JPH01233290A - Production of lysolecithin - Google Patents

Abstract

PURPOSE:To directly and rapidly obtain lysolecithin useful as a carcinostatic agent, modifier for ribosomes, etc., from lecithin in high yield with a good accuracy, by treating a lysolecithin-containing lecithin using a centrifugal liquid- liquid multistage fractionator. CONSTITUTION:Natural and synthetic lysolecithin-containing lecithin as a raw material is treated in a centrifugal liquid-liquid continuous multistage fractionator (hereinafter referred to as CPC) to separate and collect lysolecithin. Furthermore, natural lecithin concentrate or synthetic lecithin subjected to regiospecific hydrolysis treatment with snake venom phospholipase A2, etc., have a high lysolecithin content and are preferred as the raw material lecithin. The eluate of the CPC is preferably prepared by mixing and stirring a hydrophobic solvent, such as hexane, with a hydrophilic solvent, such as acetonitrile, and partitioning the mixture into two phases, and further using the respective phases as liquids for a stationary and mobile phases of the CPC.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel method for directly separating lysolecithin from natural and synthetic lecithin raw materials containing lysolecithin.

(Prior Art) Lysolecithin is obtained by removing one fatty acid from lecithin, and is known to modify the conformation of cell membranes in vivo. In particular, lysis on red blood cell membranes has such a strong physiological effect that it causes hemolysis. Lysolecithin is widely distributed in the phospholipids that constitute the cell membranes of plants and animals in nature. The content of lysolecithin in industrial phospholipid raw materials is 1 to 2 for soybean lecithin and egg yolk lecithin.
%, and even after degreasing, the amount is only around 3%.

Therefore, in order to obtain lysolecithin, it is difficult to achieve high purity, although it can be concentrated by solvent fractionation. Thin layer chromatography and column fractionation methods have been applied in the field of lipid chemistry to achieve high purity.

(Problem to be solved by the invention) Currently commercially available phospholipids as raw materials for the purpose of fractionation of lysolecithin have a low content, and it is not preferable to use them as they are from the standpoint of separation and purification. It is necessary to increase the lysolecithin content.

In purification by thin layer chromatography, even if a preparative plate is used, the amount that can be charged at one time is several tens of cubic meters.
The amount of lysolecithin that can be separated is only a few micrometers. For purification by column fractionation, open column method, medium pressure column method, high performance liquid chromatography, etc. are used. In both of these methods, the fractionation of lysolecithin in the coexistence of lecithin takes a very long time to elute with the same solvent, and it is difficult to fractionate with a solvent system that shortens the elution time or increases the throughput. Lysolecithin content is significantly reduced. In pressurized elution, the upstream time is apparently shortened, but since there is no change in retention capacity, the amount of solvent relative to the amount to be fractionated is extremely large. Therefore, in order to increase the fractionation amount, stepwise gradient elution is required. As a result, there are problems such as complicated solvent settings, a large amount of elution solvent, and an extremely long elution time.

(Means for Solving the Problems) The present invention is characterized in that natural and synthetic lecithins containing lysolecithin are used as raw materials, and lysolecithin is fractionated using a centrifugal liquid-liquid multi-stage fractionation device.

The present inventors conducted a detailed study on the composition of natural phospholipids that have been conventionally used as raw materials for lysolecithin. As a result, commercially available soybean lecithin and egg yolk lecithin contain 1 to 2% lysolecithin, and when defatted with acetone, 3%
It turned out that it was around %. It was also found that lysolecithin behaves similarly to lecithin in solvent fractionation and column fractionation, and the content of lysolecithin increases as lecithin is concentrated. For example, if the lecithin content is concentrated to 70% or more, the lysolecithin will be concentrated to 5 to 7%, so it is preferable as a raw material than commercially available products.

Furthermore, in order to increase the lysolecithin content in the raw materials, the natural lecithin concentrate and synthetic lecithin described above were partially hydrolyzed. Hydrolysis was performed by position-specific hydrolysis using snake venom phospholipase A2 and alcoholysis using low concentrations of tetrabutylammonium hydroxide or alkali metal salts. Lecithin, lysolecithin,
Fatty acids and glycerophosphocholine were observed. After extracting these mixtures with chloroform, a mixture of lysolecithin and lecithin is obtained by treatment with cold acetone. Since 30 to 70% lysolecithin is contained, snake venom phospholipase A2 treatment is more preferable from the viewpoint of lysolecithin content.

Furthermore, large amounts of lysolecithin often appear in intermediate reaction products during the production of synthetic lecithin. Lecithin synthesis is carried out by reacting glycerophosphocholine, which is a desacyl phosphoric acid, with fatty acid anhydrides or halides in a highly polar solvent in the presence of a basic esterification catalyst. At this time, if the amount of fatty acid derivative used for glycerophosphocholine is half of that used for lecithin synthesis, lysolecithin is produced. In this reaction mixture, lecithin, lysolecithin, fatty acids, glycerophosphocholine, and a complex of the basic catalyst and unreacted substances were observed. When these mixtures are treated with silica and cold acetone, a mixture of lysolecithin and lecithin is obtained. Since lysolecithin is contained in an amount of 15 to 60%, it is preferable as a raw material for lysolecithin.

The raw material lecithin used in the present invention preferably contains 4% or more of lysolecithin, and the various raw material lecithins described above can be used. If the lysolecithin content is less than 4%, it will be difficult to obtain a high concentration of lysolecithin in the subsequent centrifugal liquid continuous multi-stage fractionation process.

In the present invention, in order to precisely separate lysolecithin from partially decomposed raw material lecithin without using conventional packed columns, a centrifugal liquid-liquid continuous multi-stage fractionator (hereinafter referred to as CPC) is used.
was used.

As shown in Figure 4, in CPC, one of the two phase separated liquids is held as a stationary phase by centrifugal force, while the other is continuously passed through the stationary phase as a mobile phase. It is a countercurrent partition chromatography that continuously fractionates the injected sample. That is, CPC has different specific gravity and polarity, and is separated into two phases.One of the two solvents is used as a stationary phase and the other as a mobile phase, and the mobile phase is moved through the stationary phase by the action of centrifugal acceleration. Components are chromatographically fractionated by multistage partition equilibrium using differences in partition coefficients. Therefore, since CPC does not use silica gel as a column packing, it solves the problem of change in retention capacity due to lecithin separation for application to industrial production.

Hydrophobic solvents used in the present invention include hexane, heptane, ether, petroleum ether, chloroform, etc., and hydrophilic solvents include ethanol, methanol, hydrous alcohol,
Acetonitrile, acetic acid, and phosphate buffers are used, and addition of a metal complex is particularly preferred in terms of separation performance and yield. The eluent is prepared by premixing and stirring the hydrophobic and hydrophilic solvents containing the various solvents and additives mentioned above, and then dividing the mixture into two phases.The lower layer liquid is used as the stationary phase liquid and the upper layer liquid is used as the mobile phase liquid, or vice versa. It was used in combination.

In a CPC, a large number of cartridges (hereinafter referred to as distribution pipes) are arranged in a circumferential manner on a rotor, and the distribution pipes are connected in series with each other using tubes. The distribution pipe is made by forming thin grooves in a zigzag pattern on a fluorine-based resin plate, and stacking several resin plates to form one distribution pipe. After filling the distribution pipe with the stationary phase liquid using a pump, the mobile phase liquid is continuously pumped while rotating the rotor and applying a constant centrifugal force. The stationary phase liquid is held in the distribution pipe by centrifugal force, and the mobile phase liquid passes through the pipe continuously in the form of fine droplets, thereby performing multistage continuous liquid-liquid distribution extraction. The eluate is checked using IATROScan (TLC/FID) and fractionation is performed using a fraction collector. In addition,
After the feeding of the mobile phase liquid is completed, by reversing the liquid feeding direction and pushing out the stationary phase liquid from the inlet side, the components retained in the stationary phase liquid can be fractionated.

(Effects of the Invention) The present invention allows lysolecithin to be directly separated from synthetic and natural lecithins with high accuracy, speed, and high yield using CPC. This lysolecithin can be used as an anticancer drug or a liposome modifier by utilizing its action of modifying the conformation of cell membranes.

Furthermore, lysolecithin can be used as a choline supply agent to the brain, and since it activates peritoneal macrophages, it can also be used as a non-specific immune-enhancing agent.

(Examples) Hereinafter, the present invention will be specifically described based on Examples. still,
% in the examples indicates weight %.

Example 1 Commercially available purified soybean lecithin was defatted by cold acetone treatment. The obtained defatted soybean lecithin was subjected to solvent fractionation using ethanol and magnesium sulfate treatment to remove phospholipids other than lecithin. The obtained ethanol solution in which the soybean lecithin concentrate was dissolved was made into a thin film using a large round-bottomed evaporator, and the solvent was removed to prepare a raw material phospholipid.

The composition of the above-mentioned crude phospholipid was measured by the Yatroscan method (manufactured by Yatron) using a developing solution composition of loloform/methanol/water (65/25/4). Composition: 71% lecithin
, 6% for lysolecithin, and 24% for others.

2 g of the above phospholipid was used as a raw material. A distributed solution was prepared by mixing equal amounts of n-hexane and acetonitrile and allowing the mixture to stand until separated into two layers. The sample was dissolved in 5M of the lower layer liquid and filled into a sample tube connected to the main body. Next, the lower layer liquid of the distribution liquid prepared in advance was subjected to CPC (CPC-L B9
2-N type, manufactured by Miki Engineering ■) distribution pipe was filled with the solution. After sending the sample solution to the distribution tube, move the rotor to the 160°
Or, p, m.

While rotating the lower layer liquid at a flow rate of 5 m 11 minutes,
ml was sent overnight, and fractionated at every 100 mf using a fraction collector.

The fractionated eluate was checked with the aforementioned IATROScan, and the lysolecithin eluted portion was fractionated. The contents of lecithin and lysolecithin in the 7th to 16th fractions of the obtained lower eluate fraction are shown in FIG. The yield of the lower eluate from the 7th fraction to the 9th fraction was 0.1 g, and the yield from the 1st O fraction to the 16th fraction was 0.7 g.

Next, upper layer inversion elution was performed to separate the lysolecithin retained in the distribution tube. That is, the rotor is 160O
While rotating at r, plm, the upper layer liquid was fed in the opposite direction from the outlet side of the distribution pipe at a flow rate of 5 ml and 11 minutes at 10,100 O, and fractionated every 100 ml using a fraction collector.

FIG. 2 shows the contents of lecithin and lysolecithin in the 1st to 10th fractions of the upper elution fraction obtained by reverse elution. The yield from the second fraction of the upper eluate was 0.1 g, the yield from the third to tenth fractions was 0.9 g, and the lysolecithin content of the previous fraction of the upper eluate was 95%. . The yield from the lower eluted fraction and the upper eluted fraction is a recovery rate of 9.
It was 0%.

Example 2 6.0g (23.6mM) of palmitic acid was added with 9.8g (46.7mM) of trifluoroacetic anhydride,
The mixture was stirred at ℃ for 1 hour. Under a stream of nitrogen gas, gradually heat while stirring, and after the temperature reaches 85°C, heat for 5 to 50 mmH.
Trifluoroacetic anhydride was removed with g. 9120ml of dimethylsulfochloride was added to the total amount of palmitic anhydride obtained. 2.0 g of glycerophosphocholine anhydride (7.8 mM) and 1.6 g of N,N-dimethyl-4-aminopyridine (13
, 2mM) and stirred vigorously at 80°C for 2 hours.

After standing at room temperature, 200 d of dehydrated acetone was added and stirred to produce a white precipitate. Place this in the freezer at -20°C.
After standing for a period of time, centrifugation was performed for 10 minutes at a rotation speed of 300 Orr, p, m. Add 100ml of dehydrated acetone to the collected precipitate.
was added and centrifuged twice. Furthermore, the precipitate was repeatedly washed with dehydrated acetone and the solvent was removed under reduced pressure. The supernatant liquid was also operated in the same manner as above.

2.7 g of the obtained white precipitate was dissolved in chloroform/methanol (1/1). The solution was passed through a glass tube (2 cm φ x 3 cm) filled with silica gel, and then the solvent was removed. The composition of 2.5 g of the obtained crude phospholipid was determined by the Iatroscan method described above. The composition was 73% lecithin, 19% lysolecithin, and 8% others.

2 g of the above phospholipid was used as a raw material. The distribution liquid was mixed with chloroform/acetic acid/methanol/water (2/1/1/2),
The mixture was prepared by allowing it to stand until it separated into two layers.

The sample was dissolved in 5M upper layer liquid and filled into a sample tube connected to the main body. Next, the upper layer liquid of the distribution liquid prepared in advance was filled into the distribution tube of a CPC (CPC-L, B92-N type, manufactured by Miki Engineering (manufactured by Ibu).The upper layer liquid was also poured into the distribution tube to serve as the injection of the sample solution. After sending 2000 ml at a liquid sending rate of 2.5 ml and 11 minutes, 1000 mff of the lower layer liquid was sent to perform lower layer inversion elution.Each eluate was fractionated into 10 to 100 ml portions depending on the elution condition.The rotor at this time The rotation speed was 1500 r, p, m.

The fractionated eluate was checked with the aforementioned IATROScan, and the lysolecithin eluate was fractionated. In the upper N elution, a lysolecithin enriched section was observed in a 600 ml section from elution ff1540d, and the yield recovered from this section was 0.3 g. The contents of lecithin and lysolecithin in this section are shown in Figure 3.

The yield from the upper eluate eluted after this interval is 0.1
g, the yield from the lower eluate was 1.4 g, and the overall recovery rate was 90%.

The lysolecithin content of the pre-upper elution fraction was 89%.

[Brief explanation of the drawing]

Figure 1 is a composition diagram of each division in the lower layer elution of enzymatically decomposed crude phospholipid CPC, Figure 2 is a composition diagram of each division in upper layer inversion elution, and Figure 3 is a composition diagram of CPC of chemically synthesized crude phospholipid. FIG. 4 is a diagram showing the composition of the pre-eluting fraction in the upper layer, and FIG. 4 is a circular map showing CPC. Figure 1: Numakawa t1ml Figure 2: Elution volume: 1ml Figure 3: Elution volume (ml) Figure 4: 7q-

Claims (1)

[Claims] A method for producing lysolecithin, which comprises using natural and synthetic lecithin containing lysolecithin as raw materials and separating lysolecithin using a centrifugal liquid-liquid multi-stage fractionator.