JPH08311086A - Production of phospholipid mixture - Google Patents

Abstract

PURPOSE: To provide a process for producing an aqueous dispersion of a phospholipid mixture having uniform composition and particle size distribution and provide a process for producing a powdery phospholipid mixture having uniform composition and excellent dispersibility in water. CONSTITUTION: This process for the production of an aqueous dispersion of a phospholipid mixture comprises (a) the step of forming a solution by dissolving at least two kinds of phospholipids or a phospholipid and a lipid other than phospholipid in a water-miscible solvent, (b) the step of mixing the solution obtained by the step (a) with water to form a dispersion and (c) the step of obtaining an aqueous dispersion by removing the water-miscible solvent from the dispersion obtained by the step (b). A powdery phospholipid mixture can be produced by freeze-drying the aqueous dispersion of the phospholipid mixture produced by the above process to remove water from the dispersion.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a phospholipid mixture, and more particularly to a method for producing an aqueous dispersion and powder of a phospholipid mixture.

[0002]

The following methods (1) to (3) are available for obtaining a phospholipid mixture comprising at least two phospholipids or phospholipids and lipids other than phospholipids. (1) A method in which at least two kinds of phospholipids or phospholipids and simple lipids are dissolved in an organic solvent and then the solvent is removed under reduced pressure to form a thin film (J. Mol. Bio., No. 1).
3 pp. 238 1965). (2) An organic solvent solution of a lipid mixture is fed into a tubular heater at a constant rate, heated to evaporate the organic solvent, and this mixture is introduced into a vacuum chamber at a high speed to instantaneously volatilize the organic solvent. A method of drying the solid content to obtain a powder (Japanese Patent Laid-Open No. 2-167218). (3) At least two kinds of phospholipids or phospholipids and lipids other than phospholipids are dissolved in a water-immiscible solvent to prepare an oil-in-water dispersion, and then the solvent is evaporated together with a part of water. Method (Japanese Patent Laid-Open No. 3-207443).

[0003]

However, in the method (1), even if water is added to the obtained thin film of the phospholipid mixture,
It is difficult to hydrate and it is difficult to obtain an aqueous dispersion of the phospholipid mixture. Moreover, because it is prepared in a test tube scale,
Not suitable for industrial production. Moreover, in the method of (2),
Although this is an industrial mass production method, heat is applied when the lipid is dried, which deteriorates the lipid and impairs the stability of the product. Furthermore, in the method (3), molecules are rearranged during preparation of an oil-in-water dispersion of a phospholipid mixture,
The homogeneity of the molecule may be impaired, and lipids other than phospholipid may float on the water surface, which limits the preparation conditions and results in poor reproducibility.

In view of such circumstances, the present invention provides a method for producing an aqueous dispersion of a phospholipid mixture in which the composition of the phospholipid mixture is uniform and the particle size distribution is uniform, and the composition is uniform. And a method for producing a powder of a phospholipid mixture having excellent dispersibility in water.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies in order to achieve the above object, and as a result, at least two kinds of phospholipids or phospholipids and lipids other than phospholipids were used as water-miscible solvents. After dissolving and mixing this solution with water to obtain a dispersion, the water-miscible solvent is removed to obtain an aqueous dispersion of a phospholipid mixture having a uniform composition and a uniform particle size distribution. It was also found that by freeze-drying this water dispersion to remove water, a powder of a phospholipid mixture having a uniform composition and excellent dispersibility in water was obtained.
The present invention has been completed.

That is, the first aspect of the present invention is: a) a step of dissolving at least two kinds of phospholipids or a phospholipid and a lipid other than phospholipid in a water-miscible solvent to form a solution, and b) obtained in the step a. Water of the phospholipid mixture, comprising the steps of mixing the obtained solution with water to form a dispersion, c) removing the water-miscible solvent from the dispersion obtained in step b above to form an aqueous dispersion The present invention relates to a method for manufacturing a dispersion liquid. A second aspect of the present invention relates to a method for producing a powder of a phospholipid mixture by removing water from an aqueous dispersion of the phospholipid mixture produced by the above method by freeze-drying.

[0007]

DETAILED DESCRIPTION OF THE INVENTION Phospholipids used in the present invention include phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol. Le,
Phospholipid represented by phosphatidic acid, egg yolk,
Examples thereof include those derived from soybean and other natural materials, hydrogenated products thereof, and those obtained by synthesis.

Specifically, soybean phosphatidylcholine,
Egg yolk phosphatidyl choline, hydrogenated soy phosphatidyl choline, hydrogenated egg yolk phosphatidyl choline, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, egg yolk sphingomyelin, soy phosphine Atidyl inositol, dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanol amine, distearoyl phosphatidy ethanol amine,
Dimyristoyl phosphatidyl serine, dipalmitoyl phosphatidyl serine, distearoyl phosphatidyl serine, dimyristoyl phosphatidyl glycerol,
Dipalmitoyl phosphatidyl glycerol, distearoyl phosphatidyl glycerol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, distearoyl phosphatidyl inositol, dimyristoyl Phosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, soybean lysophosphatidylcholine, egg yolk lysophosphatidylcholine, and linoleic acid, linolenic acid, acrylic acid, methacryl as an acyl group. Also included are reactive phospholipids having a polyoxyethylene group, maleimide or the like bonded to the acyl group of a polymerizable phosphatidylcholine or phosphatidylethanolamine having a residue of an acid or vinylphenylcarboxylic acid.

The lipids other than the phospholipids used in the present invention include sterols such as cholesterol and cholesterol.
And triglycerides of natural or synthetic origin, wax ester, sphingosine, ceramide, long chain fatty acid, long chain alcohol, long chain aliphatic amine, vitamin A,
Examples include fat-soluble vitamins such as vitamin D, vitamin E, and vitamin K, monoglycosyl diglyceride, diglycosyl diglyceride, hematoside, ganglioside, and the like.

In the present invention, the phospholipid mixture is a mixture of at least two kinds of phospholipids alone or a combination of one or more kinds of phospholipids and lipids other than phospholipids. The mixing ratio of the phospholipids can be any ratio. The mixing ratio of the phospholipid and the lipid other than the phospholipid can be appropriately determined depending on the application, but in general, the former /
The molar ratio of the latter is preferably 1: 0.01 to 2.

In the step a in the present invention, first, the above phospholipid mixture is dissolved in a water-miscible solvent to obtain a solution.
The solvent used here may be any solvent that is miscible with water and can be volatilized under reduced pressure. Specifically, tetrahydrofuran, dioxane, acetonitrile, dimethoxyethane,
Methanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butanol, t-butanol
And the like, and may be used alone or in combination of two or more kinds. If a water-immiscible solvent, such as chloroform, methylene chloride, or toluene, is used instead of these water-miscible solvents, the resulting phospholipid mixture will not be uniform in water, and therefore used in the present invention. Can not do it.

[0012] Such a water-miscible solvent can be used without particular limitation as long as it completely dissolves the phospholipid mixture, but generally 1 to 1 per 1 g of the phospholipid mixture.
It is appropriate that the amount used is about 0 g. The temperature at which the phospholipid mixture is dissolved is not particularly limited, but it is preferable to stir or shake under heating at 20 to 80 ° C. to promote dissolution.

In the step b in the present invention, the solution obtained in the step a is mixed with water to obtain a dispersion liquid. The amount of water may be such that the solution becomes cloudy, and preferably the solvent concentration is 2 to 20% by weight in the mixed liquid of water and the solvent. If the amount of water is small, bubbles may be generated in the solvent removal step under reduced pressure, and the solvent may not be removed. On the other hand, if the amount of water is too large, a large amount of water, which will be finally removed during powder production, is used, resulting in poor economic efficiency.

As a method of mixing with water, water may be added to the solution of the phospholipid mixture, or the solution of the phospholipid mixture may be added to the water. The former is preferable because a dispersion having a more uniform particle size distribution can be obtained. The mixing conditions with water are not particularly limited, but it is preferable to carry out at a temperature of 20 to 80 ° C. with stirring or shaking.

In the step c of the present invention, the water-miscible solvent is removed from the dispersion obtained in the step b to obtain an aqueous dispersion. The removal of the solvent is basically performed under a pressure at which the solvent vaporizes, and it is suitable to reduce the pressure to a range of 300 to 50 mmHg. 30-70 degreeC is suitable for the temperature at the time of solvent removal. If the temperature is too high, the phospholipids are likely to deteriorate.
The solvent is removed under reduced pressure until no solvent is detected in the dispersion liquid.

In the production of such an aqueous dispersion, water may be removed under reduced pressure subsequent to the removal of the solvent under reduced pressure for the purpose of concentrating the phospholipid mixture. Alternatively, water may be added to dilute the solution to an appropriate concentration. The final concentration of the phospholipid mixture in the aqueous dispersion is appropriately determined depending on the application. The range of 5 to 25% by weight is suitable from the viewpoint of workability of powder production by subsequent freeze-drying. The aqueous dispersion of the phospholipid mixture thus obtained is milky white and uniform, and can be stably stored for a long period of time.

In the present invention, the aqueous dispersion of the phospholipid mixture is dehydrated by a freeze-drying method, that is, the water is removed by freeze-drying to obtain a powder of the phospholipid mixture. The freeze-drying may be carried out by rapidly cooling at a freezing temperature of −5 to −80 ° C., preferably −30 ° C. or less, and sublimating water under a reduced pressure of 0.1 mmHg or less. The powder thus obtained has a uniform composition of the phospholipid mixture and excellent dispersibility in water. By emulsifying and dispersing this powder in water again, the composition is uniform and stable. It can be an excellent aqueous dispersion.

[0018]

Industrial Applicability According to the present invention, an aqueous dispersion of a phospholipid mixture having a uniform composition and a uniform particle size distribution can be produced, and this aqueous dispersion can be used as an emulsion itself. Further, by removing water from this aqueous dispersion, it is possible to produce a powder of a phospholipid mixture having a uniform composition and excellent dispersibility in water, and this powder is well mixed at the molecular level,
It can be used as a raw material for liposomes, emulsions and the like in the fields of medicine, food and cosmetics.

[0019]

EXAMPLES The present invention will be described in more detail below by way of its examples.

Example 1 67 g of hydrogenated soybean phosphatidylcholine (hereinafter referred to as HSPC) and 33 g of cholesterol were placed in a reactor (5 liters) capable of stirring under reduced pressure and distilling off the solvent. It was completely dissolved in 300 g of ethyl alcohol. While stirring, 2 liters of water was added to obtain a dispersion liquid. The dispersion was decompressed to 70 mmHg with a decompression pump under the temperature condition of 60 ° C., and ethyl alcohol was distilled off with stirring. After 10 hours, the vacuum distillation was stopped. When the obtained aqueous dispersion was analyzed by gas chromatography, ethyl alcohol was not detected. Next, water was added to form an aqueous dispersion of a phospholipid mixture having a solid content concentration of 15% by weight.

An aqueous dispersion of this phospholipid mixture was observed with an optical microscope. The particles were almost uniform, and the particle size was about 2 μm. Further, the phase transition temperature was measured by DSC (differential scanning calorimeter) in order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level. No phase transition temperature (54 ° C.) derived from HSPC was observed, and it was confirmed that HSPC and cholesterol were uniformly mixed.
When this aqueous dispersion was left at room temperature, it remained stable without any change even after 30 days.

Next, the aqueous dispersion of the phospholipid mixture was freeze-dried to obtain a powder of the phospholipid mixture. The lyophilization was performed by rapidly cooling 100 g of the phospholipid mixture at a temperature of -40 ° C, and then lyophilizing it overnight with a lyophilizer (EYELA, FD-1 type) under a reduced pressure of 2 mmHg.
5 g of a phospholipid mixture powder was obtained. To 1 g of this powder, add 10 g of water and vortex mixer for 30 seconds.
Upon stirring with, a uniform aqueous dispersion was obtained. In order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, the phase transition temperature was measured by DSC. HS
No PC-derived phase transition temperature was observed, and it was confirmed that HSPC and cholesterol were uniformly mixed.

Comparative Example 1 67 g of HSPC and 33 g of cholesterol were placed in a reactor (5 liters) capable of stirring and distilling off the solvent under reduced pressure, and completely dissolved in 300 g of chloroform. While stirring, 2 liters of water was added to obtain a dispersion liquid. This dispersion liquid was heated under a temperature condition of 60 ° C. with a vacuum pump to 70
The pressure was reduced to mmHg, and chloroform was distilled off with stirring. After 10 hours, the vacuum distillation was stopped. When the obtained aqueous dispersion was analyzed by gas chromatography, chloroform was not detected. Next, water was added to form an aqueous dispersion of a phospholipid mixture having a solid content concentration of 15% by weight.

An aqueous dispersion of this phospholipid mixture was observed with an optical microscope. The particles are non-uniform and the particle size is 0.2-1
It was widely distributed to about 0 μm. Further, in order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, the phase transition temperature was measured by DSC. A peak was observed at the phase transition temperature (56 ° C) derived from HSPC,
It was inferred that C and cholesterol were not uniformly mixed. When this aqueous dispersion was left for 10 days, the aqueous dispersion separated into an aqueous phase and a precipitate, and cholesterol was floating on the water surface.

Next, the aqueous dispersion of the phospholipid mixture was freeze-dried under the same conditions as in Example 1 to obtain a powder of the phospholipid mixture. To 1 g of this powder add 10 g of water,
After stirring with a vortex mixer for 30 seconds, a uniform aqueous dispersion could not be obtained, and a precipitate was formed. Further, in order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, the phase transition temperature was measured by DSC. A phase transition temperature (56 ° C.) derived from HSPC was observed, and it was inferred that HSPC and cholesterol were not uniformly mixed.

Comparative Example 2 0.67 g of HSPC and 0.33 g of cholesterol were dissolved in 30 ml of chloroform, and then chloroform was removed by an evaporator to form a thin film. To this thin film, 5.7 g of water (solid content concentration 15% by weight) was added and stirred with a vortex mixer to obtain an aqueous dispersion. This aqueous dispersion was observed with an optical microscope. The particle size is 0.
The distribution was wide with 2 to 50 μm. When this aqueous dispersion was left for 10 days, it was separated into an aqueous phase and a precipitate, and cholesterol was floating on the water surface. Next, this aqueous dispersion of phospholipid mixture was freeze-dried under the same conditions as in Example 1 to obtain a powder of phospholipid mixture. 10g of this powder
Water was added and the mixture was stirred with a vortex mixer for 30 seconds, but precipitation occurred.

Example 2 47.6 g of dimyristoylphosphatidylcholine (phase transition temperature: 24 ° C., hereinafter referred to as DMPC) was placed in a reactor (5 liters) capable of stirring under reduced pressure and distilling off the solvent.
52.4 g of dipalmitoylphosphatidylcholine (phase transition temperature: 41 ° C., hereinafter referred to as DPPC) was added, and 5
It was completely dissolved in 00 g of dioxane. While stirring, 2 liters of water was added to obtain a dispersion liquid. The dispersion was decompressed to 70 mmHg by a decompression pump under the temperature condition of 60 ° C., and dioxane was distilled off while stirring. After 10 hours, the vacuum distillation was stopped. When the obtained aqueous dispersion was analyzed by gas chromatography, dioxane was not detected. Next, water was added to form an aqueous dispersion of a phospholipid mixture having a solid content concentration of 15% by weight.

An aqueous dispersion of this phospholipid mixture was observed with an optical microscope. The particles were almost uniform, and the particle size was about 2 μm. Further, in order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, the phase transition temperature was measured by DSC. A phase transition temperature derived from the mixture was observed at 35 ° C., and it was confirmed that DMPC and DPPC were uniformly mixed. When this aqueous dispersion was left at room temperature, it remained stable without any change even after 30 days.

Next, the aqueous dispersion of the phospholipid mixture was freeze-dried under the same conditions as in Example 1 to obtain a powder of the phospholipid mixture. To 1 g of this powder add 10 g of water,
After stirring with a vortex mixer for 30 seconds, a uniform aqueous dispersion was obtained. In order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, the phase transition temperature was measured by DSC. A phase transition temperature derived from the mixture was observed at 35 ° C., and it was confirmed that DMPC and DPPC were uniformly mixed.

Comparative Example 3 An aqueous dispersion of a phospholipid mixture was obtained in the same manner as in Example 2 except that methylene chloride was used instead of dioxane. The aqueous dispersion of this phospholipid mixture was observed with an optical microscope. The particles were non-uniform, and the particle size was widely distributed to about 0.2 to 10 μm. In order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, D
The phase transition temperature was measured by SC. Phase transition temperatures derived from DMPC and DPPC were observed at 24 ° C. and 41 ° C., respectively, and it was assumed that DMPC and DPPC were non-uniform. When this aqueous dispersion was left for 10 days, the aqueous dispersion was separated into an aqueous phase and a precipitate.

Next, the aqueous dispersion of the phospholipid mixture was freeze-dried under the same conditions as in Example 1 to obtain a powder of the phospholipid mixture. To 1 g of this powder add 10 g of water,
After stirring with a vortex mixer for 30 seconds, a uniform aqueous dispersion was obtained. In order to confirm the homogeneity of the phospholipid mixture in this aqueous dispersion at the molecular level, the phase transition temperature was measured by DSC. Phase transition temperatures derived from DMPC and DPPC were observed at 24 ° C. and 41 ° C., respectively, and it was assumed that DMPC and DPPC were non-uniform.

Example 3 100 g of hydrogenated egg yolk phosphatidylcholine (hereinafter referred to as HEPC), 12 g of cholesterol were placed in a reactor (5 liters) capable of stirring under reduced pressure and distilling off the solvent.
3 g of stearylamine was added and completely dissolved in 300 g of isopropyl alcohol. While stirring, 2 liters of water was added to obtain a dispersion liquid. This dispersion at 60 ° C
Under the temperature condition of, reduce the pressure to 70 mmHg with a vacuum pump,
Isopropyl alcohol was distilled off with stirring. 10
After a while, the vacuum distillation was stopped. When the obtained aqueous dispersion was analyzed by gas chromatography, isopropyl alcohol was not detected. Next, water was added to form an aqueous dispersion of a phospholipid mixture having a solid content concentration of 15% by weight.

An aqueous dispersion of this phospholipid mixture was observed with an optical microscope. The particles were almost uniform, and the particle size was about 1 μm. Next, this aqueous dispersion of phospholipid mixture was freeze-dried under the same conditions as in Example 1 to obtain a powder of phospholipid mixture. 1 g of this powder with 20% by weight of glucose
When 10 ml of an aqueous solution was added and the mixture was emulsified for 10 minutes, a uniform aqueous dispersion having a particle size of 1 to 2 μm was obtained. To this aqueous dispersion, a physiological saline solution having a volume 6 times that of the aqueous dispersion was added to give 3,000 rpm. p. m. The liposomes were separated three times by centrifugation for 10 minutes at 10 minutes, and the capture rate of glucose into the liposomes [(glucose in liposome / charged glucose) × 100] I asked. 10.5% by weight
It was discovered that the glucose had been captured.

Comparative Example 4 0.87 g of HEPC, 0.10 g of cholesterol,
After dissolving 0.03 g of stearylamine in 30 ml of chloroform, the chloroform was removed by an evaporator to form a thin film. To this thin film, 10 ml of 20% by weight glucose solution was added, and under the same conditions as in Example 3, 1
It was run in the emulsifier for 0 minutes. The particle size of the obtained aqueous dispersion was 0.2 to 5 μm and the distribution was wide. In this water dispersion,
Add 6 times the volume of physiological saline to the aqueous dispersion and add 3,000
r. p. m. The liposomes were separated by precipitating and separating the liposomes three times for 10 minutes at 10 minutes, and the capture rate of glucose into the liposomes [(glucose in liposome / charged glucose)] X100] was calculated. It was found that about 3.2% by weight glucose was captured.

Claims (2)

[Claims] 1. A) a step of dissolving at least two kinds of phospholipids or phospholipids and lipids other than phospholipids in a water-miscible solvent to form a solution, and b) mixing the solution obtained in the above step a with water. To obtain a dispersion, and c) a step of removing the water-miscible solvent from the dispersion obtained in the above step b to obtain an aqueous dispersion, thereby producing an aqueous dispersion of a phospholipid mixture. 2. A method for producing a powder of a phospholipid mixture, which comprises removing an aqueous dispersion of the phospholipid mixture produced by the method of claim 1 by a freeze-drying method.