JPS61129191A - Glycerophospholipid containing decosahexaenoic acid - Google Patents

Abstract

NEW MATERIAL:A compound of formula I (R<1>CO, R<2>CO are residues of natu rally occurring fatty acids where at least one is residue of docosahexaenoic acid; R<3> is H, CH2CH2N<+>(CH3)3, CH2CH2N<+>H3, formulas II-V). EXAMPLE:1,2-Didocosahexaenyl-Sn-3-glycerophosphorylcholine. USE:Liposome, cholesterol-lowering agent, preventive for thrombosis: is shows high stability to oxidation. PREPARATION:For example, a deacylated phospholipid such as glycerylphosphorylcholine or glycerylphosphorylethanolamine or its cadmium salt is allowed to react with an anhydride, chloride or imidazole salt of docosahenxaenoic acid in the presence of a catalyst such as 4- dimethylaminopyridine in a solvent such as dichloromethane at room temperature under stirring.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a glycerophospholipid containing docosahexaenoic acid in an acyl group.

[Conventional technology]

Natural phospholipids mainly consist of glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingophospholipids such as sphingomyelin.
It exists as an important component of biological membranes of cells. Among these, phosphatidylcholine is generally called lecithin and is the most abundant phospholipid in the animal and plant kingdoms.

A common feature of these phospholipids is that they have a hydrophilic group and a hydrophobic group, and they form a double layer in biological membranes, and together with proteins and cholesterol, they are essential for biological phenomena such as permeation of substances and selective transport. It performs functions that cannot be performed. Phospholipids have already been used industrially as natural emulsifiers, such as in the food industry and the cosmetics industry.

Furthermore, it has recently been known that phospholipids form very small closed endoplasmic reticulum called liposomes in aqueous solutions due to their lipid characteristics.

On the other hand, docosahexaenoic acid, a highly unsaturated fatty acid found in animal and vegetable oils, especially fish oil, has been recognized to have physiological activities such as lowering cholesterol and neutral fats, preventing arteriosclerosis, and preventing thrombosis. Recently, phospholipids containing docosahexaenoic acid are said to have anticancer effects.

[Problem that the invention seeks to solve]

However, in the past, such docosahexaenoic acid had extremely poor oxidative stability and was insoluble in water, and when used as a medicine, there were considerable restrictions such as encapsulating it with gelatin to prevent oxygen permeation.

The present invention is intended to solve these problems,
The object of the present invention is to provide a glycerophospholipid containing docosahexaenoic acid with improved oxidative stability by incorporating docosahexaenoic acid into the structure of the phospholipid.

[Means for solving problems]

The present invention is a docosahexaenoic acid-containing glycerophospholipid comprising a compound represented by the following general formula [I].

(In the formula, R'CO- and R2GO- each represent a naturally occurring fatty acid residue, one or both of which is a docosahexaenoic acid residue, and R3 is -CH2CH2N4''
(CHco)3゜[In formula 13, R'CO- or R
Docosahexaenoic acid with 2C0- as a residue is 22,
6. Omega-3 (omega-3 acid...the double bond is located at the third position from the terminal methyl group) is a highly unsaturated fatty acid in which the double bond is in the cis position, and has excellent physiological activity in vivo. show. When one of R'CO- or R2C0- is a docosahexaenoic acid residue, the fatty acid with the other as a residue is a normal saturated or unsaturated fatty acid having 10 to 20 carbon atoms such as palmitic acid, stearic acid, oleic acid, etc. can give.

The docosahexaenoic acid-containing glycerophospholipid of the present invention is
There are various compounds depending on the combination of Hl, R2, and R3 in formula (1), and representative compounds include 1-valmitoyl-2-docosahexaenyl-5n-3-glycerophosphorylcholine, 1-valmitoyl-2-docosahexaenyl, Examples include hexaenyl-S rho 3-glycerophosphorylethanolamine, (1), 2-cytocosahexaenyl-5n-3-glycerophosphorylcholine, and 1,2-cytocosahexaenyl-5n-3-glycerophosphorylethanolamine.

The docosahexaenoic acid-containing glycerophospholipid of the present invention is
Deacylated phospholipids such as glycerylphosphorylcholine (GPC) and glycerylphosphorylethanolamine (GPE), anhydride and chloride of docosahexaenoic acid,
It can be produced by reacting with an imidazole salt or the like. Another method for producing glycerophospholipids is to synthesize glycerophospholipids from diacylglycerol, etc. by total synthesis.This method allows not only natural optically active conjugated acid-type glycerophospholipids, but also optical isomers, stereoisomers, or mixtures thereof. It is also possible to synthesize

The docosahexaenoic acid used in the above production is obtained by decomposing, separating, and refining fish oil, whale oil, etc.

The obtained docosahexaenoic acid is used after being converted into an acid anhydride, acid chloride, acid imidazole salt, etc.

In addition, the deacylated phospholipid used in the above production forms the backbone of glycerophospholipid, and is mainly glycerylphosphorylcholine or glycerylphosphorylethanolamine obtained by separating, decomposing, decomposing, and recrystallizing natural phospholipids such as soybean and egg yolk. Deacylated phospholipid bodies such as the following can be used. Separation is performed using silica gel or activated alumina and elution with a mixed solvent such as chloroform/methanol. The decomposition is carried out with tetrabutylammonium t1 hydroxide, but it may also be gently decomposed with a low concentration of caustic soda or the like. Recrystallization is performed as a cadmium salt or the like. Desacylated phospholipids such as glycerylphosphorylcholine exhibit very strong hygroscopicity and can be used as they are, but it is preferable to use them in the reaction after converting them into cadmium salts, etc., as they are easier to handle in terms of reaction operations. Recrystallization is carried out in a hydroalcoholic solution in the form of a cadmium salt or the like.

The reaction between docosahexaenoic anhydride, chloride, imidazole, etc. and the deacylated phospholipid or its cadmium salt is carried out by stirring at room temperature in a dry solvent such as dichloromethane in the presence of a catalyst such as 4-dimethylaminopyridine. When carried out, docosahexaenoic acid-containing glycerophospholipids are produced.

[For production]

The docosahexaenoic acid-containing glycerophospholipid of the present invention has excellent oxidative stability and decomposes into docosahexaenoic acid and glycerophospholipid in vivo and exhibits synergistic physiological activities of each, so it is a food product.

It can be used in living organisms as cosmetics, medicines, etc., and has particularly high pharmacological effects such as lowering cholesterol and preventing blood clot symptoms.

These phospholipids can be used by living organisms as they are by ingestion or administration, but by forming liposomes and using them as an aqueous solution, they can be used as physiologically active substances with excellent oxidative stability by preventing the permeation of oxygen. I can do it.

This liposome aqueous solution can be used as a transparent injection solution or an infusion solution such as a tube feeding solution.

Since the phospholipids of the present invention are amphipathic, the liposomes are also amphipathic, and other substances can be introduced into the living body using the space of the liposomes, that is, water-soluble substances such as vitamin C. Substances can be placed in the vesicles inside liposomes, and hydrophobic substances such as vitamin E can be placed in the hydrophobic part of the liposome membrane, and then supplied to living organisms. Liposomes can be made using the ii'/'V method (ultrasonic treatment) by dissolving phospholipids in an organic solvent, evaporating the organic solvent to form a thin film, and adding an aqueous solution to it and subjecting it to ultrasound treatment. However, it can also be made by a solution injection method.

〔Effect of the invention〕

According to the present invention, since docosahexaenoic acid is included in the structure of the phospholipid, a phospholipid that has high oxidation stability, excellent physiological activity, and can be used as a liposome can be obtained.

〔Example〕

Hereinafter, the present invention will be explained in more detail based on Reference Examples and Examples.

Reference Example 1 Miscellaneous fish oil methyl ester was obtained by transesterification of sardine, mackerel, squid, etc. containing oils and fats in methanol in the presence of sodium methylate in a conventional manner. The fatty acid composition of this methyl ester is C, 4, 85, 36%, CI6
+0 14.55%, C, 6□6. 1%.

C18. 2.49%, C18,113,55%, C1
8,4,ω-32,52%, C2o,, 8.33%
, C20+41 ω-60,88%, C2g+5.
(Ll-311, 81%, C2□, I 6.63%,
C22: Srω-31,67%, C2□, 6. ω-3
13,49%, C2411,00%, other 10.81
%Met.

Next, this methyl ester was applied to a thin film type molecular distillation machine to cut off the pre-distillate. The fatty acid composition of the subsequent fraction is C+
4: o 1.31%-Cr6+o 2.12%, C
ru: +1.35%, cta+. 2.43%-C1
81111°98%, C18+41.79%, C2o,
, 11.95%-C20!4+ω-61.03%
, C20151(1)-316,64%, C22111
0.74%, C2□, 3. ω-33,01%-C221
6+ ω-321,23%, C248□ 2.77%,
Others accounted for 11.65%.

Then, 300 g of this molecularly distilled methyl ester was added to a methanol solution saturated with 1200 g of urea at 25°C.
It was cooled to 15°C and kept for 2 hours. The precipitated urea adduct was filtered, and the mother liquor was concentrated and extracted with hexane to obtain C1.
6+1 0.48%, c'ta+43.86%, C20
110・36%・C2014t ω-62・19%・C
201jj+ ω-333,77%, C2□,, 0
．． 22%, C22,5, ω-35,16%, C2□,6
．． 113 g of methyl ester having a composition of ω-348, 59% and other 5.37% was obtained. This urea-added methyl ester was distilled to 0.01 to 0.05+++ mHg using a distillation apparatus equipped with a heat-insulated packed column containing a Raschig ring.
Distilled at C2216+ ω-393, 54%.

C2G+51 (11-33, 22%, other 3.24
% methyl ester was obtained. This methyl ester was saponified in a conventional manner and converted back to acid to obtain 12.34 g of docosahexaenoic acid (93%).

Reference Example-2 50 g of commercially available egg yolk lecithin was separated using 1,5Q silica gel packed in a glass column with a diameter of 8 cm.

Elution solvent: chloroform/methanol/water = 65/2
A 5/4 mixed solvent was used. Each fraction was tracked by thin layer chromatography, and 31 g of lecithin was obtained from the lecithin portion, and 9 g of phosphatidylethanolamine was obtained from the phosphatidylethanolamine portion. The obtained lecithin was dissolved in 3500 mQ of ether, and 80 mft of 1 volume of 10% tetrabutylammonium hydroxide in methanol was added while stirring. Glycerylphosphorylcholine precipitated at the bottom is collected by decantation,
Washed with ether. The amount of glycerylphosphorylcholine obtained was 9.1 g. This glycerylphosphorylcholine was dissolved in warm water and mixed with 18.9 g of cadmium chloride 2.5 hydrate, which had been separately dissolved in warm water, and reacted. The insoluble matter was filtered out, and this glycerylphosphorylcholine
Ethanol was added to the cadmium chloride complex aqueous solution until it became cloudy, and the solution was left at 0 to 5°C overnight, and the resulting crystals were collected. These crystals were again dissolved in warm water, and ethanol was added to crystallize them in the same manner. The crystals were collected and dried to obtain 118 g of glycerylphosphorylcholine-cadmium chloride complex.

Example-1 Docosahexaenoic acid (93%) obtained in Reference Example-1 5
g was dissolved in dry chloroform, 1.7 g of dicyclohexylcarbodiimide was added, and the mixture was reacted overnight at room temperature to synthesize docosahexaenoic acid (93%) anhydride. The by-produced dicyclohexyl urea was filtered out, and the glycerylphosphorylcholine-cadmium chloride complex 1 of Reference Example-2 was obtained.
．． 1 g of dimethylaminopyridine and 0.62 g of dimethylaminopyridine were added to this solution of docosahexaenoic anhydride in chloroform.The mixture was sealed tightly and reacted for 48 hours with stirring at room temperature.After one reaction, the precipitate was filtered out, and the chloroform was evaporated to obtain a crude solution. Obtained lecithin. The crude lecithin was redissolved in a methanol/chloroform mixed solvent, 5 g each of ion exchange resins Amberlite IRC-50 and Ding RA-45 (trademarks of Rohm and Haas) were added, and residual cadmium chloride was removed in a batchwise manner. Methanol was evaporated to obtain crude lecithin again. This crude lecithin was subjected to column fractionation using a 3 cm diameter glass column filled with silica gel at 0.2° C. using a mixed solvent of chloroform/methanol/water = 65/25/4 as an eluent. The lecithin portion was recovered while being followed by thin layer chromatography, and purified by evaporation to obtain pure 1゜2-cytocosahexaenyl-3n-3.
- 1.7 g of briserophosphorylcholine was obtained.

The elemental analysis values of the obtained 1,2-cytocosahexaenyl-5n-3-glycerophosphorylcholine are as follows.

Elemental analysis value CHN (tzt%) Theoretical value 71.15 9°121°60 Actual value
70゜629゜03 1.613 The NMR spectrum of the above 1,2-cytocosahexaenyl-5n-3-glycerophosphorylcholine is as shown in Figure 1, and the position code added to Figure 1 is the one of formula (II). That's right.

1,2-cytocosahexaenyl 5 obtained above
500a+g of n-3-glycerophosphorylcholine was dissolved in benzene, and the benzene was removed under reduced pressure using a rotary evaporator in a 100 mff 1 volume eggplant flask, and the above phosphatidylcholine was adhered to the flask wall in the form of a film. Physiological saline 25IIIQ was added to the flask, and the flask was subjected to ultrasonication using a Plog-type ultrasonic generator at 35 watts for 20 minutes while cooling under a nitrogen stream. The obtained liposome solution was a blue-white, transparent liquid, and when observed under an electron microscope, it contained approximately 300 liposomes in diameter. This liposome solution is filtered and
After removing a small amount of floating matter, Im
Q was administered intravenously, but no particular immune reaction was observed.

Furthermore, the liposome solution obtained above was stable at room temperature for more than a week, and when it was azeotropically dehydrated with benzene under reduced pressure and its peroxide value (POV) was measured, no increase in POv was observed.

Example 2 7.7 g of palmitic acid was dissolved in dry chloroform, and 3.1 g of dicyclohexylcarbodiimide was reacted overnight at room temperature to synthesize palmitic acid anhydride. The by-produced dicyclohexyl urea was filtered and the glycerylphosphorylcholine-cadmium chloride complex 1.6 of Reference Example-2 was obtained.
g and 0.62 g of dimethylaminopyridine were added to this palmitic anhydride chloroform solution. Reaction and purification were carried out in the same manner as in Example 1, and 1,2-dipalmitoyl-5n-
3-glycerophosphoryl/L/-Jsu'/ (DI'
1.9 g of PC) was obtained. This DPP Cl00mg
was dissolved in 2 ml of ethyl ether, and this solution was mixed with 250 mg of snake venom phospholipase A and calcila chloride.
, 20 μmol O, LM)-Lis-HCl buffer 5 mQ
The mixture was reacted for 1 hour with shaking at 37°C. The reaction was stopped with an EDTA solution and ethanol, and the mixture was azeotropically distilled with benzene and condensed to dryness. This was separated on a silica gel column using a mixed solvent of chloroform/methanol/water: 65/35/8 to obtain 43 mg of 1-valmitoyl-5n-3-glycerophosphorylcholine.

43 mg of this lysophosphatidylcholine was mixed with a chloroform solution of docosahexaenoic anhydride (containing 150 mg of docosahexaenoic anhydride) obtained in the same manner as in Example 1 and 12 mg of dimethylaminopyridine.

The reaction and purification were carried out in the same manner as in Example 1, and 1-balmitoyl-
38111 g of 2-docosahexaenyl-5n-3-glycerophosphorylcholine was obtained.

The elemental analysis values of the obtained 1-valmitoyl-2-tocosahexaenyl-5n-3-glycerophosphorylcholine are as follows. .

Elemental analysis value CHN (tyl-%) Theoretical value 68.57 9.94 1.74 Actual value 68.16 9.89 1.77

[Brief explanation of the drawing]

FIG. 1 is an NMR spectrum diagram in Example-1.

Claims (3)

[Claims] (1) A docosahexaenoic acid-containing glycerophospholipid consisting of a compound represented by the following general formula [I]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼... [I] (In the formula, R^1CO- and R^2CO- each represent a naturally occurring fatty acid residue, and one or both of them is a docosahexaenoic acid residue. Yes, R^3 is -CH_2CH_
2N^+(CH_3)_3, -CH_2CH_2N^+
H_3, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, H, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼. ) (2) The docosahexaenoic acid-containing glycerophospholipid according to claim 1, wherein R^1CO- or R^2CO- is a palmitic acid residue. (3) Claim 1, wherein the compound of formula [I] is 1,2-didocosahexaenyl-Sn-3-glycerophosphorylcholine or 1-palmitoyl-2-docosahexaenyl-Sn-3-glycerophosphorylcholine. The docosahexaenoic acid-containing glycerophospholipid described above.