JP5062873B2 - Complex lipid fraction, human-type sphingomyelin and plasmalogen isolated therefrom, and functional food materials, pharmaceutical materials and cosmetic materials containing them - Google Patents

Description

  The present invention provides a composite lipid fraction obtained from chicken epidermis and containing human sphingomyelin and plasmalogen-type phosphatidylethanolamine, the components isolated from the composite lipid fraction, and the composite lipid fraction. A sphingolipid mainly composed of human-type sphingomyelin obtained by alkali treatment, a functional food material containing these, a pharmaceutical material, a cosmetic material, the composite lipid fraction, and a sphingolipid mainly composed of human-type sphingomyelin. The present invention relates to a method for producing the skin efficiently and industrially advantageously.

  A lipid refers to a substance having a long-chain fatty acid or a similar hydrocarbon chain in a molecule and existing in a living body or derived from an organism. These lipids can be classified into simple lipids and complex lipids. Simple lipids are composed of C, H, and O, and are generally soluble in acetone. The simple lipid triacylglycerol exists in an animal body as a reservoir of energy in adipose tissue. On the other hand, the complex lipid is a lipid group containing P of phosphate, N of base and the like. Therefore, the complex lipid is composed of a hydrophobic part (fatty acid part) and a hydrophilic part (phosphoric acid or base part) and exhibits amphipathic properties. Generally, the simple lipid is soluble in acetone. In contrast, complex lipids are insoluble in acetone. Such complex lipids are constituents of biological membranes.

  The complex lipid includes (1) glycerophospholipid [phosphatidylcholine (also known as lecithin), phosphatidylethanolamine, and the like. ], (2) sphingophospholipid (sphingomyelin, ceramide serialin, etc.), (3) glycosphingolipid (cerebrosid, sulfatide, ganglioside, etc.), and (4) gloeroglycolipid (microorganisms and higher). There are those in which various sugars are bound to diacylglycerols existing in plants, etc.). The (2) sphingophospholipid and the (3) sphingoglycolipid are collectively referred to as sphingolipids.

  The glycerophospholipid is a general term for phospholipids having glycerophosphate as a skeleton, and includes phosphatidylcholine (lecithin), phosphatidylethanolamine, diphosphitidylglycerol, and the like. Many of these glycerophospholipids are non-polar moieties that are esters of fatty acids, but there are also plasmalogen types that have vinyl ether bonds.

  This glycerophospholipid is important as a component of biological membranes. Among them, plasmalogen-type glycerophospholipid has recently attracted attention as an antioxidative phospholipid because of its high radical sensitivity of vinyl ether bond. Recently, it has been reported that plasmalogen-type glycerophospholipid contributes to oxidative stability of phospholipid membranes containing cholesterol by a mechanism different from that of α-tocopherol (vitamin E), which is an antioxidant component of cell membranes. It is pointed out that plasmalogen-type glycerophospholipids are not only involved in the antioxidant properties of cell membranes and lipoproteins, but also have an important role in cell signal transduction systems. (For example, see Non-Patent Document 2).

  Such plasmalogen-type glycerophospholipid is expected to have an effect of preventing neuronal cell death of the brain in dementia, but in reality there are no sources that are safe and available in large quantities.

  On the other hand, sphingolipid is a general term for lipids having a long-chain base such as sphingosine, and is mainly composed of glycosphingolipid and sphingophospholipid as described above. Glycosphingolipids contain long-chain base sphingosine or phytosphingosine and others in addition to sugar and long-chain fatty acids. The simplest glycosphingolipid is cerebroside, but there are sulfatides with sulfate groups, ceramide oligohexosides with several neutral sugars, and gangliosides with sialic acid. These substances are present on the cell surface and are considered to be involved in the recognition mechanism.

  Sphingophospholipids are classified into ceramide 1-phosphate derivatives and ceramide 1-phosphonic acid derivatives, and the former is well known as sphingomyelin and the latter as ceramide syratin (ceramide aminoethylphosphonic acid).

  In recent years, it has been revealed that these sphingolipids are involved in intracellular signal transduction such as ceramide, sphingosine, sphingosine-1-phosphate and the like, which are degradation metabolites thereof, and are attracting attention. Sphingolipids are also involved in the formation of membrane microdomains called “rafts” together with cholesterol, etc., and it has been clarified that these microdomains play an important role as a signal transduction field. Increasing attention.

  Such sphingolipids have been conventionally extracted and used from bovine brain, but those derived from grains and yeasts are currently used due to safety issues. However, since the sphingoid base composition constituting these sphingolipids derived from cereals and yeast is different from that of mammals, there is a problem that bioavailability is lower than that of human sphingolipids.

“J. Lipid Res.”, 44, 164-171 (2003) “J. Mol. Neurosci.”, Vol. 16, pp. 263-272; discussion 279-284 (2001)

  Under such circumstances, the present invention has developed a technology capable of easily and mass-producing human-type sphingolipid (sphingomyelin) and plasmalogen-type glycerophospholipid from a safe unused source, It is an object of the present invention to provide a functional food material containing human sphingolipid (sphingomyelin) and plasmalogen glycerophospholipid.

  As a result of intensive studies to achieve the above object, the present inventors have found that human-type sphingomyelin and plasmalogen-type phosphatidylethanolamine in the chicken epidermis are 0. It was found that about 07% by mass was contained, and that the chicken skin was subjected to a specific treatment, whereby they could be easily and mass-produced, and the present invention was completed based on this finding.

（式中、Ｒ−ＣＯは脂肪酸残基を示す。）
の構造を有し、スフィンゴイド塩基が４−トランス−スフィンゲニンのヒト型スフィンゴミエリンおよび式（ＩＩＩ）

の構造を有するプラズマローゲン型のホスファチジルエタノールアミンを含むことを特徴とする複合脂質画分、
（２）ニワトリ表皮が産卵成鶏表皮である上記（１）項に記載の複合脂質画分、
（３）上記（１）または（２）項に記載の複合脂質画分を弱アルカリ処理してなる、ヒト型スフィンゴミエリンを主体とするスフィンゴ脂質、
（４）請求項１または２に記載の複合脂質画分を溶剤抽出してなる、プラズマローゲン型のホスファチジルエタノールアミンを主体とするプラズマローゲン型グリセロリン脂質、
（５）ニワトリ表皮を加熱処理して脱脂したのち、溶剤抽出することを特徴とする上記（１）または（２）項に記載の複合脂質画分の製造方法、
（６）ニワトリ表皮を加熱処理して脱脂したのち、溶剤抽出して複合脂質画分を得、次いで弱アルカリ処理することを特徴とする、上記（３）項に記載のヒト型スフィンゴミエリンを主体とするスフィンゴ脂質の製造方法、
（７）ニワトリ表皮を加熱処理して脱脂したのち、溶剤抽出して複合脂質画分を得、次いでエタノール抽出することを特徴とする、請求項４に記載のプラズマローゲン型のホスファチジルエタノールアミンを主体とするプラズマローゲン型グリセロリン脂質の製造方法、
（８）上記（１）または（２）項に記載の複合脂質画分、上記（３）項に記載のスフィンゴ脂質および上記（４）項に記載のプラズマローゲン型グリセロリン脂質の中から選ばれる少なくとも１種を含むことを特徴とする機能性食品素材、
（９）上記（１）または（２）項に記載の複合脂質画分、上記（３）項に記載のスフィンゴ脂質および上記（４）項に記載のプラズマローゲン型グリセロリン脂質の中から選ばれる少なくとも１種を含むことを特徴とする医薬品素材、および
（１０）上記（１）または（２）項に記載の複合脂質画分、上記（３）項に記載のスフィンゴ脂質および上記（４）項に記載のプラズマローゲン型グリセロリン脂質の中から選ばれる少なくとも１種を含むことを特徴とする化粧品素材、
を提供するものである。 That is, the present invention
(1) It is obtained by heat treating chicken skin and degreasing, followed by solvent extraction . Formula (I)

(In the formula, R—CO represents a fatty acid residue.)
A human sphingomyelin having a structure of the following formula , wherein the sphingoid base is 4-trans-sphingenin and the formula (III)

A complex lipid fraction comprising a plasmalogen-type phosphatidylethanolamine having the structure :
(2) The complex lipid fraction according to (1) above, wherein the chicken epidermis is a laying hen epidermis;
(3) A sphingolipid mainly composed of human-type sphingomyelin, which is obtained by subjecting the complex lipid fraction described in (1) or (2) to a weak alkali treatment,
(4) A plasmalogen-type glycerophospholipid mainly composed of plasmalogen-type phosphatidylethanolamine, which is obtained by solvent extraction of the complex lipid fraction according to claim 1 or 2.
(5) The method for producing a composite lipid fraction according to (1) or (2) above, wherein the chicken epidermis is heat-treated and degreased, followed by solvent extraction,
(6) Mainly comprising human sphingomyelin according to (3) above , wherein the chicken epidermis is heat-treated and degreased, followed by solvent extraction to obtain a composite lipid fraction, followed by weak alkali treatment. A method for producing a sphingolipid,
(7) Mainly comprising plasmalogen-type phosphatidylethanolamine according to claim 4, wherein the chicken skin is heat-treated and degreased, followed by solvent extraction to obtain a complex lipid fraction, followed by ethanol extraction. A method for producing a plasmalogen-type glycerophospholipid,
(8) above (1) or (2) complex lipid fraction according to claim, selected from the plasmalogen-type glycerophospholipid according to above SL (3) sphingolipids and the item (4) according to claim A functional food material characterized by containing at least one kind,
(9) selected from the plasmalogen-type glycerophospholipid according to (1) or (2) complex lipid fraction according to claim upper Symbol (3) sphingolipid according to item and the item (4) A pharmaceutical material characterized in that it comprises at least one species, and
(10) above (1) or (2) complex lipid fraction according to claim, selected from the plasmalogen-type glycerophospholipid according to above SL (3) sphingolipids and the item (4) according to claim A cosmetic material characterized in that it contains at least one kind,
Is to provide.

  According to the present invention, a chicken epidermis is used as a safe unused source, a complex lipid fraction containing human sphingomyelin and plasmalogen-type phosphatidylethanolamine, and a human sphingomyelin isolated from this complex lipid fraction. And a plasmalogen-type phosphatidylethanolamine and a sphingolipid mainly composed of human-type sphingomyelin obtained by subjecting the complex lipid fraction to a weak alkali treatment.

In addition, according to the present invention, the above-mentioned complex lipid fraction and sphingolipid mainly composed of human-type sphingomyelin can be easily and mass-produced from chicken epidermis.
Furthermore, according to the present invention, it is possible to provide a functional food material, a pharmaceutical material and a cosmetic material containing the above-mentioned human-type sphingomyelin and plasmalogen-type phosphatidylethanolamine.

The complex lipid fraction of the present invention is a fraction obtained from chicken epidermis and containing human-type sphingomyelin and plasmalogen-type phosphatidylethanolamine.
An example of the types of lipids contained in the complex lipid fraction of the present invention and the quantitative ratio thereof is as follows.
Mass ratio Sphingomyelin: 1.0
Phosphatidylethanolamine: 1.7
Phosphatidylcholine: 1.2
Cholesteryl glycoside: 0.2

  Sphingomyelin is a compound in which the primary alcoholic hydroxyl group of ceramide and choline phosphoric acid are bonded by a phosphoric acid diester, and has the following formula (I)

(In the formula, R—CO represents a fatty acid residue.)
It is generally present not only in the brain tissue of an animal body but also in organ tissues.

  Since the sphingoid base constituting the sphingomyelin derived from chicken epidermis in the present invention is mostly 4-trans-sphingogenin (sphingosine), the sphingomyelin is a human-type human sphingomyelin.

  In sphingomyelin, its degradation metabolites ceramide, sphingosine, sphingosine-1-phosphate, etc. have been shown to be involved in information transmission in fat. Involved in the formation of domains, it has been revealed that this microdomain plays an important role as a field of information transmission. Furthermore, sphingomyelin is expected to have a skin moisturizing effect and a colon cancer preventing effect.

  On the other hand, about 94% by mass of phosphatidylethanolamine (PE) is a plasmalogen type, and it was unexpected that the plasmalogen type was contained at such a high concentration. Also, phosphatidylcholine (PC) contains about 14% by mass of plasmalogen type.

  The structures of diacyl glycerophospholipid and plasmalogen glycerophospholipid are shown in the following formulas (II) and (III), respectively.

A normal glycerophospholipid (lectin) has an ester bond with a fatty acid acyl group at sn-1 (position 1) of glycerol as shown in formula (II), but the plasmalogen type is shown in formula (III). As described above, glycerol sn-1 has a vinyl ether bond having an alkenyl group.
In addition, when X is an aminoethyl group, it is phosphatidylethanolamine, and when X is a trimethylaminoethyl group, it is phosphatidylcholine.

  The plasmalogen-type glycerophospholipid has attracted attention as an antioxidant phospholipid because of its high radical sensitivity of vinyl ether bond, and is known to contribute to the oxidative stability of phospholipid membranes containing cholesterol. In addition, it has been pointed out that plasmalogen-type glycerophospholipid is not only involved in the antioxidant properties of cell membranes and lipoproteins, but also has an important role in the cell information transmission system. Such a plasmalogen type glycerophospholipid is expected to have an effect of preventing brain neuronal cell death in dementia, an onset prevention effect of atherosclerosis, and the like.

  The cholesteryl glycoside is not a lipid but a cholesterol glycoside, and about 60% by mass is galactose type and about 40% by mass is glycose type. This cholesteryl glycoside is expected to have a gastrointestinal ulcer inhibitory effect.

  Thus, the complex lipid fraction obtained from chicken epidermis contains various components having physiological activity effective for the human body, and the source is chicken epidermis and is safe. It is useful as a functional food material, pharmaceutical material and cosmetic material.

  The present invention also provides human-type sphingomyelin and plasmalogen-type phosphatidylethanolamine obtained by isolating the above-mentioned complex lipid fraction by a known method such as the following method.

<Method for isolating each complex lipid from the complex lipid fraction>
(1) Ethanol method Ethanol is added to the complex lipid fraction, heated and dissolved, cooled, and the resulting precipitate is filtered and dried to obtain solid crude sphingomyelin.
Meanwhile, ethanol is evaporated to dryness to obtain a crude phosphatidylcholine and phosphatidylethanolamine mixture.
(2) Ether method The complex lipid fraction is extracted with ethyl ether, and the ether phase is evaporated to dryness to obtain a mixture of phosphatidylcholine and phosphatidylethanolamine.
On the other hand, the ether insoluble part is dried to obtain crude sphingomyelin.
(3) Purification of crude fraction (a) Sphingomyelin: Phospholipids can be removed by a weak alkali treatment to increase the purity.
(B) Phosphatidylethanolamine: Fractionation with a silica column.
(C) Phosphatidylcholine: Fractionate on a silicate column.
These lipids are useful as functional food materials, cosmetic materials, pharmaceutical materials, and the like.

  Furthermore, the present invention also provides a sphingolipid mainly composed of human sphingomyelin, which is obtained by subjecting the complex lipid fraction to a weak alkali treatment. Among the various lipids in the complex lipid fraction, sphingomyelin is more stable to alkali (not easily hydrolyzed) than phosphatidylethanolamine and phosphatidylcholine, so that sphingomyelin is not hydrolyzed as much as possible. A sphingolipid mainly composed of human-type sphingomyelin can be obtained by alkali treatment under conditions in which amine and phosphatidylcholine are hydrolyzed.

This sphingolipid mainly composed of human-type sphingomyelin is useful as a functional food material, a cosmetic material, a pharmaceutical material, and the like.
The complex lipid fraction can be easily produced with good productivity according to the method of the present invention shown below.

In the method of the present invention, first, the chicken skin is heat-treated and degreased, followed by solvent extraction to produce a complex lipid fraction.
For the degreasing treatment of chicken epidermis, a mechanical method, a hot water immersion heating method, a direct heating method, etc. can be adopted. However, in the method of the present invention, direct heating and fat treatment are performed in terms of effects and productivity. A method of removing minutes is used.

  As heat treatment conditions at this time, it is important to select conditions that effectively remove fat and that do not decompose the complex lipid as much as possible. As processing conditions, for example, when processing using a 500 W microwave, the processing time is about 3 to 20 minutes, preferably 5 to 10 minutes. This degreasing treatment preferably removes about 30 to 60% by mass of the total lipid, and particularly preferably 40 to 50% by mass.

  Next, the chicken skin thus degreased is subjected to a solvent extraction treatment. As the solvent, those which are safe for food hygiene and have good extraction efficiency are used. As such a solvent, ethanol is particularly suitable. This extraction process can be performed according to a conventional method.

  In this way, an extract containing the complex lipid fraction is obtained. This extract may be adjusted to an appropriate concentration to form a complex lipid-containing solution, or the solvent may be removed to form a complex lipid mixture.

  In addition, the extract containing the complex lipid fraction obtained as described above is used, for example, about 0.1 to 1 mol / L, preferably 0.3 to 0.6 mol / L sodium hydroxide or water. Potassium oxide, preferably potassium hydroxide, is used in an amount of 3 to 10 times the molar amount of glyceride bonds in the mixed phospholipid, and usually in the presence of a small amount of ethanol at 0 to azeotropic temperature, preferably 20 to 60 ° C. Sphingolipids mainly composed of human-type sphingomyelin can be produced by alkali treatment at a temperature of about 30 to 300 minutes, preferably 60 to 120 minutes.

After the alkali treatment, the solution containing a crude sphingolipid containing human sphingomyelin is obtained by neutralizing with an acid such as hydrochloric acid.
A purified sphingolipid containing human sphingomyelin can be obtained by subjecting this solution to purification treatment according to a conventional method.

The present invention also provides the above-described complex lipid fraction, human-type sphingomyelin and plasmalogen-type phosphatidylethanolamine isolated from this fraction, and human-type sphingomyelin obtained by subjecting the complex lipid fraction to weak alkali treatment. The functional food material containing at least 1 sort (s) chosen from sphingolipid containing is also provided.
If desired, this functional food material can be blended with stabilizers such as vitamin E and various health food materials.

EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
Example 1 Deoiling treatment of chicken epidermis (1) Cutting and quantification of lipid components after laying of laying hen's epidermis and its fat removal treatment Cutting dermis of laying hen and its degreasing treatment (mechanical fat removal and Table 1 shows the total lipid amount and the ratio of neutral lipid and complex lipid of the product (heated and immersed in warm water). The mechanical fat removal was performed by scraping with a wooden spatula, and the hot water immersion heating treatment was performed by immersing in warm water at 60 ° C. for 90 minutes.

  As can be seen from Table 1, the ratio of neutral lipids to complex lipids is improved by a mechanical fat removal method and is 1.5 times higher than the others.

  The silicic acid thin-layer chromatogram concerning the total lipid, the complex lipid, and the lipid produced by the alkali treatment (alkali treatment condition: heated at 40 ° C. for 120 minutes using 0.5 mol / L potassium hydroxide) is shown in FIG. Total lipids are almost neutral lipids. Sphingomyelin (SM), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are contained in the complex lipid fraction, and cholesterol glucoside (CG) and cerebroside are contained in the alkane-treated fraction. (Fig. 1).

(2) Degreasing treatment of broiler skin by microwave oven (a) Removal of fats and oils from broiler skin by microwave heating treatment Broiler purchased from chicken specialty store (Obihiro City) (hereinafter referred to as “chicken”) .) Is put into a beaker and the melted fat amount and residual lipid amount of the skin portion heated for 3 to 20 minutes in a microwave oven (Sharp Corporation, RE87-HSP type 500 W) are shown in Table 2. The amount of melted fat per 100 g of skin was 13 to 24 g, there was no difference depending on the length of the treatment time, and the amount of residual lipid was 13 to 16 g.

  5 minutes and 20 minutes of heat-treated melted fat / oil components were analyzed by silicic acid TLC (FIG. 2, lanes 4 and 6), but both were substantially only triacylglycerol (hereinafter sometimes referred to as “TG”). Thus, complex lipids (the origin substance under the development conditions of A) were not found. In addition, a complex lipid group was also detected from the remaining lipid components after the heat treatment (lanes 12 and 13). In this case, TG occupied the majority (lanes 5 and 7). In the test skin (lane 11), phosphatidylethanolamine (spot A), phosphatidylcholine (spot B) and sphingomyelin (spot C) were confirmed as main complex lipids.

In FIG. 2, each lane is as follows.
1: Diacylglycerol (standard product)
2: Total lipid from old chicken epidermis 3 and 11: Total lipid in chicken skin 4: Oil and fat 5 and 12 melted by heat treatment of chicken skin (5 minutes): Heat treatment of chicken skin (5 minutes) Total fat remaining after 6: Fat 7 dissolved by heat treatment of chicken skin (20 minutes) and 13: Total fat remaining after heat treatment of chicken skin (20 minutes) 8: Phosphatidylethanolamine and phosphatidylcholine (Standard goods)
9: Cerebroside (standard product)
10: Sphingomyelin (standard product)

The developing solvent and the detection reagent are as follows.
Developing solvent: hexane-diethyl ether-acetic acid (80: 30: 1, v / v) [A]
Chloroform-methanol-water (65: 25: 4, v / v) [B]
Detection reagent: 50% H ₂ SO ₄

(B) Analytical evaluation of component pattern of heat-treated melted fat and oil of broiler skin by reverse-phase HPLC and residual TG after heat treatment Figure 3 shows reverse-phase HPLC pattern (A) of TG in test broiler skin The reverse phase HPLC pattern of heat processing melted fat and oil and residual TG after heat processing is shown. There was no difference between the heat treatment for 5 minutes and 20 minutes, and the ratio of each peak of the fused TG species was almost the same for B (5 minutes treatment) and D (20 minutes treatment). Further, when the fusion TG (B and D) and the residual TG (C and E) were compared, the latter tended to have a slightly higher ratio of peaks after the region (b) in FIG. 3 than the former.
As a result, it was found that the treatment of the skin did not contain a significant amount of complex lipid, and it was proved that the treatment was effective for deoiling the skin.

In addition, in FIG. 3, each code | symbol is as follows.
A: Triacylglycerol of chicken skin B: Dissolved by heating for 5 minutes in microwave oven C: Remained after 5 minutes of heat treatment D: Dissolved by heating for 20 minutes in microwave oven E : What remained after 20 minutes of heat treatment

(C) Heat treatment conditions and assay of complex lipid component remaining in skin part Silicate TLC of the remaining complex lipid fraction after heat treatment is shown in FIG. The silicate TLC pattern (lane 3) of the remaining complex lipid after microwave treatment for 5 minutes is similar to that of the extracted total lipid from the unheated state (lane 11 in Fig. 2), and phosphatidylethanolamine as the main component (Spot A), phosphatidylcholine (spot B) and sphingomyelin (spot C) were detected.

  Unlike the case of 5 minutes treatment (lane 3), the pattern of the complex lipid fraction in the total lipid extracted from the skin heated for 20 minutes (lane 5) has a higher ratio of spot G. The spots corresponding to SPM decreased relatively. This suggests that the PE in the skin was already decomposed during the 20 minute heat treatment. On the other hand, it was suggested that PC is relatively more stable than PE for microwave processing and hardly decomposes.

In FIG. 4, each lane is as follows.
1: Phosphatidylethanolamine and phosphatidyl zircon (standard)
2: Sphingomyelin (standard product)
3: Composite skin remaining after heat treatment (5 minutes) of chicken skin by microwave oven Composite lipid 4: 3 weakly alkaline treatment product 5: Composite lipid remaining after heat treatment of microwave oven by chicken skin (20 minutes) 6: 5 weakly alkali-treated product

The developing solvent and the detection reagent are as follows.
Developing solvent: chloroform-methanol-water (65: 25: 4, v / v)
Detection reagent: 50% H ₂ SO ₄

As a result of the above, it was found that the condition for efficiently separating and preparing the complex lipid group from the chicken skin by heat treatment with a microwave oven (500 W) is preferably less than 20 minutes, and some complex lipids are degraded in 20 minutes. .
From the results of (a) and (b), it is confirmed that PE, PC, and SPM are contained in the epidermis of broilers that are the main species of poultry and spawning hens, and are used as raw materials for these extractions. It was done.

Example 2 Separation of composite lipid fraction from laying adult chicken epidermis after deoiling and composition test Extraction of ethanol from epidermis after removal of about 1/2 of oil by microwave heating treatment (heating in 500W microwave for 15 minutes) A complex lipid fraction was prepared at a concentration of 3% by mass. The composition of the fractions was phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SPM) and cholesteryl glucoside (CG).
The complex lipid fraction itself is a composition to which a stabilizer such as vitamin E is added, and is highly useful as a complex functional food factor, as a cosmetic material, or as a pharmaceutical material.

Example 3 Separation of phosphatidylethanolamine (PE) and sphingomyelin (SPM) and their structure test (1) Assay and quantification of phospholipid group of complex lipid (a) Quantification of SPM
The separated complex lipid fraction was dissolved in chloroform-methanol (mass ratio 2: 1) and subjected to normal phase HPLC with a light scattering detector to quantify sphingomyelin. The operating conditions of HPLC were Lichlorosphere Si60 (5 μ × 200 mm, manufactured by Merck) for the column, chloroform as the solvent A and methanol-water (mass ratio 95: 5) as the solvent B, A: B (Mass ratio 99: 1) at a ratio of 75:25 by 15 minutes, 10:90 by 25 minutes, then 5:95 by 35 minutes, then 99: 1 by 40 to 45 minutes A two-liquid gradient was performed. The flow rate was 1.0 ml / min.

Results of subjecting the complex lipid fraction prepared from the laying hen's epidermis and its weakly alkaline product (treatment condition: using 0.5 mol / L potassium hydroxide, heating at 40 ° C. for 120 minutes) to normal phase HPLC Is shown in FIG. The sphingomyelin isolated from the standard bovine brain was separated into two peaks on HPLC, but the peak group with the same retention time in the composite lipid fraction of the laying hen's epidermis (both peaks are shown as peak 4). ) Was observed. Since peak 4 was detected even after weak alkali treatment, it was determined to be sphingomyelin. The main peaks 2 and 3 are presumed to be phosphatidylethanolamine and phosphatidylcholine, respectively, considering the result of the two-dimensional silicic acid TLC analysis (FIG. 6). Moreover, cerebroside (corresponding to the alkaline hydrolysis product of sphingomyelin) and cholesteryl glucoside are included in the group of peaks remaining after the weak alkali treatment.
The amount of sphingomyelin in the laying adult chicken epidermis (fat-removed sample) accounted for 21% by mass of the complex lipid, calculated from the calibration curve, and 0.7 mg per 1 g of the sample (fat-removed sample).

In FIG. 5, the measurement conditions and the respective symbols are as follows.
A: Complex lipid of spawning chicken epidermis B: Alkaline processed product C: Standard sphingomyelin

Measurement condition column: LiChrosphere Si60
Column temperature: 35 ° C
Eluent: Above

Moreover, the developing solvent and detection reagent in FIG. 6 are as follows.
A: Developing solvent 1st dimension chloroform-methanol-acetic acid (65:25:10, v / v)
Second dimension chloroform-methanol-formic acid (65:25:10, v / v)
Detection reagent Dittmer reagent B: developing solvent 1st dimension chloroform-methanol-25% ammonia
(65: 35: 8, v / v)
Second dimension Chloroform-acetone-methanol-acetic acid-water
(50: 20: 10: 10: 5, v / v)
Detection reagent 50% H ₂ SO ₄

(B) Determination of PE, PC and CG Similarly, phosphatidylethanolamine (PE) (content 35% by mass), phosphatidylcholine (PC) (content 25% by mass) and CG (content 5% by mass) Obtained.

Example 4
The separated complex lipid fraction (0.5 g) was subjected to a preparative high-performance liquid chromatograph and subjected to phosphatidylethanolamine (PE) 170 mg, phosphatidylcholine (PC) 220 mg, sphingomyelin (SPM) 100 mg, and cholesterol glucoside (CG) 20 mg. , Each was separated into corresponding fractions.

Example 5 Structure analysis of PE and SPM (1) Structure identification of PE (a) Confirmation of plasmalogen type As a result of methanolysis of the separated complex lipid fraction and GC analysis of its hexane extract, at least 16 fatty acid methyl esters And three kinds of dimethyl acetal peaks were detected. The ratio of fatty acid methyl ester to dimethyl acetal was 89:11 for the entire polar lipid fraction (mass ratio, the same applies hereinafter), 54:46 for PE, and 94: 6 for PC (Table 3). This revealed that most of the dimethyl acetal was derived from PE. Assuming that PE is composed of two types, 1,2-diacyl type, 1-alkenyl and 2-acyl type, 1-alkenyl and 2-acyl type (plasmalogen) account for 94% by mass The remaining 6% by mass is diacyl type. On the other hand, with PC, 14% by mass of the whole is judged to be plasmalogen.

(B) Composition analysis and identification of dimethylacetal derived from alkenyl group of plasmalogen in chicken epidermis The methanolic polar lipid fraction, PE and PC of chicken epidermis were methanolized with methanolic 5 mass% HCl solution at 100 ° C for 3 hours. The composition of the obtained dimethyl acetal is shown in Table 4. All lipids were found to be saturated with 16 and 18 carbon atoms and monoene with 18 carbon atoms. Among them, 18: 0 accounted for the majority in PE, followed by 16: 0 and 18: 1. On the other hand, in PC, 16: 0 was 70% by mass and 18: 0 was 25% by mass.

(2) Structural analysis and identification of SPM (a) Identification of sphingolipid constituents In the analysis of constituent fatty acids, a weakly alkaline-treated product of the composite lipid fraction was methanolized with methanolic mass 5% HCl at 100 ° C. for 3 hours. The free fatty acid methyl ester is extracted with hexane, and this is subjected to silicic acid thin layer chromatography (TLC) with hexane-diethyl ether (mass ratio 85:15) to obtain normal fatty acid (unsubstituted acid) methyl and 2-hydroxy fatty acid. Fractionated into methyl, the former was used as it was, and the latter was converted into an acetyl derivative before being subjected to GC-MS analysis. The result of subjecting the hexane extract to silicic acid TLC is shown in FIG. Two major spots (1 and 2) were detected and few spots (3) of hydroxy fatty acid methyl esters. Spot 4 is derived from a constituent sterol of cholesterol glucoside. FIG. 8 shows the result of separating spots 1 and 2 and subjecting them to GC. In spot 1, a fatty acid methyl ester peak from 14: 0 to 24: 2 was detected, and a small 18: 0 dimethylacetal (DMA) peak was also observed (FIG. 8-B). In spot 2, three dimethyl acetal peaks of 16: 0, 18: 0 and 18: 1 were mainly detected (FIG. 8-C). From these, it is judged that spot 1 is a fatty acid methyl ester derived from a constituent fatty acid of sphingolipid, and spot 2 is a dimethyl acetal derived from a plasmalogen type phospholipid. Further, spot 3 was collected, acetylated, and analyzed by GC. As a result, saturated 2-hydroxy fatty acid methyl ester having 18 to 26 carbon atoms was detected (FIG. 8-D). FIG. 8-A is a gas chromatogram of the acetylated product of the entire hexane extract. As a result of the silicic acid TLC analysis in FIG. 7, it was confirmed that unsubstituted fatty acids (normal acids) accounted for most of the constituent fatty acids of sphingolipids, and that hydroxy fatty acids were minor components.

In addition, each code | symbol in FIG. 8 is as follows.
A: Total hexane extract (injected into GC after acetyl derivatization)
B: Spot 1 in FIG.
C: Spot 2 in FIG. 1 (dimethyl acetal, abbreviated as DMA)
D: Acetyl derivative at spot 3 in FIG.

  For analysis of constituent sphingoid bases, the alkali-stable complex lipid fraction was degraded with hydrous methanolic 1M HCl at 80 ° C. for 18 hours. After the decomposition solution was extracted with hexane, the residual solution was made alkaline, and then the sphingoid base released with diethyl ether was extracted. This was subjected to silicic acid TLC with chloroform-methanol-2N aqueous ammonia (80: 20: 2) to separate into dihydroxy base and trihydroxy base, then each was oxidized with periodate to induce fatty aldehyde, GC-MS analysis was performed. GC-MS was performed using a QP-2010 gas chromatograph-mass spectrometer manufactured by Shimadzu Corporation. A polar capillary column (CP-Sil 88) was used for separation of each component.

  Usually, since the constituent fatty acids of sphingolipids are saturated fatty acids and monoene fatty acids, mass spectra were analyzed to confirm the 24: 2 fatty acid peak. For comparison, FIG. 9 also shows mass spectra of 24: 0 (lignoceric acid) and 24: 1 (nervonic acid). In each spectrum, molecular ions were detected at m / z 382, 380 and 378, indicating 24: 0, 24: 1 and 24: 2 fatty acid methyl, respectively. In the unsaturated type, ions of M-32 and M-74, which are characteristic of methyl esters, were also observed.

  Table 5 shows the composition of normal and hydroxy constituent fatty acids of the sphingolipid fraction prepared from chicken epidermis. The main normal fatty acids were palmitic acid (16: 0), nervonic acid (24: 1) and stearic acid (18: 0), especially palmitic acid. On the other hand, as the hydroxy fatty acid, there are many very long chain types, and among them, the ratio of 2-hydroxylignoceric acid (24h: 0) was the highest.

(B) Identification of sphingoid bases of sphingolipids contained in the laying hen's epidermis FIG. 10 shows the results of subjecting the sphingoid base fraction constituting the sphingolipids of the epidermis to silicic acid TLC. In the region A in the figure, it is known that 3-O-methylsphingoid base, which is a byproduct of sphingoid base generated by acid decomposition, is developed. The main spot is the B region corresponding to 4-trans-sphingenin (sphingosine) and sphinganine (dihydrosphingosine) (acid degradation produces natural erythro and by-product threo forms, which are separated into dalma-like spots. No clear ninhydrin reagent positive spot was observed in the same Rf region (C) as standard 4-hydroxysphinganin (phytosphingosine).

  FIG. 11 shows the results obtained by separating the regions B and C in FIG. 10 and performing periodate oxidation to induce fatty acid aldehydes, followed by GC-MS. From the total ion chromatogram, as an aldehyde peak, three types of peaks 1 to 3 were detected from the region B in FIG. 10, and a slight peak 4 was detected from the region C. From retention time and mass spectrum, they are 16: 0 aldehyde derived from sphinganine (peak 1), 16: 1 (2t) aldehyde derived from 4-trans-sphingenin (peak 2), and C22 homologue of 4-trans-sphingenin It was determined to be 20: 1 (2t) aldehyde derived from the body (peak 3) and 15: 0 aldehyde derived from 4-hydroxysphinganine (peak 4). Although no spot could be detected in region C of FIG. 10, the ions of M-18, M-44 and M-46 indicating 15: 0 aldehyde were found in the mass spectrum of peak 4 (FIG. 11). Detected at m / z 208, 182 and 180, respectively. This means that an extremely small amount of 4-hydroxysphinganine is also present. In addition, a peak corresponding to 16: 2 aldehyde derived from plant 4-trans, 8-trans (and cis) -sphingadienin presumed to be contained in chicken feed was not detected.

In FIG. 10, each lane, developing solvent, and detection reagent are as follows.
Lane 1: Phytosphingosine (4-hydroxysphinganin, sample)
Lane 2: sphingoid base fraction derived from the laying hen's epidermis Lane 3: sphingosine (4-trans-sphingenin, preparation)

Developing solvent: chloroform-methanol-2N ammonia water
(80: 20: 2, v / v)
Detection reagent: ninhydrin

The above results confirm that the sphingomyelin is a highly bioavailable human-type sphingomyelin because the sphingoid base of the sphingolipids of chicken epidermis is mostly 4-trans-sphingenin (sphingosine). It was done.
Furthermore, in the phospholipid group, PE contains a plasmalogen type with a surprisingly high concentration of 94%, and PC also contains 14% plasmalogen type.

  The lipid component in the epidermis is mostly triacylglycerol, and if it is appropriately removed with a microwave oven or the like, a functional lipid material containing plasmalogen and sphingomyelin at a high concentration can be produced.

Example 6 SPM Functional Evaluation (1) SPM Bioavailability Differentiated Caco-2 cells were cultured for 21 days to form a monolayer, and then incubated in serum-free DMEM supplemented with 5 μmol / L sphingobase. The uptake of sphingoid bases into Caco-2 cells was compared between plant and animal sources at regular time intervals. As a result, as shown in FIG. 12, the model system experiment revealed that the sphingoid base in plants and fungi was hardly absorbed and the bioavailability was extremely low. In the figure, d18: 2 ^{4t, 8c} is 4,8-Sphingadienine,
And d18: 2 ^{4t and 8t} are 4,8-Sphingadienie and are derived from an optical isomer plant, while d18: 1 ⁴ is 4-Sphingenine (Sphingosine) and derived from an animal.

(2) Effects of sphingomyelin on skin regeneration <Materials and methods>
・ Target animal: 7-9 weeks old ICR male mouse ・ Microscopic observation: H / E staining of cryostat specimen and dermal macrophage observation by immunostaining with rat anti-mouse CD68 monoclonal antibody

(1) Experimental example 1
When the mouse dorsal shaved skin was irradiated with 50 mJ / cm ² of medium wave UV-B, the two-row cubic epithelium of the mouse epidermis changed to the stratified squamous epithelium after 3 days. When this skin was observed microscopically, many macrophages were accumulated in the dermis directly under the epidermis. The stratified squamous epithelial cells were removed by apoptosis after 5 days of irradiation, and became the original two-row cubic epithelium in 7-10 days.
When the mouse epidermis that became stratified squamous by UV-B irradiation was coated with sphingomyelin at various concentrations (1, 10, 100 mg), it was found that the recovery was accelerated by the 10 mg and 100 mg application, and at the same time, macrophage The results of histological and immunohistochemical searches for changes, etc., supported this.

(2) Experimental example 2
Irradiation with UV-B at 100 mJ / cm ² causes burns. Immediately after UV irradiation, sphingomyelin was applied at the same concentration as in Experiment 1. When sphingomyelin was applied to repair burned skin, 10 mg and 100 mg were observed.

Example 7 Application to food materials and foods: Formulation example Formulation example 1 Granule formulation of concentrated mixture 0.5 mg of complex lipid mixture prepared in Example 2, 0.1 mg of vitamin E, sucrose fatty acid ester, reduced maltose, reduced palatinose, Granules were prepared from a 1000 mg excipient such as beet oligosaccharide, dextrin, tricalcium phosphate, and other emulsifiers and binders by a fluidized bed granulation method as usual.

Formulation Example 2 Sphingomyelin Granule Formulation Emulsifiers such as sphingomyelin 0.1 mg, vitamin E 0.02 mg, sucrose fatty acid ester, reduced maltose, reduced palatinose, beet oligosaccharide, dextrin, tricalcium phosphate, etc. prepared in Example 4 Granules were prepared from 1000 mg of an excipient such as a binder by a fluidized bed granulation method as usual.

Formulation example 3 Granule formulation of plasmalogen-type phosphatidylethanolamine 0.1 mg of plasmalogen-type phosphatidylethanolamine prepared in Example 4, 0.02 mg of vitamin E, sucrose fatty acid ester, reduced maltose, reduced palatinose, beet oligosaccharide, dextrin, Granules were prepared from 1000 mg of an emulsifier / binder excipient such as tricalcium phosphate by a fluidized bed granulation method as usual.

Example 7 Prescription example of application to pharmaceuticals and cosmetics Formulation example 1 Pharmaceutical tablet; sphingomyelin Sphingomyelin 10 mg, vitamin E 2 mg prepared in Example 4, sucrose fatty acid ester, reduced maltose, reduced palatinose, beet oligosaccharide, dextrin, Granules were prepared from 988 mg of excipients such as emulsifiers and binders such as tricalcium phosphate by a fluidized bed granulation method as usual. An emulsifier is added to the granules, mixed well, and compressed into 250 mg tablets by a conventional method.
Similarly, 250 mg tablets were tableted from plasmalogen-type phosphatidylethanolamine granules.

Formulation Example 2 Cosmetic emulsion; sphingomyelin (oil phase)
Squalane 15.0%
Vaseline 7.0
Stearyl alcohol 3.0
Stearic acid 3.0
Glycerol monostearate 3.0
Polyethyl acrylate 0.5
Sphingomyelin (prepared in Example 4) 0.5
Antioxidant appropriate amount Preservative appropriate amount Fragrance appropriate amount

(Water phase)
Purified water 61.0
1,3 Butylene glycol 7.0

The oil phase part and the water phase part are each heated to 70 ° C. and dissolved. Add the oil phase to the water phase and emulsify using a homogenizer. The emulsion was cooled using a heat exchanger to obtain an O / W emulsion.
Similarly, an emulsion was obtained using the complex lipid mixture prepared in Example 2.

  According to the present invention, human-type sphingolipids and plasmalogen-type glycerophospholipids that are useful in the food field, cosmetic field, pharmaceutical field, etc. can be easily and mass-produced from chicken skin, which is an inexpensive unused resource. it can.

It is a silicic-acid thin layer chromatogram of the lipid component in an egg-laying hen's epidermis part. It is the silicic acid thin-layer chromatogram of the fat and oil which melt | dissolved by the microwave oven heating of the broiler skin part, and the lipid extracted from the skin part after heat processing. It is a reverse phase HPLC chart of triacylglycerol in a broiler skin part. It is a thin layer chromatogram of the complex lipid component in a chicken skin part. It is a HPLC chart of the complex lipid and its alkali stable lipid in the laying hen's epidermis part. It is a two-dimensional thin layer chromatogram of the complex lipid fraction prepared from the laying hen's epidermis. It is a silicic acid thin layer chromatogram which shows the constituent fatty acid of the weak alkali processed material of the composite lipid fraction prepared from the egg-laying hen's epidermis part. It is a GC-MS chart of the hexane extract after methanolysis of the weak alkali stable complex lipid fraction in the laying hen's epidermis. It is a GC-MS chart which shows the constituent fatty acid of the sphingolipid in the egg-laying chicken epidermis part. It is a silicic-acid thin-layer chromatogram of the sphingoid base fraction which comprises the sphingolipid in an egg-laying hen's epidermis part. It is a GC-MS chart of the aldehyde derived from the sphingoid base which comprises the sphingolipid in the egg-laying hen's epidermis part. It is a graph which shows the result of having taken up the sphingoid base in a differentiation Caco-2 cell, the thing derived from a chicken epidermis, and the thing of plant origin and animal origin.

Claims (10)

It is obtained by heat-treating chicken skin and degreasing, followed by solvent extraction . Formula (I)

(In the formula, R—CO represents a fatty acid residue.)
A human sphingomyelin having a structure of the following formula , wherein the sphingoid base is 4-trans-sphingenin and the formula (III)

A complex lipid fraction comprising a plasmalogen-type phosphatidylethanolamine having the structure : The complex lipid fraction according to claim 1, wherein the chicken epidermis is a laying hen epidermis. A sphingolipid mainly composed of human-type sphingomyelin, which is obtained by subjecting the complex lipid fraction according to claim 1 or 2 to a weak alkali treatment. A plasmalogen-type glycerophospholipid mainly composed of plasmalogen-type phosphatidylethanolamine, which is obtained by solvent extraction of the complex lipid fraction according to claim 1 or 2. The method for producing a complex lipid fraction according to claim 1 or 2 , wherein the chicken skin is degreased by heat treatment and then extracted with a solvent. The sphingolipid mainly composed of human-type sphingomyelin according to claim 3, wherein the chicken epidermis is heat-treated and degreased, followed by solvent extraction to obtain a composite lipid fraction, followed by weak alkali treatment. Production method.   The plasma mainly composed of plasmalogen-type phosphatidylethanolamine according to claim 4, wherein the chicken epidermis is heat-treated and degreased to obtain a complex lipid fraction by solvent extraction and then ethanol extraction. A method for producing Logen-type glycerophospholipid. Complex lipid fraction according to claim 1 or 2, functions, characterized in that it comprises at least one selected from the plasmalogen-type glycerophospholipid according to sphingolipids and claim 4 according to 請 Motomeko 3 Sex food material. Complex lipid fraction according to claim 1 or 2, medicament, characterized in that it comprises at least one selected from the plasmalogen-type glycerophospholipid according to sphingolipids and claim 4 according to 請 Motomeko 3 Material. Complex lipid fraction according to claim 1 or 2, cosmetic, characterized in that it comprises at least one selected from the plasmalogen-type glycerophospholipid according to sphingolipids and claim 4 according to 請 Motomeko 3 Material.

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