JP2793317B2 - Method for separating phosphatidylcholine or phosphatidylethanolamine - Google Patents

Description

The present invention relates to phosphatidylcholine (PC) or phosphatidylethanol which can be separated with high reproducibility and high purity from egg yolk-derived fats and oils containing two or more phospholipids. The present invention relates to a method for separating amine (PE).

<Related Art> In recent years, various studies have been made on various physiological activities possessed by phospholipids. For example, with respect to phospholipids extracted from chicken egg yolk, useful functions such as a serum cholesterol lowering action, a blood pressure lowering action, and an activation action of a neurotransmission system have been noted. However, in the state of egg yolk, for example, since cholesterol and egg yolk protein coexist, there is a problem that the useful function cannot be sufficiently exhibited. Therefore, it is necessary to fractionate the yolk phosphatidylcholine (PC) and yolk phosphatidylethanolamine (PE), which are the main components of the yolk phospholipid, from the yolk state and extract them with high purity.

There have been many reports on the method of separating and purifying the phospholipids of PC and PE described above.Liquid chromatography is suitable for obtaining high-purity phospholipids. Used in combination with a highly polar solvent (e.g., methanol, ethanol, water) and a less polar solvent (e.g., n-hexane, chloroform, benzene, acetonitrile), and the components are also, for example, acetonitrile / methanol / water Alternatively, most of them were three or more kinds such as dichloromethane / methanol / ammonia or a mixed solvent of a plurality of components to which acids and salts were added (see “Sequence Chemistry Experimental Course Vol. 3 Membrane lipids and plasma proteins (top ) ”, P3-5, edited by The Japanese Biochemical Society, published by Tokyo Doujinshi)

<Problems to be Solved by the Invention> However, in the separation of phospholipids according to the above-described conventional technology, it is necessary to use a large amount of a mobile phase solvent. , Which are disposable due to the difficulty of simple purification methods,
The cost of the product increased accordingly.

In addition, in order to use the separated and purified products as raw materials for foods and medicines, highly safe solvents must be used, but there is no such solvent combination known so far. It is the current situation.

On the other hand, it has been proposed to fractionate phospholipids by silica-based adsorption / partition chromatography using a single phase of a mobile phase solvent such as n-hexane, chloroform, methanol or the like (JP-A-62-120340). reference).

However, this publication discloses that PE and PC can be separated by dissolving a phospholipid in benzene and then flowing a mobile phase solvent (such as methanol). It is difficult to completely remove benzene from PC, and there is a high possibility that a small amount of the benzene remains in the obtained product, which is not suitable as a raw material for food and medicine. Furthermore, it is difficult to reuse a benzene-alcohol solvent in which benzene is mixed with methanol as a mobile phase solvent even if it is purified by a simple distillation method, and a large dedicated facility is required for reuse.

In view of the circumstances described above, the present invention is a method for separating phosphatidylcholine or phosphatidylethanolamine, which is sufficiently safe as a raw material for food and medicine and can efficiently separate only phosphatidylcholine (PC) or phosphatidylethanolamine (PE). The aim is to provide a method.

<Means for Solving the Problems> A method for separating phosphatidylcholine or phosphatidylethanolamine according to the present invention, which achieves the above object, comprises the steps of separating yolk-derived fats and oils containing two or more phospholipids from phosphatidylcholine or phosphatidylethanol by liquid chromatography. In the amine separation method, the mobile solvent for liquid chromatography is a mixed solvent of ethanol and water in the range of 99/1 to 50/50, and the solvent is used without heating.

 Hereinafter, the configuration of the present invention will be described in detail.

Here, the yolk-derived fats and oils containing two or more types of phospholipids (hereinafter, referred to as “phospholipid mixture”) are glycerophospholipids (PC, PE and their lyso forms) and egg yolk phospholipids of sphingolipids (SPM) Refers to egg yolk-derived fats and oils contained in two or more components. When lipids other than egg yolk-derived phospholipids (neutral lipids, etc.) are contained, phospholipids are 20% by weight.
A phospholipid mixture containing the above is desirable. Commercially available products include, for example, “Egg yolk lecithin PL-30 (phospholipid about 30
%, About 70% of neutral lipids)] [trade name; Kewpie Co., Ltd.
"Egg yolk lecithin PL-60 (phospholipid about 75%, neutral lipid about 25%)" [trade name; manufactured by KUPI Corporation], "Egg yolk lecithin PL-100H (phospholipid about 100%)" [ (Trade name; manufactured by KUP Co., Ltd.).

Liquid chromatography, which separates phosphatidylcholine (PC) or phosphatidylethanolamine (PE) from a phospholipid mixture, is a type of column chromatography that uses a column packed with an adsorbent and uses a liquid as the mobile phase. And high-pressure liquid chromatography (HPLC), as well as medium-pressure liquid chromatography and low-pressure liquid chromatography.

As the filler used in the adsorption partition chromatography, it is preferable to use silica gel obtained by three-dimensionally polymerizing monosilicic acid obtained from silicon tetrachloride or sodium silicate. This silica gel is generally called all-porous silica, and one having fine pores having a diameter of 10 to 2000 mm is used. Among them, it is preferable to use one having a diameter of about 30 to 500 mm. Specifically, the particle size is 3 to 50 μm
And porous crushed silica gel having a particle size of 5 to 350 μm.

As a mobile phase solvent for liquid chromatography, a binary mixed solvent of a lower alcohol and water can be used. In particular, when a phospholipid is used as a raw material for pharmaceuticals and foods, a mixed solvent of ethanol and water is preferable, The ethanol / water ratio is preferably in the range of 99/1 to 50/50, more preferably 95/5 to 85/15.

Incidentally, food, if not medicinal materials, C ₄ following a lower alcohol instead of ethanol, for example, can be used methyl alcohol, isopropyl alcohol, and butyl alcohol. At this time, if the concentration of the alcohol falls within the above range, there is no effect even if other components are contained.

As a method for reducing tailing during fractionation, any known method may be used.

Furthermore, these mobile phase solvents are subjected to simple distillation after use, and the concentration is confirmed with a hydrometer or the like, and the alcohol,
By adding water or the like and making appropriate adjustments, it can be easily used many times and cost can be reduced.

As a solvent for dissolving the phospholipid mixture, a new solvent need not be prepared, and a mobile phase solvent used in the liquid chromatography may be used.

<Test Examples> Hereinafter, test examples showing the effects of the present invention will be described.

920 ml of absolute ethanol and 80 ml of distilled water were mixed and degassed to obtain a mobile phase solvent.

As a sample, purified yolk lecithin
Using PL-100H (trade name, manufactured by KUPI Co., Ltd.), dissolve 0.2 g of the egg yolk lecithin PL-100H in a mobile phase solvent to make a total volume of 4 ml, pass through a filter with a pore size of 0.5 μm, and insoluble. Remove the sample and use it as a sample solution (PL-100H5% solution)
And

Using the obtained sample solution, separation was performed by high performance liquid chromatography (HPLC) under the following conditions (see FIG. 1 (a)).

HPLC conditions • Mobile phase solvent: absolute ethanol / water (92: 8 v / v) Flow rate 1 ml / min • Column: YMC-Pack SIL A003 (trade name; Co., Ltd.)
250 × 4.6mmID, silica gel particle size 5μm
Pore diameter 120Å ・ Detector: UV detector Measured at λ = 204nm (AUFS: 1.0) ・ Sample volume: 40μ (absolute amount of lipid: 2mg) ・ Chart speed: 6cm / h The effluent is collected every 2.5 minutes with a fraction collector And analyzed for lipid composition by thin layer chromatography (TLC) for each of the obtained fractions (see FIG. 1 (b)).

TLC conditions TLC plate: silica gel 60 (manufactured by Merck) Developing solution: chloroform / methanol / water (60: 25: 4 v / v / v) As a comparative example, a conventional mobile phase solvent for phospholipid separation (n
-Using hexane / absolute ethanol / water (35: 60: 5 v / v / v), inject 20 μL of the above yolk lecithin PL-100H 10% solution (sample dissolved in the same solvent as the mobile phase solvent) (2 mg absolute lipid) Then, HPLC separation of the phospholipid was performed (see FIG. 2 (a)).

TLC was similarly performed using each of the obtained fractions (see FIG. 2 (b)).

The HPLC chart (FIG. 1 (a)) and the TLC chart (FIG. 1 (b)) obtained in this test example and the H
Comparing the PLC chart (FIG. 2 (a)) and the TLC chart (FIG. 2 (b)), it was found that this test example had a separability higher than that of the comparative example.

Under conventional HPLC conditions, the mobile phase solvent used has many safety problems, and there is a possibility that the solvent will remain in the obtained product, so there is a problem in using it as a raw material for pharmaceuticals and foods However, when ethanol / water was used as the mobile phase solvent as in this test example, the separation ability was equal to or higher than the conventional one, and ethanol / water was used as the solvent. Even if the solvent remains, it can be used as a raw material for pharmaceuticals and foods.

<Embodiment> Hereinafter, a preferred embodiment of the present invention will be described in detail.

Example 1 Using the same sample solution as the test example, PL-100H was 200 m
g was injected, and HPLC separation was performed in the same manner as in the test example (see FIG. 3 (a)).

The effluent was collected every 5 minutes by a fraction collector.

The obtained effluent was subjected to TLC analysis (see FIG. 3 (b)).

In addition, fractions No. 12 to No. 18 as PC fraction components
(Effluent 55 ml to 90 ml) was collected, the solvent was removed by an evaporator, dehydrated by washing with acetone, and then dried to remove
6 mg of white powdery high purity PC was obtained.

In the analysis of the obtained high-purity PC by the TLC-FID method (Iatroscan; manufactured by Yatron Co., Ltd.) under the following conditions, the PC content was 99.6%.

Further, fractions No. 3 to No. 5 (10 to 25 ml of effluent) were collected as PE fraction components, the solvent was removed by an evaporator, dehydrated by washing with acetone, and then dried to obtain 22 mg of a yellow-white mass. Purity PE was obtained.

In the analysis of the obtained high-purity PE by TLC-FID method,
The PE content was 97.2%.

TLC-FID analysis conditions-Rod: Chromarod S-II-Primary: chloroform / methanol / water (70: 30: 3 v / v / v) 8 cm-Secondary: hexane / ether / formic acid (40: 10: 0.1 v / v / v) 10 cm From the above results, ethanol / water (9
2: 8) By using the binary solvent mixture, PC and PE could be efficiently separated and high-purity PC and PE were obtained.

Example 2 A mixture of 8.5 parts of anhydrous ethanol and 1.5 parts of distilled water was used as a mobile phase solvent. The ethanol concentration of the solvent was 86.5% as measured by a spirit analyzer.

Next, 20 g of PL-100H was dissolved in the mobile phase solvent, and HPLC separation was performed under the following conditions by the same operation as in Example 1.

HPLC conditions-Mobile phase: absolute ethanol / water (85:15 v / v)-Flow rate: 20 ml / min-Column: YMC-Pack SIL SH-046-50 (trade name; manufactured by YMC) 1000 x 20 mm ID Silical particle size 50 m Detector: UVλ = 204nm 10.5g of high purity PC was obtained as PC fraction, TLC-FID
Analysis by method revealed a PC content of 99.3%.

As a PE fraction, 1.7 g of high-purity PC was obtained, and TLC-F
Analysis by the ID method revealed that the PE content was 96.5%.

When the mobile phase solvent used for HPLC in this example was recovered by distillation, an ethanol / water mixed solvent of 8.4 was obtained.
The concentration of this recovered solvent was 85.3%
Met.

In this example, the ethanol / water ratio of the mixed solvent was 85
As a result, HPLC of phospholipid was separated as / 15, and high-purity PC and PE were obtained. The mobile phase solvent could also be recovered and used in Example 3.

Example 3 To the recovered solvent 8.4 recovered in Example 2, anhydrous ethanol 1.59 and distilled water 0.15 were added and mixed to obtain a mobile phase solvent. The concentration of the obtained mobile phase solvent was 86.3% as measured by a spirit analyzer.

The same operation as in Example 2 was performed, and 20 g of PL-100H was separated by HPLC.

As a result, as a PC fraction, 10.2 g of high-purity PC (PC content 99.
4%) and 1.8 g of high-purity PE (PE content 96.5
%).

In this example, HPLC analysis was performed using the recovered solvent.
As in Example 2, high-purity PC and high-purity PE were obtained.

<Effects of the Invention> As described above in detail together with the test examples and examples, the present invention is highly safe for the human body such as ethanol / water as a mobile phase solvent when separating a phospholipid mixture to be separated / purified. Since only the solvent is used, the following effects are obtained.

There is no residual harmful component as before, and it is sufficiently safe even when used for food and medicine.

The mobile phase solvent can be easily recovered and reused by simple distillation.

Phosphatidylcholine (PC) or phosphatidylethanolamine (PE) can be separated with high purity despite the combination of highly polar solvents such as ethanol and water.

Low-cost and high-purity phosphatidylcholine (PC) or phosphatidylethanolamine (PE) can be industrially produced.

[Brief description of the drawings]

FIG. 1 (a) is a chromatogram of HPLC according to a test example of the present invention, FIG. 1 (b) is a chromatogram of TLC of the fraction, and FIG. 2 (a) is a chromatogram of HPLC according to a conventional example. 2 (b) is a chromatogram of TLC of the fraction, FIG. 3 (a) is a chromatogram of HPLC according to Example 1 of the present invention, and FIG. 3 (b) is a fraction thereof Obtained
It is a chromatogram of TLC.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Keiji Kitagawa 68-4, Oridai 1-chome, Uji-shi, Kyoto (72) Inventor Hitoshi Narabe 1-25-2 Midoricho, Fuchu-shi, Tokyo 2 (72) Inventor Hideaki Kobayashi 761 Hyakusa, Hino-shi, Tokyo (72) Mineo Hasegawa 1-25-5 Kitanodai, Hachioji-shi, Tokyo (56) References JP-A-57-123196 (JP, A) JP-A-57-123195 (JP, A) JP-A-57-123194 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07F 9/10

Claims (1)

(57) [Claims] 1. A method for separating phosphatidylcholine or phosphatidylethanolamine from a yolk oil or fat containing two or more phospholipids by liquid chromatography, wherein the mobile solvent in liquid chromatography is in the range of 99/1 to 50/50. A method for separating phosphatidylcholine or phosphatidylethanolamine, comprising using a mixed solvent of ethanol and water without heating the solvent.