JPS6239593A - Rroduction of phospholipid containing substantially no cholesterol - Google Patents

Abstract

PURPOSE:To readily obtain the titled phospholipid useful for such as an emulsifier for midicine, by dissolving a purified phospholipid containing cholesterol in a non-polar solvent, containing the resulting solution with a silica gel and removing the silica gell and the solvent. CONSTITUTION:A purified phospholipid containing cholesterol [preferably one compound of <= about 5wt% cholesterol, <= about 5wt% triglyceride and >=90wt% phospholipid] is dissolved in a non-polar solvent (example; n-hexane, etc.) at the concentration of preferably a 0.1-30wt%. Then silica gel is added thereinto and dispersed while stirring to adsorb the cholesterol, then the silica gel is removed by, for example, filtration and the solvent is distilled off under reduced pressure to obtain the aimed phospholipid usually containing <= about 0.1wt% cholesterol. The amount of silica gel to be used is more than 5 times that of the cholesterol contained therein.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel method for producing phospholipids that are substantially free of cholesterol.

[Conventional technology]

Purified phospholipids have traditionally been used particularly in the pharmaceutical field as pharmaceutical emulsifiers and liposome forming agents. Purified phospholipids are made from ingredients other than phospholipids, such as these, from a purposeful standpoint for such use.

It is desirable that the content of sterols and the like be as low as possible.

Purified phospholipids have conventionally been obtained by purification methods using solvents, such as treating crude phospholipids with acetone to remove neutral lipids.

[Problem that the invention seeks to solve]

However, in this J:Una solvent purification method, although the operation was simple, the purified phospholipid obtained had a high residual amount of cholesterol of about 0.5 to 5% (in the present invention, % is (All figures are in percent between cars). To remove the cholesterol contained in phospholipids, for example, column chromatography or liquid chromatography can be used and can be expected to be somewhat effective, but such methods require expensive equipment and materials. up to 11
This results in higher product costs.

Therefore, an object of the present invention is to provide a novel method for producing phospholipids that is simple and substantially free of cholesterol, which can expand the use of phospholipids in the medical field.

[Means for solving problems]

As a result of extensive research in accordance with the above objective, the present inventors have found that if a cholesterol-containing phospholipid that has already been purified by a conventional method is dissolved in a nonpolar solvent and then brought into contact with silica gel, this silica gel will become phosphorous. We have discovered that only cholesterol can be selectively adsorbed and removed without changing the lipid composition.

Conventionally, as a purification method using a combination of a nonpolar solvent and silica gel, there has been known a method for removing unpleasant odors from crude egg lipid using this combination method, for example, as disclosed in Japanese Patent Application Laid-open No. 12304/1983. The crude egg lipid that is the target of purification contains neutral lipids in many colors (fats and oils 62
%, sterins: 5%, phospholipids: 33%).Therefore, when this product is purified using the above method, neutral lipids such as triglycerides are preferentially adsorbed and removed, although the unpleasant odor is reduced. This method could hardly be expected to remove cholesterol. As described above, the conventional purification method using a combination of a nonpolar solvent and silica gel did not suggest anything about the usefulness of this method for removing cholesterol.

In developing a new method for producing phospholipids that are virtually free of cholesterol, the present inventors conducted 12 rapid studies on the possibility of using the above-mentioned purification method in combination with a non-polar solvent and silica gel, and found that they were able to ) As mentioned above, we have completed the present invention by discovering that if a3 < is obtained by once purifying according to the conventional method, cholesterol in the purified phospholipid can be almost completely removed by this combined purification method.

The present invention involves dissolving purified phospholipids containing cholesterol in a non-polar solvent, contacting the resulting solution with silica gel, removing the silica gel, and then distilling off the non-polar solvent from this solution. The present invention provides a method for producing phospholipids that are substantially free of cholesterol.

The raw material phospholipid to which the method of the present invention is applied is a phospholipid purified by any conventional purification method and contains cholesterol.

Since purified phospholipids obtained by conventional purification methods usually have a residual amount of cholesterol of about 0.5 to 5%, it can be said that the purified phospholipid raw material used in the method of the present invention has a cholesterol content of about 5% or less. The purified phosphorus ffi raw material used in the method of the present invention also has a triglyceride content of approximately 5%, since it is difficult to obtain the cholesterol removal effect of the method of the present invention when the triglyceride content is high, as is clear from the results of the test examples described below. Therefore, it is preferable that the phospholipid is purified to about 90% or more.

Such purified phospholipids include, for example, egg yolk, animal brain tissue, fish eggs, etc., ethanol, dichloromethane, n
- Examples include those obtained by extracting with a solvent such as hexane or ether (crude phospholipids) and further treating with acetone to remove neutral lipids.

The solvent for purified phospholipid used in the method of the invention is a nonpolar solvent, such as n-pentane, n-hexane, n-
Mention may be made of C to C1o saturated hydrocarbons such as hebutane. (2) As is clear from the results of the water test, polar solvents such as ethanol and dichloromethane have a negligible cholesterol removal effect. The concentration of the purified phospholipid in the nonpolar solvent, ie, the concentration of the purified phospholipid in the resulting solution, is preferably about 0.1 to 30%.

If it is less than 0.1%, the amount of solvent is too large and is not economical, and if it exceeds 30%, the viscosity increases, making purification operations difficult.

The silica gel used in the method of the present invention is not particularly limited, and for example, commercially available silica gel for thin layer chromatography or column chromatography may be used as is, or it may be heated for 30 minutes at 80 to 130°C.
It may be activated for about minutes to 3 hours. The amount of silica gel to be used is usually at least 5 times the amount of remaining cholesterol. If the amount used is 5 times less than that of the mother, sufficient cholesterol removal effect will be obtained.

Ii [Q The contact between the lipid solution and the silica gel is usually carried out by adding a predetermined M of silica gel to the solution and suspending it under stirring. This contact is preferably carried out at a temperature lower than the boiling point of the solvent in order to prevent evaporation of the solvent.

According to the method of the invention, after the selective adsorption of cholesterol onto the silica gel by the above-mentioned contact, the silica gel is removed, e.g. by filtration, and the non-polar solvent used is then distilled off from the solution, e.g. under reduced pressure. .

The phospholipids obtained by the method of the present invention usually have a cholesterol content of about 0.1% or less! It is a phospholipid, and therefore it can be expected to expand its use in various fields, especially in the pharmaceutical field.

〔Effect of the invention〕

The effectiveness of the method of the invention is illustrated by the results of the following test examples.

Grave presentation 1”- This test example uses cholesterol according to the method of the present invention.

It shows how the removal effect differs depending on the raw material phospholipid applied.

Raw material phospholipids: (1) Purified egg yolk phospholipid A (triglyceride 1.0%, cholesterol 2.0%,
(phospholipid 96.0%) (2) Purified egg yolk phospholipid B (triglyceride 5.0%, cholesterol 5.0%,
phospholipid 90%) (3) Crude egg yolk phospholipid A (triglyceride 16%, cholesterol 6.8%, phospholipid 75.0%) (4) Crude egg yolk phospholipid B (triglyceride 65%, cholesterol 4.4%, (phospholipid 30.0%) For each of the above raw material phospholipids, dissolve 15 g of raw material phospholipid in 100% of n-hexane, add 7.5 g of commercially available silica gel for column chromatography to the resulting solution. After stirring at room temperature for 30 minutes, the silica gel was separated from the furnace.
The n-hexane was completely distilled off from the solution under reduced pressure to obtain the final phospholipid sample.

Next, each sample was examined for cholesterol residue using Iatroski V-Kin TH-10 (manufactured by Yatron). The results are shown in Table 1 below.

From the above results, it can be seen that unless the phospholipid raw material is purified to a triglyceride content of about 5% or less, it is difficult to achieve the cholesterol removal effect.

Test Example 2 This test example shows how the cholesterol removal effect of the method of the present invention varies depending on the type of solvent used.

15 U of purified egg yolk phospholipid (triglyceride 1.0%, cholesterol 2.0%, phospholipid 96.0%) was added to 100 ml each of n-pentane, n-hexane, dichloromethane and ethanol! 15 g of commercially available a-layer Roma 1 to silica gel for graphics was dissolved in each of the solutions obtained.
After each solution was added and stirred at room temperature for 30 minutes, the silica gel was separated in a furnace, and the solvent used in each solution was completely distilled off under reduced pressure to obtain a final phospholipid sample.

Each sample was then examined for cholesterol residue using an Iatroscan TH-10. The results are shown in Table 2 below.

Table 2 From the above results, it can be seen that only the nonpolar solvent has a greater cholesterol removal effect.

〔Example〕

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

Example 1 120y of purified egg yolk phospholipid (triglyceride 0.1%, cholesterol 1.8%, phospholipid 98.0%) was dissolved in 800ml of n-hexane, and commercially available a
Add 12g of silica gel for layered Roma 1-graphy and V
After stirring for 30 minutes under
Completely distill n-hexane from the solution under reduced pressure to obtain purified egg yolk phospholipid 115j with a cholesterol content of 0.1% or less.
(!V.

Example 2 150 g of purified egg yolk phospholipid (triglyceride 0.5%, cholesterol 2.0%, phospholipid 97.0%) was dissolved in 750 d of n-hebutane, and in the resulting solution, commercially available column chromatography grade Silica gel (pre-heated at 130°C)
After adding 303 (time-activated product) and stirring at room temperature for 1 hour, the silica gel was separated from the solution to n-
Hebutane was completely distilled off under reduced pressure to obtain 143 g of purified egg yolk phospholipid having a cholesterol content of 0.1% or less.

Example 3 Crude phospholipids selectively extracted from cow brain were treated with azetone to make purified phospholipids (triglyceride 0.1%, cholesterol 2.0%, phospholipids 95.0%) 1007
- Dissolved in 1000d of hexane, added 50y of silica gel for column chromatography to the resulting solution, stirred at room temperature for 30 minutes, and then dissolved the silica gel? going door to door,
N-hexane was completely distilled off from the solution under reduced pressure to obtain 90 purified A phospholipids having a cholesterol content of 10.1% or less.

Claims (1)

[Claims] Cholesterol, characterized in that a purified phospholipid containing cholesterol is dissolved in a non-polar solvent, the resulting solution is brought into contact with silica gel, the silica gel is removed, and then the non-polar solvent is distilled off from this solution. A method for producing phospholipids that are substantially free of phospholipids.