JPS61172890A - Fractionation of phosphatidyl choline - Google Patents

Abstract

PURPOSE:To enable the fractionation of phosphatidyl choline (PC) in a short time in high reproducibility, and to separate PC completely from other phospholipids, by carrying out the liquid chromatographic fractionation of a PC-containing specimen using an elutant produced by adding a mineral acid to a polar solvent. CONSTITUTION:A specimen containing PC (e.g. PC-60% specimen prepared by the alcohol fractionation of defatted soybean lecithin) is dissolved in a weakly polar or nonpolar solvent such as chloroform, and the solution is introduced into the column for preparative liquid chromatography. The adsorbed component is eluted with an elutant produced by adding a mineral acid to a polar solvent (e.g. a mixture of 50pts.vol. of chloroform, 50pts. of methanol and 2pts. of phosphoric acid). The eluted fractions are examined by UV light of 225nm wavelength, and the PC is separated and recovered from the PC-containing fraction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for fractionating phosphatidylcholine by fractionating a sample containing phosphatidylcholine, such as a natural lipid, by liquid chromatography.

[Conventional technology]

Phospholipids, along with glycolipids and proteins, are major constituents of biological membranes, which perform various functions and are deeply involved in life phenomena. Especially phosphatidylcholine (
A drug delivery system in which an artificial membrane (liposome) is made using PC (hereinafter referred to as PC) and various drugs are placed inside the membrane is attracting attention as a new drug administration method for treating intractable diseases. In addition, PCs are being used for medicines to treat pulmonary diseases such as neonatal respiratory distress, and for artificial heart-lung machines. Recently, it has attracted attention as a therapeutic agent for nervous system disorders, especially since choline in PC is converted to the neurotransmitter acertylcholine by choline acetyltransferase in the brain.

Conventionally, as a method for separating phospholipids from natural lipids, a method has been proposed in which fractionation is performed by liquid chromatography using a silica gel column and lower alkanol as an eluent (for example, Japanese Patent Application Laid-Open No. 123194/1982).
. Furthermore, a method has been proposed in which PC is isolated from egg yolk lecithin by preparative high-pressure liquid chromatography using a chloroform-methanol solvent, which takes 60 to 120 minutes (W, S, M, Geurts et al.
tal, Lipids 16, 58.19
81).

[Problem that the invention seeks to solve]

However, in such a conventional fractionation method using liquid chromatography, PC and other phospholipids are not completely separated, and the injected sample is not entirely eluted.
When preparative separation is repeated, insoluble parts remain at the top of the column, causing the column pressure to rise rapidly and the outflow time to fluctuate significantly. Therefore, in continuous operation, the preparative separation time must be set each time. It becomes necessary. In addition, for raw materials with low PC content such as soybean lecithin, the amount of components that do not elute and the amount of P in the aliquot
There were problems such as changes in C content.

This invention is intended to solve the above-mentioned problems.By performing fractionation using an eluent containing a polar solvent and a mineral acid, PC and other phospholipids can be completely separated, and the insoluble portion can be completely separated. The purpose of the present invention is to propose a preparative method for PC that does not leave any residue, has no fluctuation in outflow time, and can perform fractionation in a short time.

[Means for solving problems]

The present invention relates to a method for separating phosphatidylcholine by fractionating a sample containing phosphatidylcholine by liquid chromatography, which is characterized in that the fractionation is carried out using an eluent in which a mineral acid is added to a polar solvent. be.

The basic skeleton of phospholipids is the same in all molecular types, and the main difference is only in the amino acid bonded to the phosphoric acid at the 3rd position.
It has been found that PC and other phospholipids can be completely separated by using a mineral acid such as phosphoric acid, which is used as a regulator.

In the present invention, the sample to be fractionated is a sample containing PC, and components other than PC may include other phospholipids, oils, proteins, etc.

Examples of such samples include natural or synthetic lipids, natural or synthetic phospholipids such as lecithin, defatted products, or fractionated products with alcohol etc. to increase the PC concentration. Particularly preferred are those that have been degreased or fractionated.

The liquid chromatography used for fractionation is preferably one for fractional separation, particularly one for high pressure, high speed, and large volume fractionation. The column used for chromatography is preferably as large as possible; for example, a 2-inch column (diameter 55
．． 0mm, height 60cm) can be used. The stationary phase packed into the column is silica gel, alumina gel,
Materials commonly used for phospholipid fractionation, such as diatomaceous earth, can be used.

The eluent is a polar solvent with mineral acid added.

As the polar solvent, strong polar solvents used for fractionation of phospholipids, such as methanol and acetonitrile, can be used.

It is also possible to use a mixture of these and a weakly polar or non-polar solvent such as chloroform.

As mineral acids to be added to these, hydrochloric acid, sulfuric acid, phosphoric acid, etc. can be used. The amount of mineral acid added is 5% by volume or less, preferably 0.5 to 4% by volume, based on the entire eluent.

In the fractionation method, a sample is injected into a column, and then the eluent is passed through the column to perform fractionation. The sample is injected after being dissolved in a weakly polar or non-polar solvent such as chloroform. There is no need to change the concentration of the eluent, and fractionation can be carried out by flowing a constant concentration at a constant speed. Other conditions etc. are the same as in the case of general fractionation.

When fractionation is carried out using an eluent prepared by adding a mineral acid to a polar solvent, the fraction that flows out is divided into PC and other phospholipids, and the phospholipids other than PC flow out in bulk at approximately constant times. Therefore, high purity PC can be fractionated by separating only 20 sections. In addition, in the conventional method, phospholipids other than PC were leaked out as each component over a long period of time.
By adding mineral acids, these components flow out in a group in a short time, and the time required for fractionation is significantly shortened. Furthermore, the acidic phospholipid remaining in the conventional column is almost completely drained out. After performing one fractionation operation in this way, the same operation is repeated to fractionate PC.

Since the injected sample flows out almost completely from the column without any residue remaining, there is no increase in column pressure or change in flow time due to the influence of residual substances. Fractionation is performed with good reproducibility at a constant column pressure, and the flow time is The fractionation process can be repeated continuously in one step within 30 minutes, and unmanned operation is also possible.

The remaining phospholipids after fractionating PC can be separated and recovered into each molecular species by reducing the amount of mineral acid added.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

First, fully automated preparative high-performance liquid chromatography
2-inch stainless steel column packed with silica gel of 0 μm or less (column diameter x column length: 55, Omm
60ca+, cross-sectional area 23.7cd, column volume 1425
d) was installed.

The sample is PC, which is alcohol-fractionated defatted soybean lecithin.
-10% of 60% product dissolved in chloroform
The mfl sample loop was used to inject into the column. The amount of sample injected at one time was 2 g. The eluent composition was chloroform/methanol/phosphoric acid (50/50/2
vol/vol/vol), flow rate 20m 11 /
Fractionation was performed by running at min. Detection of phospholipids in the effluent was performed using UV 225 nm.

The obtained chromatogram was clearly divided into a PC section and other phospholipids (PL) sections, as shown in FIG. When the fractionation time was set in the range of 15 to 21 minutes, and one step was set to 30 minutes, 90 continuous operations were performed, and a chromatogram with good reproducibility was obtained. The total amount was 10.8 ff.

Next, the eluent was washed with 1.5 times the amount of purified water, and the emulsification was broken by centrifugation. This was repeated three times to obtain a chloroform layer of 4.5 times the volume, which was dehydrated using a column packed with sodium sulfate. Ion exchange resin Amberlite I RA-900
(Rohm and Haas Co., Ltd. trademark) column to remove residual phosphoric acid. The solvent was removed from this solution using an evaporator to obtain PC.

The obtained PC was subjected to analytical high performance liquid chromatography (Toyo Soda HL C-803D) using a column: 5ilca
60 (4, foam X 25cm), flow rate: 1.
Om Q /win, eluent niacetonitrile/methanol/phosphoric acid (900/9515, vol/vol/v
ol), detection: UV210nm (RangeX 1.
The chromatogram obtained by fractionation in step 25) is shown in FIG. 2, and 85 g (79% of the theoretical yield) of phospholipids with a PC purity of 98% or more was obtained. The purity of the fractionated PC was determined from the amount of choline using the periodic acid method, and the purity of the PC was also over 98%. The resulting material was brown, translucent, elastic and slightly plastic.

In the above treatment, when the amount of phosphoric acid was varied by 5% by volume or less, the outflow time was shortened according to the amount of phosphoric acid added, but a chromatogram similar to that in FIG. 1 was obtained in both cases. Furthermore, similar trends were observed when hydrochloric acid and sulfuric acid were used instead of phosphoric acid, although there were differences in the amounts added, and similar trends were observed when acetonitrile was used instead of methanol. In the above tests, the raw material injection amount was varied from 0.2 g to 4 g, but the same results were obtained in each case.

For comparison, we conducted a similar test using an eluent without the addition of mineral acids, and found that the history of the column is very problematic for silica-based columns (normal phase), and even if the columns are of the same type, the history may be different. In some cases, the elution pattern was different or PL did not elute.

Even with n-hexane/isopropanol-based and chloroform/methanol-based eluents, which are generally known for phospholipid analysis, when a new column is used, the PC peak position gradually moves forward, causing the column to become accustomed to the eluent. (The number of days for phospholipid measurement)

Based on the above results, the following are the conditions for preparative separation of phospholipids.

Mineral acid was required, and the chromatograms were not reproducible in the absence of mineral acid. The PL remaining in the column in which the injected raw material did not all elute or in the column overloaded with the sample was completely eluted with the eluent containing these mineral acids.

〔Effect of the invention〕

According to the present invention, since fractionation is performed using an eluent obtained by adding a mineral acid to a polar solvent, PC and other phospholipids are completely separated, there is no remaining insoluble part, and the flow time is variable. In addition, fractionation can be carried out in a short period of time with good reproducibility, and as a result, highly pure PC can be selectively collected in large quantities in a short period of time.

[Brief explanation of drawings]

FIG. 1 is a chromatogram of the effluent of the example, and FIG. 2 is a chromatogram of 20 sections thereof. Agent Patent Attorney Naruzu Yanagihara) Gin1'/I

Claims (4)

[Claims] (1) A method for separating phosphatidylcholine by fractionating a sample containing phosphatidylcholine by liquid chromatography, which is characterized in that the fractionation is carried out using an eluent in which a mineral acid is added to a polar solvent. (2) The preparative separation method according to claim 1, wherein the polar solvent is methanol or acetonitrile. (3) The preparative separation method according to claim 1 or 2, wherein the mineral acid is hydrochloric acid, sulfuric acid, or phosphoric acid. (4) The preparative separation method according to any one of claims 1 to 3, wherein the amount of mineral acid added is 5% by volume or less of the entire eluent.