JPH05163153A - Sphingolipid composition - Google Patents

Abstract

PURPOSE:To provide a new sphingolipid composition resistant to the generation of ill odor and discoloration during long-term storage and to provide a process for producing the sphingolipid composition. CONSTITUTION:The objective sphingolipid contains (a) galactosyl cerebroside, (b) galactosylcerebroside sulfate and (c) sphingomyelin at an (a+b)/c ratio of 65/35 to 85/15. The content of sphingolipid in the above three components is >=90% and that of glycerophospholipid is <=2%. The composition can be produced by hydrolyzing a lipid mixture obtained from a cerebral nerve of mammal with an alkali, distilling out the solvent used in the hydrolysis, washing with a mixture of acetone and water and collecting the insoluble component.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel sphingolipid composition and a method for producing the same.

[0002]

2. Description of the Related Art Lipids obtained by extraction from the brain (cranial nerve cells) of mammals such as cows, horses and pigs include galactosyl cerebroside (a), galactosyl cerebroside sulfate (b) and sphingomyelin (c). In addition to sphingolipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, glycerophospholipids such as lysophosphatidycholine and lipids containing cholesterol as its constituent components, these constituent components are intercellular lipids present in the skin and Since they are substantially the same, they have been conventionally used as one raw material in the field of cosmetics, for example, as an emulsion stabilizer or a moisturizer. When using them, the three components (a), (b) and (c) of the above-mentioned constituents with respect to the sphingolipid have a ratio of (a + b) / c of 65/35 to 85/15. It is said that the above-mentioned grades are preferable because these ratios are close to those of animal cranial nerve tissue, that is, natural ones. As a representative method for extracting lipids from the brain tissue of animals, a method using a hexane / isopropanol mixed solvent has been conventionally known (JP-A-64-16708). According to this method, after extracting the lipid with the above mixed solvent, the solvent was distilled off, and the obtained concentrate was then washed with ether to form an ether precipitate, which was further washed with acetone to finally precipitate the acetone. As a product, a purified sphingolipid composition is obtained.

[0003]

However, all the sphingolipid compositions obtained by the conventional methods including the above-mentioned methods produce an offensive odor after being stored for, for example, about 6 months. However, discoloration has come to be recognized, and there is a problem that the quality is unstable. Since the sphingolipid composition is used as a raw material for cosmetics as described above, it is usually required that the quality be stable over a long period of time. Therefore, the present invention provides a novel sphingolipid composition in which deterioration of the quality as described above does not easily occur even during long-term storage,
Another object of the present invention is to provide a method for producing such a sphingolipid composition.

[0004]

Means for Solving the Problems The present inventors have completed the present invention as a result of earnest studies in accordance with the above object. That is, the present invention, in the total amount, galactosyl cerebroside (a), galactosyl cerebroside sulfate (b) and sphingomyelin (c) three components sphingolipid content of 90% or more, glycerophospholipid content is 2% or less, Moreover, the ratio of (a + b) / c is 6
The present invention provides a sphingolipid composition characterized by being 5/35 to 85/15. The present inventor also hydrolyzes a lipid mixture obtained from mammalian cranial nerve tissue with alkali, then distills off the solvent used for the hydrolysis and then wash with a mixed solvent of acetone / water to remove insoluble matter. The present invention provides a method for producing a sphingolipid composition, which comprises collecting the sphingolipid composition.

The present invention will be described in detail below. In the sphingolipid composition of the present invention, first, the total content of the three components of galactosyl cerebroside (a), galactosyl cerebroside sulfate (b) and sphingomyelin (c) is 90% or more. This is because it is difficult to say that the sphingolipid composition is substantially high in purity unless it is so high. It is generally about 92 to 97%. Regarding the above-mentioned three components (a), (b) and (c), the sphingolipid composition of the present invention has a ratio of (a + b) / c of 65/35 to 85/1.
5 (about 1.9 to 5.7). If it is not at this level, it is difficult to say that this ratio in the cranial nerve tissue of the animal, that is, a ratio close to the natural one.

The sphingolipid composition of the present invention further has a glycerophospholipid content of 2% or less. This is because if the glycerophospholipid content is 2% or less, it is difficult for an offensive odor to occur or discoloration during storage, and quality deterioration is less likely to occur. It is preferably 1% or less.

As described above, in the sphingolipid composition of the present invention, the sphingolipid content of the three components (a), (b) and (c) is 90% or more, and (a + b) / The ratio of c is 65/35 to 85/15, and the glycerophospholipid content is 2% or less in terms of composition, so that the generation of off-odor during storage which the conventional sphingolipid composition has The problem of discoloration and discoloration is less likely to occur. Needless to say, the sphingolipid composition of the present invention may contain other arbitrary raw material components, such as tocophenol, as long as such effects are not impaired.

Next, a method for producing the sphingolipid composition of the present invention will be described. According to the method of the present invention, first, a lipid mixture obtained from mammalian cranial nerve tissue (neural tissue such as brain and spinal cord) is subjected to alkaline hydrolysis. The mammalian cranial nerve tissue is not particularly limited, but generally, domestic animal cranial nerve tissue such as cow, horse, pig, and sheep is used. The brain is preferred because it is easily available. The acquisition of the lipid mixture from the cranial nerve tissue is not particularly limited, but generally, an organic solvent from the dried brain or spinal cord, for example, a solvent capable of dissolving a sphingolipid such as ethanol, methanol, isopropyl alcohol, or hexane is used. The extraction method is carried out either alone or in the form of a mixed solvent. Specifically, the extraction operation may be performed at 20 to 60 ° C. for about 1 to 4 hours using an organic solvent having a volume of 10 to 30 times that of the dried cranial nerve tissue.

According to the method of the present invention, the lipid mixture thus obtained is subjected to alkaline hydrolysis. The method of alkaline hydrolysis is not particularly limited, but specifically, for example, when the one subjected to the above extraction operation is targeted, after removing the residue by filtration if necessary, Approximately 1: 1 of a mixture of an alkaline agent and water (preferably ion-exchanged water), based on the dry brain weight, is used in a ratio of 1/2 to 1/6.
It is general to add a double amount and to carry out a reaction at 20 to 80 ° C. for about 1 to 20 hours. Examples of the alkaline agent used here include sodium hydroxide, potassium hydroxide, calcium hydroxide and the like. When the organic solvent is removed after the above extraction operation, the extract is dispersed in water (preferably ion-exchanged water), sodium hydroxide is added, and the hydrolysis treatment is performed in the same manner as above. You can do it.

By alkali-hydrolyzing the lipid mixture under the conditions as described above, substantially only the glycerophospholipid in the lipid mixture can be hydrolyzed. By removing the water-soluble decomposition products (salts and the like) and the oil-soluble decomposition products (mainly free fatty acids) in the subsequent step, the glycerophospholipid content can be finally reduced to 2% or less. According to the result of the TLC method test for the final product, the phosphatidylethanolamine content is substantially zero.

According to the method of the present invention, the solvent used for the hydrolysis is then distilled off and then washed with a mixed solvent of acetone / water to obtain an insoluble sphingolipid composition. The solvent used for hydrolysis, ie the organic solvent used to extract the lipid mixture from the brain, or water, is first distilled off. The method of distillation is not particularly limited, but the solution after completion of hydrolysis is usually heated to a temperature above the evaporation temperature of the solvent under reduced pressure to remove the solvent. When the solvent is distilled off, the pH of the solution after completion of hydrolysis is preferably neutralized with hydrochloric acid or the like (pH 7 to 8) and then filtered to remove the residue.

The solid substance obtained after the distillation is washed with a mixed solvent of acetone / water. As the mixed solvent used at that time, the volume ratio of acetone / water is 70/30 to 90/1.
It is preferably about 0 for efficiency. With acetone alone,
Even if the oil-soluble decomposition product generated by alkali hydrolysis can be removed, the water-soluble decomposition product cannot be removed, making it difficult to obtain a high-purity sphingolipid composition as the final product.
Moreover, even if water alone can remove the water-soluble decomposition products, the oil-soluble decomposition products cannot be removed, and the sphingolipids also become soluble in water, resulting in a decrease in the yield of the final product and a reduction in the purity. It becomes difficult to obtain a sphingolipid composition having a high content.

Specifically, the washing operation with the above mixed solvent is carried out by adding 4 to 5 times the weight of the mixed solvent to the above solid and stirring for about 30 minutes, and then removing the washing liquid by decantation or the like. It is common to do. The operation is 4-5
Repeat about once. As a result, the alkaline hydrolysis product is removed, and in addition to salts generated by neutralization, impurities such as odorous components derived from the used cranial nerve tissue can be removed. Thus, the washing operation with the acetone / water mixed solvent of the present invention has a higher washing effect than the amount of the solvent used.

The insoluble matter obtained by washing with a mixed solvent and then filtering may be used as it is as the sphingolipid composition of the present invention, but is preferably further washed with acetone. This is because the acetone cleaning can further remove cholesterol and water. The insoluble matter obtained by washing and filtering can be obtained as a nearly odorless sphingolipid composition (white powder) by distilling off acetone under reduced pressure.

[0015]

[Function] Glycerophospholipids in lipids (particularly glycerophospholipids having amino groups such as phosphatidylethanolamine and phosphatidylserine) can be decomposed by alkaline hydrolysis of a lipid mixture obtained from cranial nerve tissue. It is speculated that the content of these compounds may be reduced in the final product, and as a result, it may be difficult for an offensive odor to occur or discoloration during storage.

[0016]

EFFECTS OF THE INVENTION According to the present invention, unlike conventional sphingolipid compositions, an offensive odor is unlikely to occur during storage, discoloration is not easily observed, and storage stability is high. A sphingolipid composition is provided that is similar to the natural one in terms of the ratio of (a + b) / c of the three components a), (b) and (c). As described above, since the sphingolipid composition of the present invention has stable quality during storage, it is used as a base material for cosmetics (for example, 0.
In addition to being preferably used (with a compounding ratio of about 001 to 5%), it can be expected to be used as an emulsion stabilizer, a percutaneous absorption enhancer, etc. in the field of medicine. Further, the present invention provides a method by which the sphingolipid composition as described above can be simply and suitably produced.

[0017]

EXAMPLES The present invention will be described in more detail below with reference to examples and test examples. In the present invention, all "%" are "% by weight" and all "proportions" are "weight percentages".
Is. Example 1 10 kg of freeze-dried bovine brain powder was subjected to a lipid extraction operation using 200 liters of ethanol at 50 to 60 ° C. for 2 hours. The filtrate was filtered to remove the residue,
A separately prepared alkaline aqueous solution (prepared by dissolving 4 kg of sodium hydroxide in 4 liters of ion-exchanged water) was gradually added, and the mixture was reacted at 40 to 55 ° C. for 5 hours while stirring. Then, after neutralizing with 12N hydrochloric acid (pH 7 to 8), the solvent (ethanol and water) was distilled off under reduced pressure. The solid substance (9.5 kg) obtained after evaporation was washed 5 times with 20 liters of a mixed solvent having a mixture ratio of acetone / water of 85/15 (1 washing was performed for 30 minutes, and the washing solution was removed by decantation). ), The insoluble matter is further washed with 10 liters of acetone while stirring for 30 minutes, and acetone is distilled off under reduced pressure to obtain 1.5 kg of the sphingolipid composition of the present invention (odorless: white powder). It was

Test Example Regarding the sphingolipid composition of the present invention obtained in Example 1 above, the sphingolipid content and the glycerophospholipid content of the sphingolipids (a), (b) and (c) were further evaluated. The ratio of (a + b) / c was examined. The quality after storage at 45 ° C. for 6 months was also examined for the occurrence of off-flavor and the presence or absence of discoloration. Furthermore, this composition was used for the production of cosmetics (cream) according to the formulation described below, and the obtained product was also examined for quality after storage at 45 ° C. for 6 months. The results are summarized in the table below. For comparison, the control sphingolipid compositions (control products 1 and 2) obtained by the following conventional method were also subjected to various tests in the same manner as above, and the results are shown in the same table.

Preparation of Control Product 1 3.5 kg of fresh bovine brain without blood was added to 1 liter of water, homogenized for 5 minutes, and then mixed with hexane / isopropanol mixed solvent (9/5 (v / v)). 1 liter was added and the mixture was stirred for 1 hour. While filtering this to obtain a filtrate,
The residue was added again with 2 liters of a mixed solvent of hexane / isopropanol in the same ratio, stirred gently, and then filtered. The filtrates were combined and the solvent was distilled off under reduced pressure at a heating temperature of 40 ° C. or lower to obtain a concentrate. The concentrate was added to 5 liters of diethyl ether, stirred for 20 minutes and then filtered. The filtrate obtained here is a portion containing a large amount of ether-soluble glycerophospholipid. Collect the residue that is insoluble in ether,
After adding 250 ml of chloroform to this and dispersing,
The mixture was added to 5 liters of acetone that had been cooled to about 4 ° C, stirred for 20 minutes, allowed to stand at 4 ° C overnight, and then filtered to obtain an insoluble matter. The solvent was removed under reduced pressure to obtain 88 g of an almost odorless pale yellow powder. This is designated as Control Product 1.

Preparation of Control Product 2 7 kg of fresh bovine brain without blood was freeze-dried to obtain 1.1 kg of bovine brain powder. 20 liters of ethanol was added to this and extraction operation was performed at 50-60 degreeC for 2 hours. The ethanol extract obtained by removing the residue by filtration was desolvated under reduced pressure to give a brown paste-like crude beef brain sphingolipid composition 29.
0 g was obtained. This product is referred to as Control Product 2.

Cream Formulation The following raw materials were uniformly mixed and emulsified by the following procedure to produce a cream. Type of raw material Mixing ratio (%) (1) Sphingolipid composition 1.5 (2) Stearic acid 2.0 (3) Stearyl alcohol 1.0 (4) Reduced lanolin 1.8 (5) Squalane 10.0 ( 6) Octyldodecanol 6.0 (7) P. O. D (25) cetyl ether 3.0 (8) Glyceryl monostearate 2.0 (9) Perfume 0.3 (10) Preservative 0.2 (11) Glycerin 5.0 (12) Water remaining amount Total 100 0.0 (%) Procedure: Raw materials (1) to (8) were mixed, melted by heating, maintained at 80 ° C., and then stirred with a homomixer and brought to the same temperature (10) to (12). Add the mixed raw material of 60 ℃
Add raw material (9) to homogenize and cool.

[0022]

[Table 1]

Note 1: In the table, a means galactosyl cerebroside, b means galactosyl cerebroside sulfate, c means sphingomyelin, PE means phosphatidylethanolamine, PC means phosphatidylcholine, and PS means phosphatidylserine. Note 2: O and X in the table have the following meanings. Occurrence of offensive odor ○: No offensive odor is observed ×: Presence or absence of discoloration with offensive odor (animal odor) ○: No discoloration is observed ×: Discoloration (coloring) is observed Any sample should be tested for storage stability. Both of them were housed and sealed in an aluminum bag at a rate of 5 g, and stored at 45 ° C. for 6 months (corresponding to storage at room temperature for about 3 years). Note 3: Composition analysis of the sphingolipid composition uses TLC and TLC-FID method (yatroscan method),
The measurement was performed under the following conditions. TLC plate: silica gel 60 (manufactured by Merck) Developing solvent: chloroform / methanol / water = 65: 2
5: 4 Detection reagent: 50% sulfuric acid (general lipid), Dittmer reagent (phospholipid) Spot amount: 100 μg The TLC result is shown in FIG. TLC-FID method Rod: Chromarod S-III Developing solvent: First time chloroform / methanol / water / acetic acid (8.5 cm) = 70: 30: 1.5: 0.5 Second time Hexane / ethyl ether / formic acid (10 cm) ) = 9
0: 10: 0.1

[Brief description of drawings]

FIG. 1 shows the TLC analysis results of the sphingolipid composition used in the test example of the present invention. In the figure, is a sample of control product 2, is a sample of control product 1, is a sample of the present invention, SF is solvent front (upper end of development), CH is cholesterol, CMH is galactosyl cerebroside, PE is phosphatidylethanolamine, CSE represents galactosyl cerebroside sulfate, PC represents phosphatidylcholine, PS represents phosphatidylserine, SPM represents sphingomyelin, and O represents origin (sample spot position).

Claims (2)

[Claims] 1. Galactosyl cerebroside (a) and galactosyl cerebroside sulfate (b) in the total amount.
And the sphingolipid content of the three components of sphingomyelin (c) is 90% or more, the glycerophospholipid content is 2% or less, and the ratio of (a + b) / c is 65/35.
The sphingolipid composition is characterized in that it is ˜85 / 15. 2. A lipid mixture obtained from mammalian cranial nerve tissue is alkali-hydrolyzed, the solvent used for the hydrolysis is then distilled off, and the mixture is washed with a mixed solvent of acetone / water to collect insoluble matter. A method for producing a sphingolipid composition, comprising: