JP2707642B2 - Method for producing lysophospholipid - Google Patents

Description

The present invention relates to a method for producing lysophospholipids such as lysophosphatidylcholine.

[Conventional technology]

Phosphatidylcholine, which is called lecithin, is known as a typical example of phospholipid. One fatty acid obtained by this choline is lysolecithin, that is, lysophosphatidylcholine. Glycerylphosphorylcholine is obtained from a single fatty acid.

Thus, the lysophospholipid has a chemical structure in which either the α-position or the β-position fatty acid of a diacylglycerophospholipid such as phosphatidylcholine has been released, which is also the glycerylphospholipid. It can also be said that glycerophospholipid having both hydroxyl groups at the α-position and β-position such as rucholine has a chemical structure in which a fatty acid is ester-bonded to the one hydroxyl group.

As a method for producing such a lysophospholipid, conventionally, a diacylglycerophospholipid as a natural product containing phosphatidylcholine or the like as a main component as a starting material, and only the β-position acyl group of the lysophospholipid is used as a phospholipase. A
_{2. A} method for obtaining a lysophospholipid having a fatty acid only at the α-position by selective hydrolysis according to the method [2], edited by Biochemical Society, Biochemistry Experimental Lecture 9, “Metabolism of lipids”
P.319 (1981) Tokyo Chemical Doujinshi].

[Problems to be solved by the invention]

However, the above known methods, in addition disadvantageous that the phosphine Oh lipase A ₂ used in this is a special and expensive, very determined by the kinds of natural materials α- position of fatty acids obtained lysophospholipids used connexion (Usually a mixed fatty acid), and it is difficult to introduce any other fatty acid, especially a single fatty acid.

In general, lysophospholipids are used as pharmaceutical products, as final products having fatty acids only at one of the α-position and β-position, and as products obtained by using this as an intermediate and further introducing another fatty acid. It is expected to be used in various fields such as cosmetics and food. When applying to these materials, α, based on basic research such as physical property values,
It is desired that the fatty acid to be introduced at the -position and / or the β-position be as high as possible and a single fatty acid.

In view of the above, the present invention provides a novel method for producing a lysophospholipid capable of optionally and economically introducing a desired fatty acid which is highly pure and single at only one of the α-position and the β-position. It is intended to provide law.

[Means for solving the problem]

In order to achieve the above object, the present inventors first employ a conventional method of partially hydrolyzing a diacylglycerophospholipid as a raw material component, which would result in a desired fatty acid regardless of the means of hydrolysis. Glycerophospholipid having both α- and β-hydroxyl groups such as glycerylphosphorylcholine, which can be said to be a total hydrolyzate thereof, instead of the above diacylglycerophospholipid, is used as a raw material component. An attempt was made to esterify one of the above hydroxyl groups of the lipid with the desired fatty acid.

However, even if this esterification is performed by an organic chemical reaction using an organic catalyst, there is no difference in the reaction characteristics between the α-position hydroxyl group and the β-position hydroxyl group.
It was not possible to esterify only one hydroxyl group at the -position. In place of the organic catalyst, where the conventional phosphine O lipase A _2, known as hydrolases of phospholipids tried using ignores the economic disadvantages, this enzyme is no contribution as an esterification catalyst Turned out not to be.

Therefore, the present inventors further repeated trial and error experimental study to achieve the above esterification,
By using an inexpensive and easily available general lipase as an esterification catalyst, that is, a normal lipase known as a triglyceride hydrolase, it is possible to obtain a desired ester having only one of the α-position or β-position esterified. The inventors have found the surprising fact that lysophospholipids can be selectively produced, and have finally completed the present invention.

That is, the present invention relates to a method for producing lysophospholipid, characterized in that only one of two hydroxyl groups of glycerophospholipid is esterified by lipase.

[Structure and operation of the invention]

Glycerophospholipids used as a raw material component in the present invention, as shown in Examples below, directly hydrolyze natural diacylglycerophospholipids such as soybean lecithin to form free hydroxyl groups at both the α-position and β-position. Or a highly purified diacylphosphatidylcholine once purified from the above phospholipid, and then hydrolyzed to convert its α-position and β-position to free hydroxyl groups. It may be what was done.

The fatty acid used in the present invention can be variously selected depending on the purpose of use of the lysophospholipid, but usually a saturated or unsaturated fatty acid having 6 to 24, preferably 12 to 22 carbon atoms is preferably used. As described above, this fatty acid is generally used as a single fatty acid with high purity, but may be used as a mixture of two or more kinds if necessary. Specifically, lauric acid, myristic acid, palmitic acid, stearic acid, palmitooleic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid,
Eicosapentaenoic acid, docosahexaenoic acid, and the like.

Lipases used in the present invention include microorganisms,
Those of animal, plant and plant origin can be used alone or in combination of two or more. Depending on the type of lipase,
It is possible to esterify only the α-position hydroxyl group or to esterify only the β-position hydroxyl group.

Specific examples include those derived from microorganisms such as Rhizopus and
Lipase from Rhizopus chinensis, lipase from Rhizopus delemar, Candida cylindra
cea), lipase from Pseudomonas fluorescens, Geotrichum candidum
Lipase, Penicillium cyclopium (Pe
lipase from nicillium cyclopium, lipase from Mucor miehei, lipase from Aspergillus niger, Chromobacterium viscum
osum).

In addition, animal-derived lipases include pancreatic lipases of humans, pigs, cows, and the like, and plant-derived lipases include castor seed lipase.

Commercially available lipases particularly suitable for the present invention include lipase P from Amano Pharmaceutical Co., Ltd. and Paratase M from Novo Japan Ltd.
And lipozyme, lipase LP of Toyo Brewery Co., Ltd., and lipase of Tanabe Seiyaku Co., Ltd.

Esterification reaction of the present invention, a lipase aqueous solution, a powder lipase or immobilized lipase is added to a mixture of an aqueous solution of glycerophospholipid and a fatty acid as a raw material component,
It may be carried out while continuously removing excess water under reduced pressure so that the amount of water in the reaction system becomes a necessary minimum. The reaction temperature may be near the optimal temperature of the lipase to be used, and generally is 40 ° C.
~ 60 ° C is preferred.

In this reaction, the amount of the fatty acid per mol of glycerophospholipid is generally 0.2 to 5.0 mol, preferably 0.5 to 5.0 mol.
The range is preferably 2.0 moles. If the amount is too small, the progress of the reaction is slow and a large amount of unreacted glycerophospholipid remains. Conversely, if the amount is too large, a large amount of excess fatty acid remains, which is unfavorable because it hinders purification.

The amount of the lipase varies depending on the type and shape (powder, granule, immobilized product, etc.) of the lipase to be used, but is usually 500 to 5,000 international units (IU), preferably 2,000 to 1 g of glycerophospholipid. ~ 4,000 IU. If the amount is too small, the reaction is extremely slow. Conversely, if the amount is too large, the effect corresponding to the reaction cannot be obtained, and further, the cost is increased, and both are disadvantageous.

After the esterification reaction according to the present invention, unreacted glycerophospholipids and fatty acids are removed by, for example, column chromatography to obtain lysophospholipids in which a fatty acid has been introduced into only one of the α-position and the β-position. Can be Whether the fatty acid is introduced at the α-position or the β-position can be easily confirmed by a known NMR method.

〔The invention's effect〕

As described above, according to the method of the present invention, a highly pure lysophospholipid in which a desired fatty acid is introduced into only one of the α-position and the β-position using an easily and inexpensively obtainable lipase Can be easily produced.

Therefore, as described above, the lysophospholipid obtained by this method can be used as a final product itself, or as a product obtained by using this as an intermediate and further adding another fatty acid to pharmaceuticals, cosmetics, foods, etc. Is expected to be applied to various fields. Especially when used as the latter intermediate,
For example, while a fatty acid such as palmitooleic acid which may be targeted to cancer cells is esterified at the α-position, a fatty acid such as docosahexaenoic acid having an anticancer property is introduced at the β-position to thereby obtain an anticancer drug. Effective phospholipids can be obtained.

〔Example〕

Hereinafter, only examples using a lipase in which only the hydroxyl group at the α-position is selectively esterified as an example of the present invention will be shown for convenience, but the present invention is limited to these examples. It goes without saying that it is not. In the following, “%” means “% by weight”. Glycerophospholipid A and glycerylphosphorylcholine used as raw material components were prepared in the following manner.

<Glycerophospholipid A> The method of H. Brocherhoff et al. From soybean lecithin [Can. J. Bio
chem., 43, 177 (1965)], a brown viscous liquid obtained by washing with acetone and vacuum drying after hydrolyzing the acyl group, and having a purity of 95.1%.

<Glycerylphosphorylcholine> Purified by column chromatography from soybean lecithin to obtain 99.0% of diacylphosphadylcholine, then hydrolyzed with an acyl group in the same manner as above, and further purified. is there.

Example 1 A mixture of 10 g (35.5 mmol) of oleic acid having a purity of 90% and 10 g (42.6 mmol) of glycerophospholipid A dissolved in 10 g of water was added thereto, and 25,000 IU of lipase LP powder was added thereto. The reaction was carried out at 40 ° C. for 20 hours while stirring.

After completion of the reaction, the mixture was extracted with n-hexane to obtain a mixture of unreacted fatty acid and lysophospholipid. This was purified by column chromatography to obtain 12.4 g (23.6 mmol, yield 55.2%) of lysophospholipid.

Example 2 20 g (71.4 mmol) of 99% pure linoleic acid and 10 g (38.9 mmol) of glycerylphosphorylcholine dissolved in 10 g of water were mixed, and a paratase M aqueous solution 2 was added.
After adding 0.000 IU, the mixture was reacted at 50 ° C. for 20 hours with stirring under a reduced pressure of 20 to 30 mmHg.

After completion of the reaction, the reaction was carried out in the same manner as in Example 1 to obtain lysophosphatidylcholine 10.0 g (19.2 mmol, yield 49.2%).

Example 3 20 g (62.5 mmol) of docosahexaenoic acid having a purity of 65%
And glycerylphosphorylcholine 10 g (38.9 mmol)
Was dissolved in 5 g of water, and lipozyme was added.
While adding 35,000 IU and stirring under reduced pressure of 25 to 35 mmHg,
The reaction was performed at 60 ° C. for 10 hours.

After completion of the reaction, the reaction was carried out in the same manner as in Example 1 to obtain lysophosphatidylcholine 7.8 g (15.0 mmol, yield 38.6%).

Example 4 10 g (50.0 mmol) of 99% pure lauric acid and 10 g (38.9 mmol) of glycerylphosphorylcholine dissolved in 5 g of water were mixed, and 30,000 IU of lipase P immobilized on an ion exchange resin was mixed. The mixture was added and reacted at 60 ° C. for 10 hours while stirring under a reduced pressure of 25 to 35 mmHg.

After completion of the reaction, the reaction was carried out in the same manner as in Example 1 to obtain 7.0 g (13.4 mmol, yield 34.4%) of lysophosphatidylcholine.

Example 5 10 g (35.5 mmol) of oleic acid having a purity of 90% and a solution prepared by dissolving 10 g (42.6 mmol) of glycerophospholipid A in 10 g of water were mixed, and 40,000 IU of lipase was added.
The reaction was carried out at 50 ° C. for 20 hours while stirring under a reduced pressure of 2525 mmHg.

After completion of the reaction, the mixture was treated in the same manner as in Example 1 to obtain 10.8 g (20.8 mmol, 48.8% yield) of lysophospholipid.

Example 6 10 g (36.0 mmol) of 99% pure γ-linolenic acid and 10 g (38.9 mmol) of glycerylphosphorylcholine were added to water 1
And 0 g of lipase P.
Add 00IU and stir under reduced pressure of 20 mmHg at 40 ° C for 20
Reacted for hours.

After completion of the reaction, the same treatment as in Example 1 was carried out to obtain 9.0 g (17.4 mmol, yield 45.0%) of lysophosphatidylcholine.

Comparative Example 1 Phospholipase A ₂ was replaced with 30,000 I instead of lipase P
The reaction was carried out in the same manner as in Example 6 except that U was added, and the reaction was further processed. However, the yield of lysophosphatidylcholine was trace.

As is clear from comparison of the above Examples and Comparative Examples, according to the method of the present invention, only one of the two hydroxyl groups at the α-position and the β-position has high purity. It turns out that the desired lysophospholipid into which a single fatty acid is selectively introduced can be economically produced.

Claims (1)

(57) [Claims] 1. A method for producing lysophospholipids, wherein only one of two hydroxyl groups of glycerophospholipid is esterified with a lipase.