of 1 to 10. , ketones, and polyhydric alcohols, among which ethanol is preferred from the viewpoint of safety. Furthermore, as the polyvalent metal salt used as the polyvalent metal salt aqueous solution, Mg, Ca
Group IIA elements such as , Sr, and Ba, IIIB elements such as Al
Examples include hydrochlorides, sulfates, carbonates, phosphates, etc. of Ca group elements, preferably hydrochlorides, more preferably Ca
Salts may be mentioned, with calcium chloride being particularly preferred. The optimal concentration of the polyvalent metal salt aqueous solution is at least 1 mole, preferably 1 to 7 times the mole of the phospholipid mixture. A phospholipid mixture containing polyvalent metal salts of PA and L-PA can be obtained by stirring the above composition under alkaline conditions at pH 7.0 or higher, preferably at pH 10 to 12 for 0.5 hours or more.

Step (b) of the present invention is the step (a) above.
This is a step of selectively precipitating the polyvalent metal salt of PA and/or L-PA obtained in . In this step, PA is separated from the phospholipid mixture containing the polyvalent metal salt of PA and/or L-PA by known techniques such as static separation and centrifugation.
and/or separating a non-polar solvent phase containing a phospholipid mixture containing a polyvalent metal salt of L-PA. In a dispersion system using water, it is dissolved in a nonpolar solvent after dehydration and solvent removal. As the nonpolar solvent used, those used in the above step (a) can be used. Next, to a non-polar solvent containing a phospholipid mixture containing a polyvalent metal salt of PA and/or L-PA,
The polyvalent metal salt of PA and/or L-PA is added by adding a substance that precipitates the polyvalent metal salt of PA and/or L-PA at least 0.1 times the volume, preferably 0.1 to 2.0 times the volume. Selective precipitation of salts. If the amount added is less than 0.1 times the volume, precipitation of polyvalent metal salts of PA and/or L-PA will not occur, and if it exceeds 2.0 times the volume, impurities dissolved in the non-polar solvent will become polar. The purity of the polyvalent metal salt of PA and/or L-PA that is extracted by the solvent and produced is reduced.

[0009] As the substance that selectively precipitates polyvalent metal salts of PA and/or L-PA, lipids or substances having 1 to 1 carbon atoms are used.
Polar solvents having 10 polar groups are mentioned. Lipids include one or more mixtures of monoglycerides, diglycerides, and triglycerides. Examples include monoglycerides, diglycerides, and triglycerides containing saturated fatty acid residues and unsaturated fatty acid residues having 8 to 24 carbon atoms.
It is desirable that the temperature is below 0°C. More preferred are edible fats and oils from the viewpoint of availability of raw materials, particularly edible liquid fats and oils from the viewpoint of safety. Specifically, there are vegetable oils such as soybean oil, rapeseed oil, sunflower oil, corn oil, rice oil, and cottonseed oil, and animal oils and fats such as fish oil. In addition, examples of polar solvents having a polar group having 1 to 10 carbon atoms include liquid organic compounds with a dielectric constant exceeding 10, and examples of liquid organic compounds having a dielectric constant exceeding 10 include monohydric alcohols, ketones, and polyhydric alcohols. Monohydric alcohols include methanol, ethanol, isopropanol, etc., ketones include acetone, methyl ethyl ketone, etc., and polyhydric alcohols include ethylene glycol, glycerin, etc. Monohydric alcohols are particularly preferred.

[0010] In order to improve the purification purity of PA and L-PA, it is desirable to repeat the above step (b) two or more times. That is, the obtained precipitate is dissolved again in a nonpolar solvent such as n-hexane, and purification is performed by adding a substance that selectively precipitates polyvalent metal salts of PA and/or L-PA. It is.

PA and/or L thus obtained
- Polyvalent metal salts of PA can be converted into various salt compounds such as sodium salts by conventionally known salt exchange methods, and metal-free free PA and/or L-P can be obtained by desalting treatment.
A is obtained.

0012

[Examples] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 PA, L treated with enzymes such as phospholipase D
- 10g of natural phospholipid containing PA at 10% (w
100 m of n-hexane to a concentration of
Dissolved in l. 20 ml of ethanol was added to this hexane solution, 30 ml of an aqueous calcium chloride solution with a molar ratio of 5 times the amount of phosphorus (P) in the hexane solution was added, and 1N NaOH was added dropwise with stirring until the pH reached 10. After 4 hours, the stirring was stopped and the speed was increased to 3000 rpm (
Centrifugation was performed at 25° C. for 10 minutes (50 ml centrifuge tube), and the upper hexane layer was removed to obtain 90 ml of a phospholipid calcium salt fraction. Add 45 ml of ethanol to this, and after stirring, add 3000 ml of ethanol.
Centrifugation was performed at 25° C. for 10 minutes at rpm to obtain calcium salts of PA and L-PA as precipitates. Further, 81 ml of n-hexane was added to the obtained precipitate and dissolved,
Add 40.5ml of ethanol and stir at 3000 rpm.
By centrifuging for 10 minutes at 25°C, precipitates of purified PA and calcium salts of L-PA were obtained.

The obtained precipitate was again diluted with 100% n-hexane.
After dissolving in 1 ml of the solution, insoluble matters were removed, and hexane was further removed using an evaporator to obtain 6 g of calcium salts of PA and L-PA.

Example 2 10g of natural phospholipids containing PA and L-PA were added at 10% (
n-hexane 10 to give a concentration of wt/vol)
Dissolved in 0ml. Add 20ml of ethanol to this hexane solution, and add phosphorus (P) in the hexane solution.
30 ml of calcium chloride aqueous solution of 5 times the molar amount
1N NaOH was added dropwise with stirring until the pH reached 10. After 4 hours, stop stirring and turn to 3000 rpm.
Centrifugation was performed for 10 minutes at 25° C., and the upper hexane layer was removed to obtain 90 ml of a phospholipid calcium salt fraction. To this, 36 g of triglyceride derived from rapeseed oil was added and stirred at 3000 rpm for 10 minutes at 25°C.
Centrifugation was performed at the bottom to obtain calcium salts of PA and L-PA as precipitates. Furthermore, 81 ml of n-hexane was added to the obtained precipitate to dissolve it, and then 32.4 g of triglyceride derived from rapeseed oil was added and stirred at 3000 rpm.
The purified PA and L-PA calcium salts were obtained as precipitates by centrifugation for 10 minutes at 25°C. Furthermore, after dissolving the precipitate in 100 ml n-hexane,
Insoluble materials were removed, and hexane was further removed using an evaporator to obtain 6 g of calcium salts of PA and L-PA.

Comparative Example A case where PA was purified using a silica gel column will be shown as a comparative example. PA treated with enzymes such as phospholipase
73.5g of natural phospholipids containing 5g of n-hexane
0 ml and added dropwise to 250 ml of acetone, which had been previously ice-cooled in an acetone-dry ice bath. Stirrer 3000 rpm in 500 ml centrifuge tube
After centrifugation for 10 minutes, 200 ml of ice-cold acetone was added to the resulting paste-like precipitate, and then centrifuged again at 3000 rpm.
After centrifugation at pm x 10 minutes, the precipitate was solvent-removed using an evaporator, and 55.41 g was obtained as an acetone-insoluble fraction.

The acetone-insoluble matter was dissolved in 70 ml of chloroform, divided into 52 g portions, and fractionated using three columns. One column below (WAKOGEL C-20
0,800g). PA was eluted by using a chloroform-methanol mixed solvent as the elution solvent and changing the solvent ratio. The solvent ratio and solvent amount are shown below.

1. sample charge
52 g 2.
Chloroform:methanol=5:1
200 ml 3. Chloroform:methanol=2:1 4500 ml 4.
Chloroform:methanol=3:2
4200 ml 5. Chloroform:methanol=1:1 1800 ml 6
．． Chloroform:methanol=2:3
After separating and analyzing 600 ml and 450 ml, PA was chloroform-
Elutes from the latter half of the methanol ratio of 2:1 to the first half of the methanol ratio of 1:1, with the highest purity chloroform-methanol ratio of 3:
3,600 ml of the fraction eluted in step 2 was obtained, and purified PA was obtained after removing the solvent using an evaporator. A total of 19 by this method
．． 67 g of purified PA was obtained. Furthermore, the total amount of solvent used for purification was 34,770 ml (chloroform 2069 ml).
8 ml, methanol 13572 ml, acetone 45
0 ml, n-hexane 50 ml).

Regarding Example 1 and Comparative Example, PA and/or
Alternatively, the amount of solvent used to purify 1 g of L-PA was compared. The results are shown in Table 1.

[0020]

[Table 1]

As is clear from the above results, the method of the present invention can produce highly purified PAs and/or L-
PAs can be produced.

[0022]

Effects of the Invention According to the purification method of the present invention, unlike the conventional purification method using adsorption chromatography, the operation is simple and highly pure PA containing no impurities can be obtained.
It is an industrially useful method that can obtain L-PAs and L-PAs in good yield.

Claims (11)

[Claims] 1. A method for purifying phosphatidic acids and/or lysophosphatidic acids, which comprises the following step (a). (a) A phospholipid mixture containing phosphatidic acid and/or lysophosphatidic acid is dispersed or dissolved in water or a non-polar solvent, and then reacted with an aqueous polyvalent metal salt solution in the presence of a polar solvent under alkaline conditions to obtain phosphatidic acid and/or lysophosphatidic acid. Or a step of producing a phospholipid mixture containing a polyvalent metal salt of lysophosphatidic acid. 2. The method for purifying phosphatidic acids and/or lysophosphatidic acids according to claim 1, which comprises the following steps (a) and (b). (a) A phospholipid mixture containing phosphatidic acid and/or lysophosphatidic acid is dispersed or dissolved in water or a non-polar solvent, and then reacted with an aqueous polyvalent metal salt solution in the presence of a polar solvent under alkaline conditions to obtain phosphatidic acid and/or lysophosphatidic acid. Or a step of producing a phospholipid mixture containing a polyvalent metal salt of lysophosphatidic acid. (b) After dissolving the phospholipid mixture obtained in (a) in a non-polar solvent, a substance that precipitates the polyvalent metal salt of phosphatidic acid and/or lysophosphatidic acid is added, and phosphatidic acid and/or lysophosphatidine is added. A process of precipitating polyvalent metal salts of acids. [Claim 3] The polyvalent metal constituting the polyvalent metal salt is II
The purification method according to claim 1 or 2, wherein the element is a group A element or a group IIIB element. 4. The purification method according to claim 1, wherein the nonpolar solvent is a liquid organic compound having 5 to 16 carbon atoms and a dielectric constant of 10 or less. 5. The purification method according to claim 4, wherein the liquid organic compound having a dielectric constant of 10 or less is a hydrocarbon or a hydrocarbon having a substituent. 6. The purification method according to claim 1, wherein the polar solvent is a liquid organic compound having 1 to 10 carbon atoms and having a dielectric constant exceeding 10. 7. The purification method according to claim 6, wherein the liquid organic compound having a dielectric constant exceeding 10 is a monohydric alcohol, a polyhydric alcohol, or a ketone. 8. The purification method according to claim 1 or 2, wherein the substance that precipitates the polyvalent metal salt of phosphatidic acid and/or lysophosphatidic acid is a lipid or a polar solvent having a polar group having 1 to 10 carbon atoms. . 9. The purification method according to claim 8, wherein the lipids are a mixture of one or more of monoglycerides, diglycerides, and triglycerides. 10. The purification method according to claim 8, wherein the polar solvent having a polar group having 1 to 10 carbon atoms is a liquid organic compound having a dielectric constant of more than 10. 11. The purification method according to claim 10, wherein the liquid organic compound having a dielectric constant exceeding 10 is a mixture of one or more of monohydric alcohols, ketones, and polyhydric alcohols.