JP4887674B2 - Method for producing phospholipid composition - Google Patents

Description

The present invention relates to a method for efficiently producing a phospholipid composition containing both phosphatidylinositol and a phospholipid obtained by a base exchange reaction of phosphatidylcholine and phosphatidylethanolamine at a high concentration.

Phospholipids are known to have various medicinal effects depending on their types, and many studies have been conducted so far. For example, it has been reported that phosphatidylserine (PS), which is one of phospholipids, has an effect of improving brain function such as memory disorder recovery effect (see Patent Document 1), and phosphatidylinositol (PI) is It has been reported that it is effective in treating nervous system disorders and improving metabolism (see Patent Documents 2 and 3).
Thus, since these phospholipids are all important for health, in recent years, a preparation containing two or more phospholipids, particularly a preparation containing both PI and PS at a high concentration has been demanded in the market. ing.

A method is known in which PS or the like is obtained by base exchange reaction of phosphatidylcholine (PC) with alcohols in the presence of phospholipase D.
For example, as a method for obtaining a high concentration of PS, a method of reacting PC with serine in the presence of phospholipase D is known (see Patent Document 4). When natural materials such as soybean lecithin and egg yolk lecithin are used as raw materials for PC, these lecithins contain about 10% of PI. However, in order to increase the yield of PS, the raw materials are refined to increase the concentration of PC. Since it raises, PI becomes very trace amount. In addition, PI does not react with serine in this reaction like PC, but rather is one of the factors that inhibit the reaction between PC and serine. Therefore, it was necessary to purify the raw material and perform the reaction.

As a method for obtaining a high concentration of PS, a method using phospholipase D having high phosphatidyl group transfer activity has been reported (see Patent Document 5). In this method, low-purity lecithin can be used as a raw material without purification, but the PI content of the resulting composition is not sufficient.

On the other hand, as a method for obtaining a high concentration of PI, a method has been reported in which plant-derived lecithin is treated with a solvent containing a water-containing lower alcohol and further treated with an aqueous solution containing ethanol to concentrate (see Patent Document 6). . In this method, PI can be obtained at a high concentration, but the concentration of PS or the like in the obtained composition was about several percent.

In order to use phospholipids and PI such as PS obtained by base exchange reaction as pharmaceuticals and foods, it is necessary that the composition contains PI of 15% or more and PS or the like of 20% or more. Therefore, a composition containing both PS and PI at a high concentration has been prepared by mixing PS and PI obtained by different production methods so far. In addition, since another material and process are required, there is a problem that the manufacturing cost becomes high.
Japanese Patent No. 3053537 Japanese Patent Laid-Open No. 5-97875 JP 2000-300196 A Japanese Patent Publication No. 5-42917 JP-A-9-173092 Japanese Patent Laid-Open No. 9-165

The present invention provides a method for efficiently producing a phospholipid composition containing a high concentration of both phosphatidylinositol and a phospholipid such as phosphatidylserine obtained by a base exchange reaction in view of the above-mentioned present situation.

As a result of intensive studies to solve the above-mentioned problems, the present inventors compared the phosphatidylinositol with the raw material phospholipid by stirring the phospholipid and the solvent in advance when performing the base exchange reaction of the phospholipid. The base exchange reaction proceeds efficiently even if it contains a large amount, and a phospholipid composition containing both phosphatidylinositol and a phospholipid such as phosphatidylserine obtained by the base exchange reaction can be easily obtained. I found it.

That is, in the present invention, a phospholipid containing 30% by weight or more of phosphatidylcholine and phosphatidylethanolamine in total and 10% by weight or more of phosphatidylinositol was stirred in a two-phase system composed of an organic solvent and water. Then, it is a manufacturing method of a phospholipid composition which consists of the process of adding a hydroxyl-group containing compound and phospholipase D, and performing the base exchange reaction of at least 1 sort (s) of phosphatidylcholine and phosphatidylethanolamine.

The present invention is described in detail below.
In the method for producing a phospholipid composition of the present invention, the phospholipid used as a raw material includes a total of 30% by weight or more of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Using 10% by weight or more. The raw material phospholipid may contain 30% by weight or more of either PC or PE, but may contain 30% by weight or more of both PC and PE. Preferably, the total of at least one of PC and PE is 30 to 60% by weight, and PI is 10 to 40% by weight.

The phospholipid used as the raw material may be of any origin, and examples include natural products containing phospholipids, extracts from natural products, purified products, and synthetic phospholipids. Specific examples include soybean, rapeseed, sunflower, palm-derived phospholipids, and soybean-derived ones are preferred. Examples of commercially available phospholipids containing a relatively large amount of PI include, for example, SLP-WHITE (PC 25.6%, PE 24.1%, phosphatidylic acid (PA) 8%, PI 12%, other about 30%), SLP-PI powder (an example: PC18%, PE22%, PA8%, PI17%, other about 35%) (all are made by Sumi Oil).
In the conventional base exchange reaction, in order to increase the yield, the phospholipid used as a raw material is purified to increase the concentration of PC and PE. However, in the production method of the present invention, it is obtained by the base exchange reaction of PC and PE. In order to obtain a phospholipid composition containing a large amount of PI together with phosphatidylserine (PS) and the like, a phospholipid containing a relatively large amount of PI is used as a raw material. As such a phospholipid containing a large amount of PI, a by-product generated when PC and PE are purified is also inexpensive and suitable.

The concentration of the phospholipid is preferably 5 to 40% by weight and more preferably 10 to 30% by weight during the base exchange reaction described below. If the amount is less than 5% by weight, the amount of phospholipid obtained is small, and if it exceeds 40% by weight, the transfer rate in the base exchange reaction decreases.

In the production method of the present invention, the phospholipid is stirred in a two-phase system composed of an organic solvent and water before the hydroxyl group-containing compound and PLD are added to perform the base exchange reaction of the phospholipid.
The organic solvent is not particularly limited, and aliphatic hydrocarbons such as n-heptane, n-hexane, and petroleum ether; cycloaliphatic hydrocarbons such as cyclopentane and cyclohexane; ethers such as diethyl ether and tetrahydrofuran; methyl acetate And esters such as ethyl acetate; halogenated hydrocarbons such as carbon tetrachloride and chloroform; ketones such as acetone, methyl ethyl ketone, and diethyl ketone. These can use 1 type (s) or 2 or more types. As said organic solvent, what contains a polar solvent and a nonpolar solvent is preferable.

Examples of the water include ion-exchanged water, purified water, distilled water, tap water, and the like. Acetic acid or the like may be contained in these to make a buffer solution for pH adjustment. As the water, a buffer solution is preferably used. The concentration of the buffer solution is preferably 0.1 to 2.0M, and more preferably 0.4 to 1.0M.
In the above two-phase system, the mixing ratio of water is preferably 20% by weight or less, more preferably 2 to 15% by weight, and further preferably 5 to 10% by weight with respect to the organic solvent.

In the above two-phase system, when the organic solvent contains a polar solvent and a nonpolar solvent, the mixing ratio is not particularly limited, but preferably the polar solvent: nonpolar solvent is 0.5: 9.5 to 5: 5 is there.
Examples of such a two-phase system composed of an organic solvent and water include a combination of hexane: acetone: water, heptane: acetone: water, hexane: ethyl acetate: water, and the like.

Stirring of the phospholipid is preferably performed at 10 to 40 ° C, more preferably 20 to 30 ° C. The stirring is preferably performed for 30 minutes to 2 hours. For the stirring, a general stirrer can be used. The strength of stirring is not particularly limited, but may be such that the liquid can be mixed vertically.
In the method of the present invention, the phospholipid is usually in a state dissolved in an organic solvent, and the phospholipid, the organic solvent, and water may form an emulsion by the stirring.

In the production method of the present invention, the phospholipid is stirred in a two-phase system composed of an organic solvent and water, and then a hydroxyl group-containing compound and phospholipase D (PLD) are added to perform a base exchange reaction of PC and / or PE. Do.
Examples of the hydroxyl group-containing compound include alcohols, nitrogen-containing alcohols, saccharides, polyols, and hydroxy cyclic compounds.
Examples of alcohols include methanol, ethanol, propanol, ascorbic acid and the like.
Examples of nitrogen-containing alcohols include amino acids such as serine; 1-amino-2-propanol.
Examples of the saccharide include nucleosides such as adenosine, guanosine, inosine, xanthosine, deoxyadenosine, deoxyguanosine; glucose, trehalose, N-acetyl-D-glucosamine and the like.
Examples of polyols include glycerol, ethylene glycol, propylene glycol and the like.
Examples of the hydroxy cyclic compound include succinic acid and arbutin.

The concentration of the hydroxyl group-containing compound in this reaction system is preferably 20 to 40% by weight, more preferably 25 to 30% by weight.

The PLD is not particularly limited as long as it can hydrolyze the phospholipid base moiety, and examples thereof include plant-derived PLD and microorganism-derived PLD. Examples of plant-derived PLD include cabbage and soybean-derived PLD, and examples of microorganism-derived PLD include actinomycetes, filamentous fungus-derived PLD, and the like.
The concentration of PLD in this reaction system is preferably 5 to 200 U, more preferably 20 to 100 U, with respect to 1 g of phospholipid. In addition, 1U uses 95% soybean phosphatidylcholine as a substrate, 37 M 0.2M acetate buffer (pH 4.0, 10 mM CaCl ₂ , 1.3% Triton X-100) 37%. It is the amount of enzyme that liberates 1 μmol of choline per minute when reacted at ° C.

The base exchange reaction is preferably performed under conditions of pH 3.5 to 10, and more preferably pH 4 to 9. Moreover, 10-40 degreeC is preferable and, as for reaction temperature, 20-30 degreeC is more preferable.

By the base exchange reaction, PC and PE in the phospholipid react with a hydroxyl group-containing compound such as serine to obtain PS and the like. Since PI generally does not easily react with a hydroxyl group-containing compound in the base exchange reaction, PI contained in the raw material phospholipid is contained in the phospholipid composition almost as it is.
Examples of the phospholipid composition obtained by the production method of the present invention include a phospholipid composition containing PI and phosphatidic acid, a phospholipid composition containing PI and phosphatidylethanol, and the like in addition to the phospholipid composition containing PI and PS. Is mentioned.

After performing the base exchange reaction, for example, the PLD is deactivated by a treatment such as heating, and the organic solvent layer and the aqueous layer are separated by a centrifugal method or the like to obtain the organic solvent layer. Concentrate by removing. Next, the phospholipid composition of the present invention can be isolated by crystallization with acetone or ethanol, obtaining a solid by solid-liquid separation, and drying.

In the method for producing a phospholipid composition of the present invention, the base exchange reaction is carried out by stirring the phospholipid and the solvent before the base exchange reaction of the phospholipid, even if the raw material phospholipid contains a relatively large amount of PI. Phospholipid composition containing a high concentration of both PI and phospholipids such as PS obtained by base exchange reaction, preferably containing 15% by weight or more of PI and 20% by weight or more of PS A phospholipid composition can be produced efficiently. In the method of the present invention, since it is not necessary to separately prepare phospholipids such as PI and PS, the phospholipid composition can be produced industrially at low cost.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples. “%” In the examples indicates “% by weight”.
Example 1
After dissolving SLP-PI powder (manufactured by Sumi Seiyaku Co., Ltd.) in 90 ml of a heptane: acetone mixed solvent (85:15) to a concentration of 10%, 6 ml of 1 M acetate buffer (pH 4.0) was added, and 30 The mixture was stirred at 0 ° C. for 2 hours (pretreatment).
Then, 25% of serine was added to the aqueous layer so as to be PLD80U / lecithin (g), and the amount of the aqueous layer was adjusted to 17 ml. The mixture was stirred at 30 ° C. overnight for reaction, and allowed to stand for 2 hours. After the lower aqueous layer was separated, the upper solvent layer was concentrated under reduced pressure. Three times the amount of ethanol was added to the concentrated solution to cause precipitation. The precipitate was dried and then dissolved in a chloroform: methanol (2: 1) solution, and the phospholipid content was analyzed by the high performance liquid chromatography method (HPLC method) under the following conditions. The phospholipid composition shown in Table 1 was obtained.
HPLC conditions <br/> Column used: manufactured by GL Sciences Inc. _{Unisil Q NH 2 (4.6mm I.D. ×} 250mm)
Mobile phase: acetonitrile / methanol / 50 mM ammonium dihydrogen phosphate = 1856/874/270
Flow rate: 1.3 mL / min Detection: UV 205 nm
The transfer rate is shown in FIG. The transfer rate is a value determined by the following formula.
Transfer rate (%) = PS concentration after reaction / Before reaction (PC + PE) concentration × 100

Comparative Example 1
After dissolving SLP-PI powder (manufactured by Sumi Seiyaku Co., Ltd.) in 90 ml of a heptane: acetone mixed solvent (85:15) so as to have a concentration of 10%, 6 ml of 1M acetate buffer solution (pH 4.0) and serine are 25%. PLD80U / lecithin (g) was added to adjust the volume of the aqueous layer to 17 ml, and the mixture was stirred overnight at 30 ° C. for reaction. After the lower aqueous layer was separated, the upper solvent layer was concentrated under reduced pressure. Three times the amount of ethanol was added to the concentrated solution to cause precipitation. After the precipitate was dried, it was dissolved in a chloroform: methanol (2: 1) solution, and the phospholipid content was analyzed by the HPLC method under the same conditions as in Example 1. The results are shown in Table 1 and FIG.

As is clear from Table 1 and FIG. 1, Example 1 in which stirring (pretreatment) was performed before the base exchange reaction was compared with Comparative Example 1 in which pretreatment was not performed, and from PC, PE to PS. The PI concentration in the phospholipid composition obtained in Example 1 exceeded 15% by weight, and the PS concentration exceeded 20% by weight.

Example 2
SLP-WHITE (manufactured by Tsuji Oil) was dissolved in 90 ml of a heptane: acetone mixed solvent (75:25) to a concentration of 20%, and then 6 ml of 1 M acetate buffer (pH 4.0) was added, and the mixture was heated to 30 ° C. And stirred for 2 hours (pretreatment).
Then, 25% of serine was added to the aqueous layer so as to be PLD80U / lecithin (g), and the amount of the aqueous layer was adjusted to 17 ml. The mixture was stirred at 30 ° C. overnight for reaction, and allowed to stand for 2 hours. The upper layer solution was diluted with chloroform: methanol (2: 1), and the transfer rate was determined by HPLC analysis. The results are shown in FIG.

Comparative Example 2
Except for carrying out the reaction without stirring (pretreatment), the reaction and analysis were carried out under the same conditions as in Example 2 to obtain the transfer rate. The results are shown in FIG.

As is clear from FIG. 2, the transition from PC, PE to PS is more significant in Example 2 in which stirring (pretreatment) was performed before the base exchange reaction than in Comparative Example 2 in which pretreatment was not performed. The rate was good. Further, the difference in the transfer rate was larger in Example 1 and Comparative Example 1 having 10% by weight than in Example 2 and Comparative Example 2 having a phospholipid concentration of 20% by weight.

In the method for producing a phospholipid composition of the present invention, the base exchange reaction is carried out by stirring the phospholipid and the solvent before the base exchange reaction of the phospholipid, even if the raw material phospholipid contains a relatively large amount of PI. Phospholipid composition containing a high concentration of both PI and phospholipids such as PS obtained by base exchange reaction, preferably 15% by weight or more of PI and 20% by weight or more of PS The containing phospholipid composition can be produced efficiently. Examples of such compositions include, in addition to phospholipid compositions containing PI 15 wt% or more and PS 20 wt% or more, phospholipid compositions containing PI 20 wt% or more and phosphatidic acid 20 wt% or more, PI 18 wt% or more and Examples thereof include a phospholipid composition containing 20% by weight or more of phosphatidylethanol. In the method of the present invention, since it is not necessary to separately prepare phospholipids such as PI and PS, the phospholipid composition can be produced industrially at low cost.

2 is a graph showing the transfer rate of phospholipids in Example 1 and Comparative Example 1. 4 is a graph showing the transfer rate of phospholipids in Example 2 and Comparative Example 2.

Claims (3)

A phospholipid containing at least one phosphatidylcholine and phosphatidylethanolamine in a total amount of 30% by weight or more and phosphatidylinositol in an amount of 10% by weight or more is stirred in a two-phase system composed of an organic solvent and water, and then serine and phospholipase D. A process for producing a phospholipid composition comprising the step of adding at least one base exchange reaction of phosphatidylcholine and phosphatidylethanolamine. The method according to claim 1, wherein the organic solvent includes a polar solvent and a nonpolar solvent. The method according to claim 1 or 2, wherein the concentration of the phospholipid during the base exchange reaction is 5 to 40% by weight based on the total compounds.

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