JPH0759586B2 - Method for producing docosahexaenoylphosphatidylcholine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing phosphatidylcholine containing docosahexaenoic acid, and more specifically, a method for producing phosphatidylcholine having docosahexaenoic acid at the sn-2 position from eggs of aquatic animals. Regarding

(Prior Art) Docosahexaenoic acid, like eicosapentaenoic acid,
Cholesterol and neutral lipid lowering action is also large, and its physiological importance has been recognized. On the other hand, it has been suggested that docosahexaenoic acid controls some action in the phospholipids, which are constituent lipids of the brain and nervous system, due to advances in biochemistry. In addition, docosahexaenoic acid in phospholipids, which governs the physical properties of plasma membranes, has attracted attention as a cell differentiation factor, and phosphatidylcholine containing docosahexaenoic acid, which is present in these, has attracted a great deal of interest.

Polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid are small in triacylglycerol and cholesterol ester in adipose tissue, which is rich in fat in living tissues, and become polar lipids in the constituent components of cell membranes, which are minor components. It is unevenly distributed. In particular, docosahexaenoic acid is known to be abundant in ester type phosphatidylcholine and phosphatidylethanolamine and ether type phosphatidylcholine and phosphatidylethanolamine in the cranial nervous system such as the myelin sheath of the brain and the rod of the optic nerve. The egg yolk lipids of chicken eggs contain several% of the phospholipids phosphatidylcholine and phosphatidylethanolamine.

(Problems to be solved by the invention) As described above, phosphatidylcholine containing docosahexaenoic acid exists in a specific part of the living body, but it is difficult to specifically collect only that part, and its content is very high. There are few suitable ingredients. For example, it is known that phosphatidylcholine and phosphatidylethanolamine, which are phospholipids in commercially available yolk lecithin that are easily available, contain several% of docosahexaenoic acid (Tokyo Kagaku Dojinsha Co., Ltd., Biochemistry Laboratory 3, Chemistry 257). But,
When phosphatidylcholine and phosphatidylethanolamine were individually isolated and the amount of each docosahexaenoic acid was quantified, the docosahexaenoic acid present in the phospholipid of egg yolk lecithin was localized in phosphatidylethanolamine and was not contained in phosphatidylcholine so much. Therefore, a special device is required to efficiently fractionate phosphatidylcholine containing docosahexaenoic acid from phosphatidylcholine in egg yolk lecithin.

In order for phosphatidylcholine containing docosahexaenoic acid to play a physiological role in biological membranes, for example, the expression of hormone action (Toyoaki Akino, Oil Chemistry 30 , 705 (1981)), docosahexaenoic acid is present at the sn-2 position of phosphatidylcholine.
It is important that a saturated or monoenoic acid such as palmitic acid or oleic acid is attached to the sn-1 position of phosphatidylcholine. However, in order to bind docosahexaenoic acid to the sn-1 and sn-2 positions of phosphatidylcholine, a cadmium chloride complex of glycerophosphorylcholine may be reacted with an acid anhydride or halide of docosahexaenoic acid. At present, it is difficult to bond a fatty acid other than docosahexaenoic acid to the sn-1 position with an acid bonded by such a direct reaction.

Therefore, an object of the present invention is to provide a method for producing a phosphatidylcholine containing docosahexaenoic acid at the sn-2 position in an economically high yield and by the simplest possible process.

(Means for Solving the Problems) The present invention, in the production of docosahexaenoylphosphatidylcholine having docosahexaenoic acid at the sn-2 position of phosphatidylcholine, phosphatidylcholine is separated from a marine animal egg as a raw material, and then reverse phase partition is performed. It is characterized in that the main peak is collected by column chromatography.

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

The present inventors have investigated in detail the egg lipids of aquatic animals, which are rich in lipid and contain a large amount of docosahexaenoic acid in the constituent fatty acids.

It is known that the fat composition of the natural lipid raw material is not related to the phylogenetic system and environmental factors are important, and that the composition is remarkably similar especially when the feeding environment is the same in animals. That is, docosahexaenoic acid is hard to find from plant raw materials, and even animal raw materials are found in aquatic animals rather than land animals, so eggs of these animals are preferable as raw materials. As natural raw materials, eggs of marine animals such as salmon and herring are easily available, and eggs such as yellowtail, carp, eel, rainbow trout, and prawns that are actively cultivated are also preferable as raw materials.

These ingredients should be as fresh as possible,
If you use raw materials that have been frozen, freeze-dried or vacuum-dried immediately after collection, you can avoid the deterioration of quality due to the increase of acid value in the raw materials and the increase of peroxide value of the target product, and obtain high quality target product. You can The presence of peroxide in the target substance causes cell damage in the living body, and the peroxidation reaction of the polyunsaturated fatty acid accompanies the production of a conjugated diene, which causes a double bond transfer. Since these phenomena cause physiologically unfavorable effects, suppression of peroxide production is an important condition in the production method of the present invention.

To purify the fertilized egg material, it is preferable to carry out solvent extraction of the fresh material under anoxic conditions. At that time, for example, distilled water and a solvent such as methanol-chloroform solvent, acetone, ether, hexane, etc. are added to the material, and the mixture is rolled with a Rouledes homogenizer, if necessary.
Solver Omni Mixer, Waring Blender,
Homogenize with a potter, elve gem, glass homomixer, etc. As the anoxic state, for example, under vacuum, under nitrogen stream and carbon dioxide stream, 0 ℃ ~ 60
Solvent extraction can be performed at ℃ for 10 to 180 minutes. It is usual to add 1 to 5 parts of the solvent to 1 part of the roe.

As a method for separating phosphatidylcholine from solvent-extracted total lipids, for example, the following method is available. A method in which total lipids are treated with cold acetone to fractionate phospholipids and then separated by column chromatography, and total lipids are directly subjected to silica gel column chromatography, and the mixing ratio of hexane → chloroform → methanol and solvent is changed from nonpolar. It is a method of separating while changing the polarity.

20% or more of docosahexaenoic acid was contained in the total lipid extracted from fish eggs with the Folch solvent according to the present invention. Among the total lipids, the neutral lipids extracted with a hexane-ether solvent were mainly acylglycerols. Triacylglycerol is 37% in total lipid and docosahexaenoic acid content is 12%, diacylglycerol is 3% in total lipid.
%, The content of docosahexanoic acid was 9%, monoacylglycerol was 1% or less in the total lipid, and the content of docosahexaenoic acid was 7%.

In addition, the content of cholesterol esters and free fatty acids in neutral lipids other than acylglycerol was very small, and docosahexaenoic acid was widely present in neutral fat, but its distribution was not biased.

The solvent for extracting total lipids can reduce the chances of glycolipid contamination by washing with water several times, eliminating the possibility that docosahexaenoic acid in fertilized eggs is derived from glycolipids.

The amount of phospholipids fractionated from the total lipids by the cold acetone method was 47% of the total lipids. The phospholipid fraction was subjected to methyl esterification by the boron trifluoride methanol method and analyzed by gas chromatography. As a result, the amount of docosahexaenoic acid was 36% in the phospholipid fatty acid. The results showed that the content of docosahexaenoic acid in phospholipids was obviously higher than that of other lipids in total lipids.

In order to quantify the phosphatidylcholine fraction in phospholipids, two-dimensional development was performed using a high performance thin layer chromatography plate. After the development, the phosphatidylcholine spot was scraped off, and the phosphatidylcholine content was determined by measuring the phosphorus content with each phospholipid fraction by the phosphomolybdenum blue absorptiometry. The amount of phosphatidylcholine was 67% of the phospholipid fraction. On the other hand, the phospholipid fraction was eluted with a silica gel column in a chloroform-methanol system to obtain a phosphatidylcholine fraction, which was subjected to methyl esterification by the boron trifluoride-methanol method and subjected to gas chromatography analysis in the same manner as above, showing that docosahexaenoic acid. The amount was 46% in phosphatidylcholine fatty acid. Based on the amount of phosphatidylcholine in the phospholipid fraction and the amount of docosahexaenoic acid in it, docosahexaenoic acid in total lipids was distributed more in phospholipids, more specifically in phosphatidylcholine.

Finally, in the method of the present invention, the main peak is collected by performing reverse phase partition chromatography. As reversed-phase partition chromatography, high-performance liquid chromatography equipped with an ODS (silica gel in which octadecyl group is chemically bonded) column is used.
It is preferable because it can be mass-produced.

To investigate the molecular species of phosphatidylcholine fractionated from fish eggs, high performance liquid chromatography equipped with an ODS column was developed with methanol (1 ml / min), and UV 210 nm was applied.
Monitor by RI (refractive index). The chromatogram obtained by the UV monitor has one main peak and several minute peaks, the main peak position corresponds to the single peak obtained by the RI monitor, and the molecular species of this phosphatidylcholine is very small. It is shown that.

The phosphatidylcholine obtained from the main peak was 806 (equivalent to C _{16: 0} -docosahexaenoylphosphatidylcholine) as m / z corresponding to the molecular ion when measured by the direct introduction method of a mass spectrometer FAB-MS (Pos.). , 832 (C _{18: 1}
-Corresponds to docosahexaenoylphosphatidylcholine)
And 834 (corresponding to _{C18: 0} -docosahexaenoylphosphatidylcholine) have high strength. To determine the fatty acid composition at the sn-2 position of this phosphatidylcholine, for example, after hydrolyzing the ester bond of the fatty acid at the sn-2 position of phosphatidylcholine with phospholipase A _z , methyl esterification by the boron trifluoride methanol method is performed. Measured by capillary gas chromatography and compared with a standard of methyl docosahexaenoate.

Regarding the amount of peroxides from raw materials to final products, a large amount of measurement sample is required in the conventional iodometry method, but the number of measurement samples can be small when measured by potentiometric titration.

From fish phase egg phosphatidylcholine, reverse phase partition column chromatography, mass spectrometry, and analysis of phospholipase A _z revealed that the molecular species of the main component of this phosphatidylcholine is phosphatidylcholine that binds docosahexaenoic acid to the sn-2 position. Therefore, in order to separate the phosphatidylcholine that binds docosahexaenoic acid at the sn-2 position from the fish egg phosphatidylcholine,
The main peak was continuously collected from the fish egg phosphatidylcholine using a fully automatic high performance liquid chromatography equipped with a large-scale preparative ODS column to produce the target substance in units of g.

(Effects of the Invention) According to the method of the present invention, attention is paid to the constituent lipids of the brain / nerve system, the lipids that control the physical properties of the plasma membrane involved in cell differentiation, etc. by subjecting the eggs of aquatic animals to purification treatment. The phosphatidylcholine containing docosahexaenoic acid, which is useful as a physiologically active substance, can be obtained in high yield without special synthesis and while maintaining the natural stereospecificity. Furthermore, when treated under anoxic conditions, docosahexaenoic acid-containing phosphatidylcholine can be obtained while suppressing the production of lipid peroxide that causes cell damage.

(Example) Example 1 A fertilized rainbow trout fertilized egg immediately after egg collection was thawed under a nitrogen stream. 1300 g of this raw material was put in 2 liters of acetone, homogenized roughly with a TH homomixer (manufactured by Tokushu Kiko Kogyo) under a nitrogen stream, and then homogenized for 30 minutes under ice-cooling with an Excel Auto Homogenizer (manufactured by Nippon Seiki Seisakusho).

The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 10 g of an acetone extract was obtained from the filtrate.

830 g of milky gray wet cake was put in 3 L of ethyl ether, and extracted for 30 minutes while rotating at 200 rpm with a three-one motor type 600GM (manufactured by Shinto Kagaku). The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 48 g of ethyl ether extract was obtained from the filtrate.

770 g of milk gray wet cake was added to chloroform-methanol (1 /
1, vol / vol) Mixture 1 was extracted twice in a separatory funnel.
The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 55 g of chloroform-methanol extract was obtained from the filtrate. 690 g of milky gray wet cake was extracted twice with 1.5 l of a chloroform-methanol (2/1, vol / vol) mixture in a separatory funnel. The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 5 g of chloroform-methanol extract was obtained from the filtrate. Each lipid extract was combined. The total amount of lipid was 118 g (yield of raw material was 9.1%).

After total amount of total lipids was applied to silica gel column chromatography (silica 60, Wako Pure Chemical Industries: 8φ × 40cm column, 2l), neutral lipids were removed by eluent system in which the ratio of chloroform in hexane was increased. . Recovery amount of neutral lipids 69g
In the composition, thin-layer chromatography (developing solvent: hexane-ethyl ether-acetic acid 50/50/1, vol / vol / vol) analysis showed that triacylglycerol was the main component.

An eluent system in which the ratio of methanol to chloroform was increased was passed through the column without neutral lipids. In a ratio of chloroform to methanol of 35:65 to 25:75, a group containing phosphatidylcholine as a main component was eluted, and 28 g after desolvation
To obtain phosphatidylcholine. The classification of phosphatidylcholine was determined by thin layer chromatography (developing solvent: chloroform-methanol-water 65/25/4, vol / vol / vol). Rf value 0.20 to 0.30 on thin layer chromatography
Fractions showing only a single spot in (phosphatidylcholine) were collected and desolvated under a nitrogen stream to obtain 19 g of pure phosphatidylcholine.

The obtained phosphatidylcholine was used as a 10% w / v benzene solution, and an ODS packed column (φ2 inch x 60) was applied to a fully automatic mass preparative liquid chromatography HLC-837 manufactured by Toyo Soda Co., Ltd.
cm) was mounted, methanol as an eluent was caused to flow at 40 ml / min, and automatic fractionation was repeated 28 times continuously to fractionate the main peak of the target substance. The preparative fraction of methanol was removed to obtain 12.2 g of the desired product, phosphatidylcholine.

The separated phosphatidylcholine was methyl esterified by the boron trifluoride methanol method and subjected to capillary column gas chromatography (liquid phase: carbowax-20M, 25
The fatty acid composition was measured at m, 170 ° C). As a result, docosahexaenoic acid is 40%, palmitic acid, stearic acid,
Oleic acid accounted for about 10% each.

The phosphatidylcholine was hydrolyzed with phospholipase A ₂ , methylesterified by the boron trifluoride methanol method, and the fatty acid composition was measured by capillary column gas chromatography. As a result, docosahexaenoic acid is 82
%, And several other small peaks were observed.

Then, to analyze the molecular species of phosphatidylcholine, FA
The m / z corresponding to the molecular weight M was measured by B-MS (Pos.). As a result, m / z 806 (corresponding to palmitoyl docosahexaenyl phosphatidylcholine), m / z 832 (corresponding to oleoyl docosahexaenyl phosphatidylcholine), m / z
834 (corresponding to stearoid cosahexaenylphosphatidylcholine) was strongly recognized.

To analyze the distribution of alkyl chains in phosphatidylcholine, high performance liquid chromatography (Shordex OD
Spak F-611 A) developed with methanol (1 ml / min) and UV2
Monitored at 10 nm. As a result, only one main peak and several small peaks were observed, and the distribution of the alkyl chain of phosphatidylcholine was very small.

The amount of lipid peroxide of phosphatidylcholine was measured by a potentiometric titration method (automatic titrator GT-05, manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). The amount of peroxide in total lipid of raw fish egg is 12.3 meq./kg
On the other hand, the phosphatidylcholine fraction recovered from the main peak was 29.3 meq./kg.

Example 2 Fertilized rainbow trout fertilized eggs immediately after egg collection were thawed under a nitrogen stream. 1500 g of this raw material was placed in 6 l of a chloroform / methanol (2/1, vol / vol) mixture, and homogenized for 30 minutes in an ice-cooled state while shear-extracting at high speed with a TH homomixer (manufactured by Tokushu Kiko Kogyo) under a nitrogen stream.

The suspension was filtered on a Buchner funnel and separated into a filtrate and a milky wet cake. 910 g of milky gray wet cake was added to 2 l of the above solvent, and treated under the same conditions as above. Similarly, the suspension was separated into a filtrate and a wet cake. 780 g of milky gray wet cake was put in 2 l of the above solvent and treated under the same conditions. Further, the filtrate and the wet cake were separated by filtration from the third suspension. All the filtrates were collected and centrifuged, and 3 l of chloroform and 3 l of distilled water were added to the supernatant liquid, washed well with water, and then separated into two layers by centrifugation.

The lower chloroform layer was collected and concentrated at 30 ° C. under a nitrogen stream using a rotary evaporator, and 100 ml of benzene was added at the end of the solvent distillation to remove the solvent. The weight of total lipids obtained by drying the solvent-distilled extract in a vacuum desiccator overnight was 127 g (8.
5%).

Put all the obtained lipids in 1.3 liters of ice-cold acetone and use a three-one motor type 600 GM (Shinto Kagaku) under a nitrogen stream.
It extracted for 10 minutes, rotating at 200 rpm. The acetone suspension was filtered through a cold Buchner funnel and the precipitate was collected.
The same cold acetone treatment as above was repeated 4 times on this precipitate to obtain 57 g (45.2% of total lipids) of the phospholipid fraction in which neutral lipids were completely removed.

Silica gel column chromatography of all phospholipids (Fuji gel CG-3, manufactured by Mito Kagaku Co., Ltd.
c), followed by chloroform-methanol (4/1, vol / v
The phospholipid such as phosphatidylethanolamine, which was eluted before the phosphatidylcholine, was removed by the mixed solvent eluent system.

Further, in order to fractionate pure phosphatidylcholine, it was eluted with an eluent system of a chloroform-methanol (3/2, vol / vol) mixture. Fractions of 500 ml each of the eluent were fractionated, and each fraction was measured by thin layer chromatography (developing solvent: chloroform-methanol-water 65/25/4, vol / vol / vol). Rf value 0.20-0.35 by thin layer chromatography in each fraction
(Phosphatidylcholine) 19 g of a fraction having only a single spot was obtained.

Then, the obtained phosphatidylcholine was used as a 10% w / v benzene solution, and an ODS packed column (φ2 inch × 60 cm) was attached to a fully automatic mass preparative liquid chromatography HLC-837 manufactured by Toyo Soda Co., Ltd. 40% methanol as eluent
The main peak of the objective substance was collected by repeating automatic collection 32 times at a flow rate of ml / min. The solvent of the separated fraction was desolvated to obtain 13.9 g of the desired product, phosphatidylcholine.

The separated phosphatidylcholine was methyl esterified by the boron trifluoride methanol method, and then subjected to capillary column gas chromatography (liquid phase: carbowax-20M, 25
The fatty acid composition was measured at m, 170 ° C). As a result, docosahexaenoic acid accounted for 41%, and palmitic acid, stearic acid and oleic acid accounted for about 10% each.

This phosphatidylcholine was hydrolyzed with phospholipase A ₂ , methylesterified by the boron trifluoride methanol method, and the fatty acid composition was measured by capillary gas chromatography. As a result, docosahexaenoic acid was found to have several minute peaks at 82%.

Next, to analyze the molecular species of phosphatidylcholine,
FAB-MS (Pos.) Was used to measure m / z corresponding to the molecular weight (Fig. 1). The mass spectrum obtained was strong at m / z 806 (corresponding to palmitoyl docosahexaenyl phosphatidylcholine), m / z 832 (corresponding to oleoyl docosahexaenyl phosphatidylcholine) and m / z 834 (corresponding to stearoyl docosahexaenyl phosphatidylcholine). Admitted. To analyze the distribution of alkyl chains in phosphatidylcholine, an ODS column (Shordex ODSpa
High-performance liquid chromatography equipped with k F-611 A, manufactured by Teruko Tsusho, developed with methanol (1 ml / min), UV210nm
I monitored it at. The obtained chromatogram showed only one main peak and several minute peaks, and the distribution of the alkyl chains of this phosphatidylcholine was very small.

The lipid peroxide amount of phosphatidylcholine was measured by a potentiometric titration method (automatic titrator GT-05, manufactured by Mitsubishi Kasei Co., Ltd.) and was 25.6 meq./kg.

Example 3 Salmon eggs (salmon roe) frozen immediately after egg collection were thawed under a nitrogen stream. Chloroform / methanol (2/1, vol / vol) mixture 2 l was added to 500 g of this raw material, thoroughly mixed with a homomixer, filtered, and the residue was treated with the same mixture 1 twice under a nitrogen stream. It was After combining the filtrates and washing with water,
The solvent was distilled off, and the weight of the lipid obtained by drying for 3 hours in a vacuum desiccator was 73.5 g (yield to raw material: 14.7%).

The obtained total lipid was put in 0.4 l of ice-cold acetone and extracted for 10 minutes while rotating under a nitrogen stream. The acetone suspension was filtered through a cold Buchner funnel and the precipitate was collected. To this precipitate, cold acetone treatment similar to the above was repeated 4 times,
23.7 g of phospholipid fraction completely depleted of neutral lipids (3% of total lipids)
2.2%) was obtained.

Silica gel column chromatography (Fuji gel CG-3, manufactured by Mito Kagaku Co., Ltd.)
c), followed by chloroform-methanol (4/1, vol / v
The phospholipid such as phosphatidylethanolamine, which was eluted before the phosphatidylcholine, was removed by the mixed solvent eluent system.

In addition, to fractionate pure phosphatidylcholine,
It was eluted with an eluent system of chloroform-methanol (3/2). Fractions of 500 ml each were eluted and each fraction was analyzed by thin layer chromatography (developing solvent: chloroform-methanol-
Water 65/25/4, vol / vol / vol). Of each fraction
Fraction with a single spot at Rf value of 0.20 to 0.35 (phosphatidylcholine) by thin layer chromatography 6.
I got 1g.

Then, the obtained phosphatidylcholine was made into a 10% w / v benzene solution, and was applied to an ODS packed column (φ2) on a fully automatic mass-preparative liquid chromatography HLC-837 manufactured by Toyo Soda Co., Ltd.
(Inch x 60 cm) with methanol as the eluent
Flowing at 40 ml / min, continuous automatic collection was repeated 11 times to collect the main peak of the target substance. The preparative fraction of methanol was desolvated to obtain 4.5 g of the desired product, phosphatidylcholine.

The separated phosphatidylcholine was methyl esterified by the boron trifluoride methanol method and subjected to capillary column gas chromatography (liquid phase: carbowax-20M, 25
The fatty acid composition was measured at m, 170 ° C). As a result, docosahexaenoic acid was 39%, and palmitic acid, stearic acid and oleic acid accounted for about 10% each.

This phosphatidylcholine was hydrolyzed with phospholipase A ₂ , methylesterified by the boron trifluoride methanol method, and the fatty acid composition was measured by capillary gas chromatography. As a result, docosahexaenoic acid was 80%, and several other small peaks were observed.

Next, to analyze the molecular species of phosphatidylcholine,
FAB-MS (Pos.) Was used to measure m / z corresponding to the molecular weight. The mass spectrum obtained was m / z 806 (corresponding to palmitoyl docosahexaenyl phosphatidylcholine), m / z 832
(Corresponding to oleoyldocosahexaenylphosphatidylcholine) and m / z 834 (corresponding to stearoyldocosahexaphenylphosphatidylcholine) were strongly observed.
In order to analyze the distribution of alkyl chains in phosphatidylcholine, high performance liquid chromatography equipped with an ODS column (SHOdex ODSpak F-611 A, manufactured by Shoko Tsusho) was developed with methanol (1 ml / min) and monitored at UV210 nm. The obtained chromatogram showed only one main peak and several minute peaks, and the distribution of the alkyl chain of this phosphatidylcholine was very small.

The lipid peroxide content of phosphatidylcholine was measured by a potentiometric titration method (automatic titrator GT-05, manufactured by Mitsubishi Kasei Co., Ltd.) and was 17.2 meq./kg.

Example 4 500 g of herring eggs was treated with a solvent in the same manner as in Example 1 to give a lipid.
14 g (yield of 2.8% of raw material) was obtained.

Silica gel column chromatography (Kieselgel 60, Merck & Co., 4φ × 40 cm column 0.4
After the reaction with l), chloroform was flowed until neutral lipid was not discharged, and further methanol was flowed to distill off the solvent of the phospholipid fraction. The obtained phospholipid fraction was 8.3 g (59.2% of total lipids).

Pure phosphatidylcholine fraction 3.1 showing a single spot with an Rf value of 0.20 to 0.35 by thin layer chromatography using an eluent of a chloroform-methanol (3/2 vol / vol) mixture in the above-mentioned column excluding neutral lipids. got g.

Then, this pure phosphatidylcholine was subjected to fully automatic mass preparative liquid chromatography in the same manner as in Example 1, and the main peak was fractionated by repeating the automatic fractionation 6 times consecutively.
2.0 g of the desired phosphatidylcholine was obtained.

When this phosphatidylcholine was analyzed in the same manner as in Example 1, it contained 43% of docosahexaenoic acid as a fatty acid composition, and the sn-2 position fatty acid composition obtained by hydrolysis with phospholipase A ₂ was docosahexaenoic acid. It contained 85%.

The amount of lipid peroxide was 13.3 meq./kg.

Example 5 Eggs of prawns that were frozen immediately after egg collection were thawed under a nitrogen stream. 1000 g of this raw material was put in 1.5 l of acetone, and homogenized roughly with a TH homomixer (made by Tokushu Kiko Kogyo) under a nitrogen stream, and then homogenized for 30 minutes under ice-cooling with an Excel Auto Homogenizer (made by Nippon Seiki Seisakusho). .

The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 7 g of acetone extract was obtained from the filtrate.

630g of milk gray wet cake is put into 2.5l of ethyl ether, and 200rpm with three one motor type 600GM (Shinto Kagaku)
It was extracted for 30 minutes while rotating with. The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 35 g of an ethyl ether extract was obtained from the filtrate.

580 g of milky gray wet cake was added to chloroform-methanol (1 /
1, vol / vol) Mixture 1 was extracted twice in a separatory funnel.
The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. 40 g of chloroform-methanol extract was obtained from the filtrate. 535 g of a milky gray wet cake was extracted twice with 1.5 l of a chloroform-methanol (2/1, vol / vol) mixed solution in a separating funnel. The suspension was filtered on a Buchner funnel and separated into a filtrate and a wet cake. Chloroform-methanol extract from filtrate
I got 4g. Each lipid extract was combined. The total amount of lipids was 86 g (yield to raw material 8.6%).

After total amount of total lipids was applied to silica gel column chromatography (silica 60, Wako Pure Chemical Industries: 8φ × 40cm column, 2l), neutral lipids were removed by eluent system in which the ratio of chloroform in hexane was increased. . Recovery amount of neutral lipid 54g
In the composition, thin-layer chromatography (developing solvent: hexane-ethyl ether-acetic acid 50/50/1, vol / vol / vol) analysis showed that triacylglycerol was the main component.

An eluent system in which the ratio of methanol to chloroform was increased was passed through the column without neutral lipids. When the ratio of chloroform to methanol was 35:65 to 25:75, the segment containing phosphatidylcholine as the main component was eluted and 24.7
g of phosphatidylcholine was obtained. Thin layer chromatography (developing solvent: chloroform-methanol-water 65/25/4, vol / vol / vol) was used to determine the classification of phosphatidylcholine.
I went there. Rf value 0.20 to 0.30 on thin layer chromatography
Fractions showing only a single spot in (phosphatidylcholine) were collected and desolvated under a nitrogen stream to obtain 15.1 g of pure phosphatidylcholine.

As a result, docosahexaenoic acid accounted for 30%, and palmitic acid and oleic acid accounted for about 20% each.

The phosphatidylcholine was hydrolyzed with phospholipase A ₂ , methylesterified by the boron trifluoride methanol method, and the fatty acid composition was measured by capillary column gas chromatography. As a result, docosahexaenoic acid was 61
%, Oleic acid was 30%, and several other small peaks were observed.

Application Example A carcinogenicity test confirming the carcinostatic activity of the phosphatidylcholine obtained in Example 1 will be described. A test was performed on Friend leukemia cells (mouse erythroblastic leukemia cells (B8) cells). 2.5% of HAM F-12 medium (GIBCO) plus 15% fetal bovine serum and 60 mg / l kanamycin
B8 cells are inoculated at a concentration of × 10 ⁵ cells / ml, and a predetermined amount of the test compound is added to this (final volume 5 ml).

After culturing in 8.0% carbon dioxide gas at 37 ° C for 7 days, the number of cells stained by Orkin's benzine staining method, that is, the number of cells that started to produce hemoglobin by differentiation into erythrocytes Was measured, and the differentiation induction rate was calculated from the ratio to all cells (Table 1).

[Brief description of drawings]

FIG. 1 is a mass spectrum of the phosphatidylcholine obtained in Example 1 by a mass spectrometer FAB-MS (Pos.).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kenichi Asahi 1-4-108 Suwahara housing complex, Wako City, Saitama Prefecture (72) Inventor Nobutaka Takahashi 4-27-2 Ogikubo, Suginami-ku, Tokyo

Claims (1)

[Claims] 1. When producing docosahexaenoylphosphatidylcholine having docosahexaenoic acid at the sn-2 position of phosphatidylcholine, total lipids obtained by solvent-extracting aquatic animal eggs as a raw material under anoxic conditions to obtain total lipids. Is treated with a cold solvent, or the total lipid obtained by solvent extraction is directly separated by column chromatography to separate phosphatidylcholine, and then reverse phase partition column chromatography is performed to collect the main peak. Method for producing noylphosphatidylcholine.