JP2008125365A - Method for preparing high-purity plasmologen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide high-purity plasmologen derived from a marine product. <P>SOLUTION: A method for preparing the plasmologen is carried out as follows: a marine oil and fat is reacted with a 1,3-specific lipase and the plasmologen is then separated. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for preparing a high-purity plasmalogen.

  Phospholipid is a lipid component that is widely present in the living world and is known to be one of the main components constituting a biological membrane. Phospholipids are classified into glycerophospholipids having glycerol as a basic skeleton and sphingophospholipids having a sphingosine base as a basic skeleton. Glycerophospholipids include diacyl phosphates in which long-chain fatty acids are ester-bonded at the 1- and 2-positions of glycerol, as well as alkenyl acyl-type phospholipids in which long-chain alcohols instead of fatty acids are vinyl-ether-bonded to 1-position of glycerol (plasmalogens) Similarly, there are ether-type phospholipids having a long-chain alkyl group at the 1-position of glycerol.

  All animals, including fish, have diacyl phospholipids as the major phospholipids, but also reliably have small amounts of plasmalogens. Plasmalogens are generally abundantly contained in tissues that consume a large amount of oxygen, such as brain, heart muscle, and nerve cells, and are recognized to have antioxidant activity. Although there are still many unclear points about the function of plasmalogens, it has recently been reported that plasmalogens derived from the sea squirt of marine animals have a preventive effect on Alzheimer-type dementia. In addition, plasmalogens have been pointed out to suppress DNA fragmentation due to apoptosis of nerve cells and to reduce brain learning function degradation due to β-amyloid peptide accumulation (Non-patent Document 1). Thus, plasmalogens, which are physiologically active phospholipids, are of great interest.

  Since plasmalogens and diacyl-type phospholipids have similar chemical structures and properties, it is extremely difficult to isolate only plasmalogens from the lipid fraction in their intact form (intact form). As a method for quantifying plasmalogens in lipid fractions, an iodine method and a derivatization method have been established (Non-patent Documents 2 and 3). In these methods, however, plasmalogens can be isolated as they are. Impossible. Currently, the purity of plasmalogens supplied to the market is about 20%, and there is a demand for providing plasmalogens with higher purity.

  On the other hand, conventional methods for producing glycerophospholipids include an acid anhydride method and an acid chloride method. Furthermore, Patent Documents 1 and 2 have a desired fatty acid composition by transesterifying a phospholipid having a single fatty acid composition, a fatty acid, and the like under a slight water condition using a 1,3-position specific lipase. A method for producing diacyl phospholipids is disclosed. However, plasmalogens cannot be produced by these methods, and development of a method capable of providing high-purity plasmalogens is still required.

  By the way, in recent years, attention has been focused on utilization of marine oils and fats such as fish oil. Marine fats and oils have a common component characteristic of containing a large amount of highly unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, and are distinguished from vegetable oils and terrestrial animal fats in this respect. Since these highly unsaturated fatty acids have various physiological functions such as a reduction in neutral fat and a platelet aggregation inhibitory effect, marine fats and oils are expected to be useful in health. However, since highly unsaturated fatty acids exist in the form of neutral fats and phospholipids in vivo, further development of methods for preparing these lipid components is indispensable for more effective utilization of highly unsaturated fatty acids. is there. Marine oils and fats are expected to be used in terms of safety because they are considered to have less risk of contamination with infectious agents than conventional animal fats and oils.

JP-A-4-71495 JP-A-4-71496 By Hidehiko Hibino, supervised by Morihiko Sakaguchi and Takashi Hirata, "Development of marine-derived fat-soluble materials, advanced effective use of marine resources-Aiming for zero emissions", (2005), NTS, Tokyo, pp 133-148 J.N.Williams, C.E.Anderson, and A.D.Jasik, J. Lipid Res., (1962) 3, p.378-381 K. Waku, H. Ito, T. Bito, and Y. Nakazawa, J. Biochem. (1974) 75, p.1307-1312

  An object of the present invention is to provide a high-purity plasmalogen derived from a marine product.

  As a result of intensive studies to solve the above problems, the present inventors have used diacyl phospholipids without degrading plasmalogens (alkenyl acyl phospholipids) using the 1,3-position specific lipase. Based on this finding, the present invention has been completed.

That is, the present invention includes the following.
[1] A method for preparing a plasmalogen, comprising reacting a marine oil and fat with a 1,3-position specific lipase and then separating the plasmalogen.
[2] The method according to [1] above, wherein the marine fat is fish oil.
[3] The method according to [1] or [2] above, wherein the 1,3-position specific lipase is derived from Rhizopus oryzae.
[4] The method according to [1] to [3] above, wherein the plasmalogen is a choline-type plasmalogen.

  According to the method for preparing plasmalogens of the present invention, high-purity plasmalogens can be easily produced from marine oils and fats.

Hereinafter, the present invention will be described in detail.
In the present invention, by reacting a marine oil and fat with a 1,3-position specific lipase, the diacyl-type phospholipid is specifically decomposed, and the plasmalogen derived from the marine fat is separated by separating the plasmalogen from the reaction product. It relates to a method of preparing with higher purity.

  Aquatic fats and oils used in the method of the present invention may be any animal that inhabits the water (for example, fish such as tuna, skipjack, saury, sardine, sea animals such as whales and seals, molluscs such as squid and octopus, starfish, jellyfish, The oil may be derived from sea urchins or the like. Although not limited, as fishery fats and oils, fish oil, liver oil, sea animal oil (such as whale oil), squid oil, and fish wax are mentioned, for example. The aquatic fats and oils used in the method of the present invention are more preferably fats and oils derived from tissues rich in plasmalogens, such as the heart (myocardium) and brain. Plasmalogens contained in these marine oils and fats have a relatively high content of highly unsaturated fatty acids. Preparation of aquatic fats and oils from biological tissues may be carried out according to a conventional method. For example, it can be prepared by adding several times the amount of chloroform-methanol (2: 1) mixture to a sample, shaking and mixing to perform lipid extraction. (Refer to Yasuhiko Fujino, “Introduction to Lipid Analysis”, Society of Science Publishing Center, 1978). The marine fats and oils used in the present invention may be a lipid fraction obtained by extracting total lipids from an organ or tissue of any animal living in water using an organic solvent or the like by a conventional method. The aquatic fats and oils used in the present invention may also be a phospholipid fraction further separated from the lipid fraction thus obtained, and any lipid fraction or any phospholipid fraction prepared to contain the phospholipid fraction. It may be a test sample.

  The aquatic fats and oils prepared as described above contain phospholipids such as plasmalogens (alkenyl acyl phospholipids) and alkylacyl phospholipids in addition to the main phospholipid diacyl phospholipids. In the method of the present invention, diacyl phospholipids are specifically decomposed so as not to decompose plasmalogens in marine fats and oils, thereby reducing diacyl phospholipids mixed in the plasmalogen fraction, and the degree of plasmalogen purification. Can be improved.

  By the way, various reports have been made so far regarding analysis results of phospholipid fractions in marine fats and oils. For example, the choline-type phospholipid fraction prepared from walleye pollock contains 54.8%, 7.2%, and 38.0% of diacyl-type phospholipid, plasmalogen (alkenylacyl-type phospholipid), and alkylacyl-type phospholipid, respectively. On the other hand, the ethanolamine phospholipid fraction contained 64.3%, 29.7%, and 6.0% diacyl phospholipid, plasmalogen (alkenyl acyl phospholipid), and alkylacyl phospholipid, respectively ( Kikuchi K. et al., Comp. Biochem. Physiol., (1999) 124B, p.1-6). In addition, analysis results on the phospholipid content and phospholipid composition in the heart as shown in Table 1 below have been reported (Kikuchi K. et al., Comp. Biochem. Physiol., (1999) 124B, p.1). -6).

  Thus, it is known that the proportion of plasmalogen in the marine oil is usually relatively small.

  On the other hand, the 1,3-position specific lipase used in the method of the present invention is a lipase capable of specifically decomposing the ester bond at the 1-position (sn-1 position) and 3-position (sn-3 position) of lipids. Say. For example, triacylglycerol, which is a main component of neutral fat, has ester bonds to which fatty acids are bonded at the 1st, 2nd and 3rd positions. In the diacyl phospholipid, an ester bond to which a fatty acid is bonded is present at the 1-position and the 2-position. Therefore, the 1,3-position specific lipase acts on the ester bond at the 1-position and 3-position of the glycerol skeleton of triacylglycerol and the 1-position of the glycerol skeleton of diacyl phospholipid, and can decompose them. On the other hand, the 1- and 3-position specific lipases do not act at all on plasmalogens having fatty acid ester-bonded only at the 2-position and no ester bonds at the 1- and 3-positions, and cannot be decomposed. Specific examples of the 1,3-specific lipase include 1,3 derived from microorganisms (fungi or bacteria) such as Rhizopus sp., Mucor sp., Aspergillus sp., Eurotium sp., Chromobacterium sp., Penicillium sp., Pseudomonas sp. Examples include position-specific lipases, but are not limited thereto. The 1,3-position specific lipase may be lipase F, pancreatic lipase, or the like. The preferred 1,3-position specific lipase is a lipase derived from Rhizopus oryzae (lipase F), for example, manufactured and sold by Amano Enzyme as lipase F-AP15.

The marine oil and fat and the 1,3-position specific lipase may be reacted by a conventional method according to the properties such as optimum pH and optimum temperature of each lipase. Usually, a 1,3-position specific lipase may be added to a solution containing aquatic fats and oils and incubated under conditions where the activity of the lipase is observed. Specifically, the examples described later can be referred to, but in brief, taking a lipase derived from Rhizopus oryzae as an example, an organic solvent (such as a benzene / ethanol (1: 1) mixture) is added to the marine oil. Solubilize the lipid, add lipase-specific lipase derived from Rhizopus oryzae to the lipid solution, pH 5.0-8.0 (more preferably pH 7.0), 25 ° C-45 ° C (more preferably Incubating for 30 minutes to 2 hours (more preferably 30 minutes) under the condition of 37 ° C.), the marine oil and fat can react with its 1,3-position specific lipase. The addition amount of the 1,3-position specific lipase is not limited, but typically, 0.25 milligram to 1 milligram per 100 micrograms of fat is used. By this reaction, the diacyl phospholipid in the marine oil can be specifically decomposed. In this reaction, it is preferable that 50% or more of the diacyl phospholipid contained in the marine oil is decomposed. By this reaction, diacyl phospholipid is decomposed into 2-lyso form (2-acyl lysophospholipid) and free fatty acid, but plasmalogen (particularly preferably choline-type plasmalogen) remains without being decomposed (FIG. 1).
Here, the diacyl type phospholipid in the present invention is represented by the formula 1.

In the formula (1), R ₁ CH ₂ and R ₂ CO represent fatty acid residues, and X represents a nitrogen-containing alcohol group such as choline, ethanolamine, or serine.

  The plasmalogen (alkenyl acyl type phospholipid) in the present invention is represented by the formula 2.

In the formula (2), CH = CHR ₁ represents a vinyl ether-bonded long chain alcohol residue, R ₂ CO represents a fatty acid residue, and X represents a nitrogen-containing alcohol group such as choline, ethanolamine or serine. For example, choline-type plasmalogen (also referred to as choline plasmalogen) is represented by the formula 2 in which X is —CH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ .

  As described above, diacyl phospholipids have fatty acid ester bonds at the 1st and 2nd positions, whereas in all plasmalogens, long chain alcohols are vinyl ether bonded at the 1st position and fatty acids are ester bonded at the 2nd position. ing. In the method of the present invention, the diacyl phospholipid is decomposed into a lyso form by the ester bond being degraded by the 1,3-position specific lipase, but the plasmalogen is not degraded by the 1,3-position specific lipase. Diacyl phospholipids and plasmalogens are very similar in structure and properties, so it is difficult to separate them into separate fractions, but diacyl phospholipids are degraded by the 1,3-position specific lipase. The lyso form can be easily separated from the plasmalogen.

  After reacting marine oil and fat with 1,3-position specific lipase as described above, if necessary, the lipase is deactivated to stop the reaction, and then plasmalogen is separated from the lipase reaction product. . Separation of plasmalogen from the lipase reaction product means separation of the plasmalogen fraction from at least a part of other lipid components contained in the lipase reaction product. Plasmalogens may be separated according to a conventional technique capable of fractionating or separating lipids. For example, lysophospholipids derived from diacyl phospholipids and free fatty acids are separated by thin layer chromatography (TLC), high performance liquid chromatography, etc. Can be removed from plasmalogen. The plasmalogen fraction thus separated contains a high content of plasmalogen. In this plasmalogen fraction, components other than plasmalogen (such as alkylacyl-type phospholipids and diacyl-type phospholipids) may remain, but the plasmalogen is compared with the phospholipid composition of the marine oil used as the material. It is preferable that the purity (content rate) of is higher. This plasmalogen fraction may be collected by elution from a chromatography column by a conventional method and then further purified. The plasmalogen concentrated in the plasmalogen fraction in this method may be a choline-type plasmalogen, ethanolamine-type plasmalogen, serine-type plasmalogen, or the like, more preferably a choline-type plasmalogen.

  Plasmalogens (fractions) prepared in this way from marine oils and fats are highly unsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), preferably in the 2nd position of the glycerol skeleton, with a high content. May be included. Accordingly, the plasmalogen (fraction) derived from aquatic fats and oils obtained by the method of the present invention is also useful as a source of highly unsaturated fatty acids known to have various advantageous physiological activities. The fact that plasmalogens derived from aquatic fats and oils contain a large amount of highly unsaturated fatty acids has been reported by Kikuchi K. et al., "Fatty acid composition of choline and ethanolamine glycerophospholipid subclasses in heart tissue of mammals and migratory and demersal fish." Comp. Biochem. Physiol. (1999) 124B, p.1-6. In this document, the main fatty acid composition (mol%) of myocardial plasmalogen is shown. For example, myocardial cholinergic plasmalogens derived from bovine (control), tuna and skipjack are fatty acid 18: 2 (n-6) 52.5, 1.2, 0.4 (mol%), 20: 4 (n-6) 16.2 9.7, 6.7 (mol%), 20: 5 (n-3) 1.0, 4.1, 5.0 (mol%), 22: 6 (n-3) 3.8, 45.0, 52.9 (mol%), 24: 1 (Not detected), 17.0, 14.9 (mol%) are reported. On the other hand, myocardial ethanolamine plasmalogens derived from bovine (control), tuna and skipjack are fatty acid 18: 2 (n-6) 40.8, 0.8, 0.2 (mol%), 20: 4 (n-6), respectively. 37.7, 13.6, 8.4 (mol%), 20: 5 (n-3) 0.6, 2.8, 7.3 (mol%), 22: 6 (n-3) (not detected), 65.3, 66.4 (mol%) ), 24: 1 (not detected), 5.5, 5.0 (mol%).

  On the other hand, it is also known that as a part of the function of plasmalogen derived from marine oils and fats, there is an effect of preventing Alzheimer-type dementia (Japanese Patent Laid-Open No. 2004-26803). Plasmalogens derived from marine oils and fats have a low risk of contamination with infectious agents like those derived from conventional bovine brain, and are highly safe and useful in that respect.

  By using the plasmalogen preparation method of the present invention as described above, plasmalogen can be prepared with high purity from marine resources. As a specific application example, first, total lipids are extracted from various animal organs or tissues inhabiting underwater, for example, the heart or head of fish as fishery waste, using an organic solvent or the like. The fishery waste is not limited, but industrially, bonito and tuna, which are large fishes, and the heart and head of unused fish are preferably used. Subsequently, the extracted lipid fraction can be separated into a phospholipid fraction (including plasmalogen), a neutral lipid fraction, and the like by any conventional lipid fractionation method such as silicic acid column chromatography. it can. This phospholipid fraction may be further separated into various phospholipid fractions such as choline type and ethanolamine type by a separation operation based on a nitrogen-containing alcohol group, for example. The various phospholipid fractions thus obtained usually contain a mixture of diacyl phospholipid and plasmalogen. For example, the choline-type phospholipid fraction includes (diacyl-type) phosphatidylcholine and choline-type plasmalogen. The ethanolamine type phospholipid fraction contains (diacyl type) phosphatidylethanolamine and ethanolamine type plasmalogen. Therefore, the 1,3-position specific lipase is allowed to act on the lipid fraction or each phospholipid fraction prepared as described above according to the method of the present invention, whereby the diacyl-type phospholipid contained in those fractions is converted into the above-mentioned fraction. It can be degraded by lipase while plasmalogens can remain. From the solution after this reaction, plasmalogens can be easily separated and purified by using a lipid fractionation method such as silicic acid column chromatography.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

  In this example, lipase F-AP15 (generic name: lipase F; manufactured by Amano Enzyme) derived from the fungus Rhizopus oryzae, which is a lipase specific for position 1 and 3, was used. Furthermore, egg yolk phosphatidylcholine (PC) (manufactured by Wako Pure Chemical Industries, Ltd.) as diacyl phospholipid, and 1-O-1 '-(Z) -octadecenyl-2-oleonyl-sn-glycero-3-phosphocholine (synthesis) as plasmalogen Choline-type plasmalogen (Avanti Polar Lipids, Inc.) was used.

First, 100 micrograms of PC or 1-O-1 '-(Z) -octadecenyl-2-oleonyl-sn-glycero-3-phosphocholine in chloroform was collected in a small glass test tube and evaporated. After drying, 10 microliters of a benzene / ethanol (1: 1) mixture was added to the residue to solubilize the lipid. To the lipid solution thus prepared, 88 microliters of a reaction mixture composed of 50 mM Tris-HCl (pH 7.0), 2.7 mM sodium deoxycholate and 5 mM CaCl ₂ was added. Finally, lipase F-AP15 solution was added to adjust the total amount of the reaction solution to 130 microliters, and then the reaction was performed at 37 ° C. for 30 minutes. The amount of lipase F-AP15 added was 250 micrograms, 500 micrograms, 750 micrograms and 1000 micrograms.

  After the reaction, the lipase reaction was stopped by adding 650 microliters of a chloroform / methanol (2: 1) mixture to each test tube and stirring by vortexing. After the test tube was allowed to stand, the lower chloroform layer was collected with a Pasteur pipette, concentrated with an evaporator, and lipid was collected therefrom. The concentrated lipid was dissolved by adding 13 microliters of a chloroform / methanol (3:10) mixed solution, and then 10 microliters was spotted on a thin layer chromatography (TLC) plate coated with silica gel as an adsorbent. For the development of the TLC plate, a solvent system of chloroform / methanol / acetic acid / water (55: 17: 3: 2) and n-hexane / diethyl ether / acetic acid (80: 20: 1) was used. After development, the plate was sprayed with a 50% sulfuric acid solution and heated on a hot plate for 5 minutes to record the appearing lipid spots. The result is shown in FIG. Similarly, the prepared TLC plate was separately developed using a solvent system of n-hexane / diethyl ether / acetic acid (80: 20: 1) mixed solution. The result is shown in FIG.

  As shown in FIG. 2, phosphatidylcholine (PC) was hydrolyzed to lysophosphatidylcholine (lysoPC) by the reaction with lipase F-AP15. This lysophosphatidylcholine was clearly distinguished in terms of mobility and phosphatidylcholine and plasmalogen spots. The amount of lysophosphatidylcholine increased with increasing amount of lipase F-AP15. Furthermore, it was shown that phosphatidylcholine (PC) was hydrolyzed by reaction with lipase F-AP15 to produce free fatty acids (FIG. 3).

  In contrast, unlike phosphatidylcholine, 1-O-1 ′-(Z) -octadecenyl-2-oleonyl-sn-glycero-3-phosphocholine was not hydrolyzed by lipase F-AP15. As shown in FIGS. 2 and 3, it is a degradation product of 1-O-1 ′-(Z) -octadecenyl-2-oleonyl-sn-glycero-3-phosphocholine (choline type plasmalogen) on the TLC plate. There were no lysoplasmalogens or free fatty acid spots. That is, this choline-type plasmalogen was not degraded at all by lipase F-AP15.

  From the above results, it is possible to degrade diacyl phospholipids while maintaining plasmalogens in an intact form by using a 1,3-position specific lipase, and a lyso form produced by degradation of diacyl phospholipids It was shown that can be clearly separated from plasmalogens.

  The method for preparing plasmalogens of the present invention can be used to obtain plasmalogens with high purity. In the method of the present invention, a plasmalogen containing polyunsaturated fatty acids (EPA, DHA, etc.) that are characteristic of marine oils and fats and have various physiological functions can be prepared.

FIG. 1 is a diagram showing a lipase reaction used in the plasmalogen separation technique of the present invention. FIG. 2 is a photograph showing the results of thin-layer chromatography showing the production of lyso-form phospholipid accompanying the degradation reaction of phosphatidylcholine (PC) and choline-type plasmalogen using lipase F-AP15. FIG. 3 is a photograph showing the results of thin-layer chromatography showing the production of free fatty acids accompanying the degradation reaction of phosphatidylcholine (PC) and choline-type plasmalogen by lipase F-AP15.

Claims (4)

  A method for preparing a plasmalogen, comprising reacting a marine oil and fat with a 1,3-position specific lipase and then separating the plasmalogen.   The method according to claim 1, wherein the marine oil is fish oil.   The method according to claim 1 or 2, wherein the 1,3-position specific lipase is derived from Rhizopus oryzae.   The method according to any one of claims 1 to 3, wherein the plasmalogen is a choline-type plasmalogen.

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