JP2016023137A - Method for producing ganglioside gm2-containing extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing ganglioside GM2-containing extract conveniently and efficiently.SOLUTION: A method for producing ganglioside GM2-containing extract comprises the steps (a) and (b): (a) step of extracting lipid from the muscle of rays; and (b) saponifying the lipid extract obtained in step (a) and then neutralizing it.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing an extract containing ganglioside GM2, and an extract containing ganglioside GM2 produced by the method.

  Gangliosides are one type of glycosphingolipid containing sialic acid as a sugar, and various types having different sugar chain sequences, sialic acid binding sites and the like are known. For example, ganglioside GM2 has a structure of GalNAcβ1 → 4Galβ1 → 4 [α3 → 2NeuAc] Glcβ1 → 1Cer (GalNAcβ1 → 4Gal is composed of a hydroxyl group at position 1 of N-acetyl-D-galactosamine and 4 of galactose. This shows that the hydroxyl group at the position is linked by a β-glycosidic bond. GM3 has a structure of Galβ1 → 4 [NeuAcα2 → 3] Glcβ1 → 1Cer.

  Gangliosides are known to be concentrated in lipid rafts on the surface of cell membranes and regulate cell signaling. In particular, for ganglioside GM2, the antibody is useful for diagnosis and treatment of cancer (Patent Document 1), and when ganglioside GM2 is inoculated as a vaccine, it significantly increases the production of IgG and IgM antibodies in melanoma patients. (Non-Patent Document 1) has been reported to be useful.

  Ganglioside GM2 is very expensive, although commercially available mainly from human brain. Moreover, although there is a report that ganglioside GM2 is contained in mammalian muscle (Non-patent Document 2), the amount is about the trace amount.

JP-A-10-257893

Cancer Res. 49, 7045-7050 (2014) J. Biochem. 94, 1359-1365 (1983)

  An object of the present invention is to provide a simpler and more efficient method for producing an extract containing ganglioside GM2.

  As a result of intensive studies in view of the above problems, the present inventors have found that a large amount of ganglioside GM2 is contained in muscles of ray. Previously, ganglioside GM2 was thought to be almost absent from animal muscles, but this finding was completely unexpected. We also found that ganglioside GM2 can be efficiently purified from ray muscle by adopting specific column purification. Furthermore, when purifying using a silicic acid column, it discovered that ganglioside GM2 could be eluted more efficiently if elution from the column was performed by isocratic elution. The present invention has been completed as a result of further research based on these findings. That is, the present invention includes the following configurations.

  That is, the present invention includes the following aspects.

  Item 1. A method for producing an extract containing ganglioside GM2, characterized in that it comprises steps (a) and (b): (a) a step of extracting lipids from ray muscles, and (b) obtained in step (a). After neutralizing the saponified lipid extract.

  Item 2. Item 2. The production method according to Item 1, wherein the extraction solvent in the step (a) is a mixed solvent of chloroform and alcohol.

  Item 3. Item (1) or (2), further comprising a step (c): (c) fractionating the neutralized product obtained in step (b) by chromatography to obtain a ganglioside GM2-containing fraction. Method.

  Item 4. Item 4. The production method according to Item 3, wherein the fractionation is performed by fractionation by anion exchange column chromatography, fractionation by silicic acid column chromatography, or a combination thereof.

  Item 5. Item 4. The production method according to Item 4, wherein the step (c) includes the steps (c3-1) and (c3-2): (c3-1) The neutralized product obtained in the step (b) is converted into an anion. Fractionation by exchange column chromatography to obtain a ganglioside GM2-containing fraction, and (c3-2) The ganglioside GM2-containing fraction obtained in step (c3-1) is fractionated by silicic acid column chromatography. And obtaining a ganglioside GM2-containing fraction.

  Item 6. In fractionation by the anion exchange column chromatography, elution is performed using a solution containing ammonium acetate at a concentration of 5 to 15 mM, and a fraction eluted with the solution is obtained as a ganglioside GM2-containing fraction. Item 6. The manufacturing method according to Item 4 or 5.

  Item 7. In the fractionation by the silica column chromatography, elution is performed using a mixed solution of chloroform / alcohol / water, and the fraction eluted with the solution is obtained as a ganglioside GM2-containing fraction. The manufacturing method in any one of.

  Item 8. Item 8. The production method according to any one of Items 4 to 7, wherein elution is performed by isocratic elution in the fractionation by the silicic acid column chromatography.

  Item 9. Item 9. A ganglioside GM2-containing extract produced by the production method according to any one of Items 1 to 8.

  According to the present invention, an extract containing ganglioside GM2 can be more easily and efficiently produced by using ray muscle as a raw material. In addition, since muscles occupy most of ray bodies, it is possible to secure sufficient muscles. Moreover, according to the present invention, it is also possible to purify ganglioside GM2 from this abundant raw material more efficiently with less loss. Rays prey on seafood, cephalopods, crustaceans, etc., and enter the fishing net to crush the fishing target, causing damage to the fishery. Since the present invention can effectively use ray species, it can contribute to the reduction of these fishery damages.

The TLC analysis result (Example 1 (1-1)) of a crude glycosphingolipid extract is shown. The TLC analysis result (Example 1 (1-2)) of an ion exchange column refinement | purification fraction is shown. The TLC analysis result (Example 1 (1-3)) of a silicic acid column refinement | purification fraction is shown. The TLC analysis result (Example 1 (1-4)) of refined ganglioside GM2 is shown. The TLC analysis result of the enzyme digest of purified ganglioside GM2 is shown (Example 1 (1-5)). The ESI-MS analysis result of purified ganglioside GM2 is shown (Example 1 (1-7)).

  The present invention relates to a method for producing an extract containing ganglioside GM2, comprising steps (a) and (b).

Step (a)
Step (a) is a step of extracting lipids from ray muscles.

  The ray is not particularly limited, and examples thereof include a terrestrial animal, a terrestrial animal, a saw-toothed animal, and an eleganid animal. Among these, preferably, Cretaceous animals are mentioned.

  The animal species is not particularly limited, and examples thereof include animals belonging to the family Tephroaceae, Tobieiidae, Stingrayidae, Prickly shark, Mutsueraei, Useiidae, Hirateiidae, Urotrigonidae, Potamotrigonidae, and the like. Among these, from the viewpoint that the ganglioside GM2-containing extract can be more efficiently produced, preferably animals belonging to the family Tephroaceae, Tobieiidae, Stingrayidae, etc. are mentioned, and more preferably, animals belonging to the Department Taceae, etc. are mentioned. It is done. Examples of the animal belonging to the family Thorneye Ray include the Thorneye Ray. Examples of animals belonging to the family Tobiei include Narutobei, Madarabi, and Onimachiei, and preferably include Narutobei. Examples of animals belonging to the stingray family include stingrays.

  Although it does not specifically limit as an animal of the order, for example, the animal which belongs to the Yamato Shibirei department, the Thai Sibireibeidae, etc. is mentioned. Among these, Preferably, the animal which belongs to the Yamatoshibirei family etc. is mentioned. Examples of the animals belonging to the family Yamatoshibirei include Yamatoshibirei.

  The scorpionidae animal is not particularly limited, and examples thereof include animals belonging to the scorpionidae, Shinonomesa shark, Tongarisakata shark, Sakata shark, and the like. Among these, animals belonging to the family Gangiidae are preferred. Examples of animals belonging to the Gangiidae include Gangiei.

  The sawtooth animal is not particularly limited, and examples thereof include animals belonging to the family Sawrididae. Examples of animals belonging to the sawtooth family include sawtooth and the like.

  The above ray may be used alone or in combination of two or more.

  The muscle is not particularly limited as long as it contains the muscle tissue of the animal, but from the viewpoint of more efficient production of the ganglioside GM2-containing extract, the muscle tissue is, for example, 70% by weight or more, Preferably, it may contain 85% by weight or more, more preferably 95% by weight or more, and still more preferably 99% by weight or more.

  Ray muscles may be subjected to the lipid extraction process described later without being processed, but from the viewpoint of more efficient production of the ganglioside GM2-containing extract, the processed muscle is subjected to the lipid extraction process. It is preferable. The processing mode is not particularly limited. For example, drying treatment (preferably lyophilization) such as room temperature drying, heat drying, freeze drying, etc., or removal of acetone-dissolved fraction (by this, neutral lipid is removed). And the like. The acetone-dissolved fraction can be removed according to a known method. For example, after adding acetone of 0.5 to 100 times (preferably 1 to 50 times) weight to a muscle dry product and homogenizing with polytron or the like, The residue can be obtained by filtration, centrifugation, or the like.

  One type of muscle may be employed alone, or two or more types may be employed in combination.

  Lipid extraction is performed by extraction using various solvents (extraction solvents) that can dissolve lipids. The extraction method is not particularly limited, and various known methods can be employed. For example, a method of adding an extraction solvent to muscles and homogenizing, and then removing an insoluble residue can be mentioned. The obtained lipid extract is used in the following step (b).

  Examples of the extraction solvent include organic solvents. Examples of organic solvents include alcohols such as methanol, ethanol, propanol and isopropanol, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, halogenated hydrocarbons such as acetonitrile and chloroform, pentane and hexane. Aliphatic hydrocarbons, aromatic hydrocarbons such as benzene and toluene, ethers, pyridines, polyethers, supercritical carbon dioxide and the like. Among these, a mixed solution of chloroform and alcohol is preferable, more preferably a solution in which chloroform and methanol are mixed at a volume ratio of 1: 0.2 to 5, more preferably 1: 0.4 to 3 (chloroform: methanol). Illustrated. The extraction solvent may be used alone or in combination of two or more.

  The weight of the extraction solvent is not particularly limited as long as it is sufficient to extract lipid from muscle. Specifically, for example, the weight can be 0.5 to 100 times, preferably 1 to 50 times the dry weight of muscle. Although the temperature conditions during extraction depend on the boiling point of the extraction solvent, for example, when ethanol is used, 0 to 80 ° C. is preferable. If the extraction temperature is lower than this range, the extraction efficiency is lowered. If the extraction temperature is higher than this range, the solvent is volatilized and only the energy consumption is increased. The extraction time is not particularly limited, but is preferably 10 minutes to 24 hours from the viewpoint of extraction efficiency and workability.

  The insoluble residue can be removed according to a known method. For example, insoluble residues can be removed by suction filtration, filter press, cylinder press, decanter, centrifuge, filtration centrifuge, and the like.

  The obtained lipid extract is preferably concentrated to dryness from the viewpoint of the efficiency of the saponification reaction in the following step (b). Examples of the method for concentrating and drying include various known methods such as freeze drying and evaporation drying.

Step (b)
Step (b) is a step of neutralizing the saponified lipid extract obtained in step (a).

  By saponifying the lipid extract obtained in the step (a), neutral lipids and proteins in the lipid extract can be destroyed and lipid components other than gangliosides can be removed.

  Saponification can be performed by various known methods. For example, it is carried out by mixing the lipid extract obtained in the step (a) and an alkaline solution and allowing to stand for a certain time.

  The alkali solution is not particularly limited as long as it causes a saponification reaction, and examples thereof include a solution in which sodium hydroxide and / or potassium hydroxide is dissolved in alcohol, preferably a solution in which sodium hydroxide is dissolved in methanol. Is mentioned. The alkaline solution may be used alone or in combination of two or more.

  The addition amount of the alkaline solution is not particularly limited as long as neutral lipids and proteins in the lipid extract can be destroyed, and is appropriately selected according to the amount of the lipid extract. It is preferable to add such that the pH of the mixture of the extract and the alkaline solution is in the range of 10 to 14, preferably 11 to 14, more preferably 12 to 14.

  The standing time after mixing the lipid extract and the alkaline solution is not particularly limited as long as neutral lipids and proteins in the lipid extract can be destroyed, but for example, 0.1 hour or more, preferably The standing time is 1 hour or longer, more preferably 10 hours or longer, and still more preferably 24 hours or longer.

  Neutralization can be performed by various known methods, for example, by adding an acid solution to the solution after saponification (saponification solution) and mixing. The obtained neutralized product is used in the following step (c).

  The acid solution is not particularly limited as long as the saponification solution can be neutralized, and examples thereof include acetic acid, hydrochloric acid, nitric acid, sulfuric acid, formic acid, and citric acid. The acid solution may be used alone or in combination of two or more.

  The addition amount of the acid solution is not particularly limited as long as the saponification solution can be neutralized, and is appropriately selected according to the pH of the saponification solution.

  The solution after neutralization is preferably concentrated to dryness from the viewpoint of easy preparation of the mobile phase solvent in the following step (c). Examples of the method for concentrating and drying include various known methods such as freeze-drying. Further, instead of concentrating and drying the neutralized solution, or after concentrating to dryness, the concentrated dried product is dialyzed and removed from the viewpoint of selecting a solvent suitable for the chromatography in the following step (c). It is preferable to salt. The dialysis method is not particularly limited as long as it can be desalted, but it may be dialyzed against distilled water.

Step (c)
According to the present invention, the purity of ganglioside GM2 can be further increased by further combining step (c).

  Step (c) is a step in which the neutralized product obtained in step (b) is fractionated by chromatography to obtain a ganglioside GM2-containing fraction.

  The method of fractionation by chromatography is not particularly limited. As an example, the neutralized product obtained in step (b) is mixed with a mobile phase solvent, and the mixed solution is mixed with a chromatography column. The components in the mixed solution are adsorbed onto the carrier of the column by flowing in, and the column is washed with a washing solvent, and then the adsorbate is eluted with an eluent. Fractionation is performed by collecting the liquid eluted with the eluate in a plurality of fractions in the order of elution.

  The chromatography column is not particularly limited as long as ganglioside GM2 can be adsorbed, and a known column can be appropriately selected. Examples of such a column include an anion exchange column, a silicic acid column, a reverse phase distribution column, a normal phase distribution column, -RP, -CN, or -Diol obtained by chemically modifying silica gel. Among these, from the viewpoint that ganglioside GM2 can be purified more efficiently, an anion exchange column, a silicate column and the like are preferable.

  The mobile phase solvent is not particularly limited as long as it can adsorb ganglioside GM2 to the column, and can be appropriately selected according to the type of column. When the column is an anion exchange column or a silicate column, for example, a mixed solvent of chloroform / alcohol / water is used. When the column is a silicic acid column, a mixed solvent of chloroform / alcohol is exemplified. Various alcohols can be used as the alcohol, and methanol is preferable. The mixing ratio of the mixed solvent of chloroform / alcohol / water is not particularly limited, but as one preferred embodiment, mixing in which chloroform: alcohol: water is 100: 200 to 800: 20 to 90 (volume ratio) As a more preferable embodiment, a mixing ratio in which chloroform: alcohol: water is 100: 300 to 500: 40 to 70 (volume ratio) can be mentioned. In a more preferable embodiment, chloroform: alcohol: water is 100: 300. The mixing ratio which is 350-450: 50-65 (volume ratio) is mentioned. The mobile phase solvent may be used alone or in combination of two or more.

  The washing solution is not particularly limited as long as it does not desorb the ganglioside GM2 adsorbed on the column, and can be appropriately selected according to the type of the column. When the column is an anion exchange column or a silicate column, for example, a mixed solvent of chloroform / alcohol / water is used. When the column is a silicic acid column, a mixed solvent of chloroform / alcohol is exemplified. Various alcohols can be used as the alcohol, and methanol is preferable. The mixing ratio of the mixed solvent of chloroform / alcohol / water is not particularly limited, but as one preferred embodiment, mixing in which chloroform: alcohol: water is 100: 200 to 800: 20 to 90 (volume ratio) As a more preferable embodiment, a mixing ratio in which chloroform: alcohol: water is 100: 300 to 500: 40 to 70 (volume ratio) can be mentioned. In a more preferable embodiment, chloroform: alcohol: water is 100: 300. The mixing ratio which is 350-450: 50-65 (volume ratio) is mentioned. The cleaning liquid may be used alone or in combination of two or more.

  The elution is performed by a known method, for example, isocratic elution, linear gradient elution, or stepwise elution. Isocratic elution is performed with one type of eluate having the same composition. In linear gradient elution, elution is performed while gradually changing the concentration of a specific component (one or more) in the eluate. Usually, A liquid with a certain composition and composition different from A liquid Elution is performed by mixing the B liquid having the ratio of B with increasing the ratio of the B liquid to the A liquid. More specifically, linear gradient elution is performed with, for example, 100% solution A, further increasing the amount of solution B added to solution A, and finally eluting with 100% solution B. Done by doing. Stepwise elution is performed by sequentially using a plurality of types of eluates having different concentrations of specific components (one or more) in the eluate for elution. More specifically, for stepwise elution, prepare 1st liquid, 2nd liquid, ... nth liquid (n = 2 or more), elute first with 1st liquid, then elution with 2nd liquid And finally, by eluting with the nth liquid.

  The eluate is not particularly limited as long as it can desorb ganglioside GM2 adsorbed on the column, and can be appropriately selected according to the type of column and the elution method. The eluate may be used alone or in combination of two or more.

  When the column is an anion exchange column, examples of the eluent include a solution containing ammonium acetate and / or ammonium bicarbonate, and the solvent of the solution is a mixed solvent of chloroform / alcohol / water. It is done. Various alcohols can be used as the alcohol, and methanol is preferable. The mixing ratio of the mixed solvent of chloroform / alcohol / water is not particularly limited, but as one preferred embodiment, mixing in which chloroform: alcohol: water is 100: 200 to 800: 20 to 90 (volume ratio) As a more preferable embodiment, a mixing ratio in which chloroform: alcohol: water is 100: 300 to 500: 40 to 70 (volume ratio) can be mentioned. In a more preferable embodiment, chloroform: alcohol: water is 100: 300. The mixing ratio which is 350-450: 50-65 (volume ratio) is mentioned. The concentration of ammonium acetate in the eluate is not particularly limited as long as it is a concentration at which ganglioside GM2 can be eluted. Such a concentration includes, for example, a concentration of 3 to 20 mM, preferably 5 to 15 mM, more preferably 7 to 12 mM. In the case where the column is an anion exchange column, from the viewpoint that ganglioside GM2 can be purified more efficiently, impurities other than ganglioside GM2 have a low concentration of ammonium acetate (0 to 3 mM, preferably 0 to 5 mM). The elution is preferably performed after removing the eluate.

  When the column is a silicate column, examples of the eluent include a mixed solvent of chloroform / alcohol / water. When the column is a silicic acid column, a mixed solvent of chloroform / alcohol is exemplified. Various alcohols can be used as the alcohol, and methanol is preferable. The mixing ratio of the mixed solvent of chloroform / alcohol / water is not particularly limited, but as one preferred embodiment, mixing in which chloroform: alcohol: water is 100: 20-80: 5-25 (volume ratio) A ratio is mentioned, and a more preferred embodiment includes a mixture ratio of chloroform: alcohol: water of 100: 40 to 70:10 to 20 (volume ratio), and a more preferred embodiment is chloroform: alcohol: water 100: 40. A mixing ratio of 50 to 60:12 to 18 (volume ratio) can be mentioned, and a more preferable aspect is a mixing ratio of chloroform: alcohol: water of 100: 54 to 68:14 to 17 (volume ratio). It is done. Further, when the column is a silicate column, isocratic elution using the eluate is preferable from the viewpoint that ganglioside GM2 can be purified more efficiently. When employing other elution methods (linear gradient elution or stepwise elution), the elution method is not particularly limited as long as the eluate is used.

  Other conditions for chromatography and fractionation (temperature, column size, amount of mobile phase, amount of washing solution, amount of eluate, volume of each fraction, etc.) can be appropriately selected based on known information. .

  Obtaining a ganglioside GM2-containing fraction refers to obtaining a fraction containing ganglioside GM2 by selecting from a plurality of fractions obtained by chromatography fractionation. This selection is performed by determining whether or not ganglioside GM2 is contained in each fraction by a known method. This determination is not particularly limited, and can be performed using various methods capable of analyzing the components in the fraction. Examples of such a technique include thin layer chromatography, MASS spectrometry, NMR, and the like. Among these, thin layer chromatography is mentioned from a viewpoint of the ease of a method.

The thin layer chromatography can be performed by a known method, and is not particularly limited. For example, the solution of each fraction is concentrated to dryness, and a mixed solvent of chloroform / methanol / 12 mM MgCl ₂ solution ( 50/40/10, volume ratio) and the like, and the mixture is developed on a carrier and then stained with a reagent for detecting sugar or lipid (DPA reagent or copper sulfate reagent). Is called. At this time, it is possible to determine whether or not ganglioside GM2 is contained in each fraction by simultaneously developing commercially available ganglioside GM2 and comparing the detected band positions.

  In the case of anion exchange column chromatography, as a preferred embodiment, elution is performed using a solution containing ammonium acetate at a concentration of 5 to 15 mM (preferably 7 to 12 mM), and the fraction eluted with the solution is used. The aspect which acquires a part as a fraction containing ganglioside GM2 is mentioned.

  In the case of silicic acid column chromatography, a preferred embodiment includes an embodiment in which elution is performed using a mixed solvent of chloroform / alcohol / water, and a fraction eluted with the solution is obtained as a ganglioside GM2-containing fraction. .

  Chromatographic fractionation is repeated multiple times from the viewpoint of increasing the purity of ganglioside GM2, that is, the fraction containing ganglioside GM2 obtained by chromatography fractionation is further fractionated by the following chromatography. It is preferable. In this embodiment, the chromatography column may be the same type of column or a different column. When different columns are used, a combination of an anion exchange column and a silicate column is preferred. One preferred embodiment of such embodiments includes embodiments in which step (c) is steps (c3-1) and (c3-2).

Step (c3-1)
Step (c3-1) is a step of fractionating the neutralized product obtained in step (b) by anion exchange column chromatography to obtain a ganglioside GM2-containing fraction.

  Step (c3-1) is performed in the same manner as in step (c) except that an anion exchange column is used as the chromatography column.

Step (c3-2)
Step (c3-2) is a step of fractionating the ganglioside GM2-containing fraction obtained in step (c3-1) by silicic acid column chromatography to obtain a ganglioside GM2-containing fraction.

  Step (c3-2) is performed in the same manner as in step (c) except that a silicate column is used as the chromatography column.

Ganglioside GM2-containing extract According to such a method for producing an ganglioside GM2-containing extract, a ganglioside GM2-containing extract can be obtained more simply and efficiently. According to the production method of the present invention, an extract containing from 1 g of raw muscle of ray, for example, 0.1 mg or more, preferably 0.2 mg or more, more preferably 0.5 mg or more, and even more preferably 0.8 mg or more ganglioside GM2. Can be obtained. Furthermore, it is possible to obtain an extract containing ganglioside GM2 with high purity, which is almost free from other glycolipids and other impurities.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1: Production of ganglioside GM2-containing extract from camellia muscle <1-1. Extraction of crude glycosphingolipid>
1000 ml of acetone was added to 50 g of the freeze-dried product of camellia muscle and homogenized with Polytron, followed by suction filtration to obtain residue 1. 1000 ml of a mixed solvent of chloroform / methanol (2/1, v / v) was added to residue 1 and homogenized with polytron (room temperature (20 to 25 ° C., 5 minutes)), followed by suction filtration. Residue 2 was obtained. Add 1000 ml of a mixed solvent of chloroform / methanol (1/2, v / v) to residue 2 and homogenize with polytron (room temperature (20 to 25 ° C.), 5 minutes), and then suction filter to obtain filtrate 2. Obtained.

  The filtrates 1 and 2 were concentrated to dryness, dissolved in 100 ml of 0.6 N NaOH / methanol, and left overnight at room temperature (this operation destroys neutral lipids and proteins). The mixture was neutralized with 3.5 ml of concentrated acetic acid, concentrated and dried to remove methanol, suspended in distilled water and dialyzed against distilled water. The dialyzed solution was freeze-dried to obtain a dry powder (crude glycosphingolipid extract).

A part of the obtained crude glycosphingolipid extract was dissolved in a developing solvent (a mixed solvent of chloroform / methanol / 12 mM MgCl ₂ solution (50/40/10, v / v / v)), and TLC (Merck The lysate was developed using a silica gel 60 plastic plate (manufactured by Komatsu). The developed pattern was detected with a DPA reagent (for sugar detection), a copper sulfate reagent (for lipid detection), or a ninhydrin reagent (for amino acid detection). Detection with the DPA reagent is performed according to the method described in Reference 1 (Anal Biochem., 287, 337-339, 2000), and detection with the copper sulfate reagent is Reference 2 (J. Lipid Res., 5, 126-127, 1964). The detection with a ninhydrin reagent was performed according to the method described in Reference 3 (J. Liquid Chromatogr., 7, 2793-2801, 1984). The results are shown in FIG.

  In FIG. 1, the developing solvent is developed from the bottom to the top (the same applies to the following drawings). The lane labeled GM1 is a lane in which commercially available ganglioside GM1 (manufactured by Funakoshi, catalog number 8G16-2b) is developed (the same applies to the following figures). The lane labeled Gal-Cel is a lane in which a commercially available galactosylceramide (manufactured by Sigma-Aldrich, catalog number C4905) is developed (the same applies to the following figures).

  In FIG. 1, in the lane (DPA) in which the crude glycosphingolipid extract was developed and the sugar was detected, a band having a slightly longer movement distance than GM1 was detected (a band indicated by an asterisk). GM2 has a structure obtained by removing galactose from GM1, and its molecular weight is smaller than that of GM1, suggesting that this band may be GM2. Therefore, this fraction was further purified as a material (1-2 below).

<1-2. Purification by anion exchange column chromatography>
All the crude glycosphingolipid extracts obtained in 1-1 above are dissolved in 9 ml of a mixed solvent of chloroform / methanol / water (15/60/8, v / v / v) and equilibrated with the mixed solvent. The sample was sampled on an activated QMA cartridge (anion exchanger, column volume 3 ml). Non-adsorbed material was washed away with 9 ml of the mixed solvent. As an eluent, use a solution (9 ml each) of ammonium acetate dissolved in the mixed solvent at a concentration of 5 mM, 10 mM, 20 mM, 50 mM, or 100 mM, and gradually increase the ammonium acetate concentration. Stepwise elution was performed and fractions were each 1 ml.

The resulting fraction is divided into 6 fractions:
"FT and Wash fractions"
"1st to 7th fraction of 5 mM (5 mM (1) -5 mM (7))"
"5 mM 8th to 10 mM sixth fraction (5 mM (8) -10 mM (6))"
“10 mM 7th to 20 mM second fraction (10 mM (7) -20 mM (2))”
"20mM third to 100mM first fraction (20mM (3)-100mM (1))"
"The second fraction of 100 mM to the ninth fraction of 100 mM (100 mM (2)-100 mM (9))"
A portion of each fraction was lyophilized, and development and detection of the development pattern by TLC were performed as in 1-1 above. All detections were performed with DPA reagent. The results are shown in FIG.

  In Fig. 2, in the lane where the 8th to 10 mM sixth fraction of 5 mM (5 mM (8) -10 mM (6)) was developed, the band with a slightly longer movement distance than GM1 is the main band. As detected. It was speculated that this was a band of GM2, and further purification was performed using this fraction as a material (1-3 below).

<1-3. Purification by silica column chromatography>
From the 5 mM (8) -10 mM (6) fraction obtained in 1-2 above, ammonium acetate and the organic solvent were volatilized by drying (40 ° C., overnight). The fraction after the treatment was sampled on a silicic acid column (column volume: 3 ml) equilibrated with a mixed solvent of chloroform / methanol / water (180/100/24, v / v / v). Isocratic elution was performed using a mixed solvent of chloroform / methanol / water (180/100/24, v / v / v) solution (20 ml) as an eluent, and fractionated in 0.3 ml fractions.

  A part of each of the obtained fractions was freeze-dried, and development by TLC and detection of the development pattern were performed in the same manner as in 1-1 above. All detections were performed with DPA reagent. The results are shown in FIG.

  In FIG. 3, the lane labeled GM2 is a lane in which commercially available ganglioside GM2 (manufactured by Funakoshi, catalog number 8G16-3b) is developed (the same applies to the following figures). Each number indicates a fraction number.

  In FIG. 3, in the lane where fraction numbers 26 to 45 are developed, a band having a movement distance slightly longer than GM1 and the same movement distance as GM2 was detected as the main band. Therefore, these fractions, the crude glycosphingolipid extract of 1-1 described above, which was a purification raw material of these fractions, and the 5 mM (8) -10 mM (6) fraction of 1-2 above. Minutes were shown to contain GM2. Fractions Nos. 26 to 38 were collected and concentrated to dryness, and this was used as “purified ganglioside GM2” for the following analysis.

<1-4. Evaluation of purification by TLC>
The purified ganglioside GM2 obtained in 1-3 above was developed by TLC and the development pattern detected in the same manner as in 1-1 above. Detection was performed with a DPA reagent, a copper sulfate reagent, and a ninhydrin reagent. The results are shown in FIG.

  In FIG. 4, no band was detected in the ninhydrin reagent detection lane in which impurities such as protein were detected. In addition, no bands other than GM2, which is a glycolipid, were detected in the DPA reagent detection lane in which sugar was detected and the copper sulfate reagent detection lane in which lipid was detected. This suggested that the purity of purified ganglioside GM2 was very high.

<1-5. Analysis of fragments produced by enzymatic digestion>
Clostridium perfringens-derived sialidase is known to cleave ganglioside GM1 and GM2 sialic acid in the presence of sodium cholate (FEBS LETTERS, 65, 229-233, 1976). Therefore, it was investigated whether the purified ganglioside GM2 obtained in 1-3 above was also digested by the sialidase in the presence of sodium cholate. Specifically, it was performed as follows.

  Purified ganglioside GM2 (100 μg), commercially available ganglioside GM2 (100 μg), commercially available ganglioside GM1 (100 μg), or commercially available ganglioside GM3 (100 μg) (manufactured by Funakoshi, catalog number 8G16-4b) and 1% sodium cholate solution (1 μl), Clostridium perfringens-derived sialidase solution (0.0125 unit, 5 μl), and 0.02 M acetate buffer (pH 5.22, 20 μl) were mixed to prepare a reaction solution. In addition, a reaction solution containing neither sodium cholate nor sialidase and a reaction solution not containing sodium cholate were also prepared. These reaction solutions were incubated at 40 ° C. for 2 days. The solution after the reaction is lyophilized and dissolved in a developing solvent (a mixed solvent of n-butanol / acetic acid / water (2/1/1, v / v / v)) and TLC (Merck, silica gel 60 plastic plate). ) To develop the solution. The developed pattern was detected with DPA reagent (for sugar detection). The results are shown in FIG.

  In FIG. 5, the purified glycolipid means the purified ganglioside GM2 obtained in 1-3 above. The lane labeled GM1, GM2, purified glycolipid, or GM3 indicates that neither sodium cholate nor sialidase is included, the lane labeled + E indicates that no sodium cholate is included, and + E + C The displayed lane shows the case containing both sodium cholate and sialidase. The lane labeled NeuAc indicates a lane in which a commercially available sialic acid (Sigma-Aldrich, catalog number A2388) is developed. EB indicates Enzyme Blank.

  In FIG. 5, the mobility of the band detected in the lane (purified glycolipid) in the case where neither sodium cholate nor sialidase is contained in 3 lanes using purified ganglioside GM2 (purified glycolipid) is shown in FIG. It was consistent with the mobility of the band. In 3 lanes using purified ganglioside GM2 (purified glycolipid), no sialic acid was detected in lane (+ E) when sodium cholate was not included, and lane when both sodium cholate and sialidase were included Sialic acid was detected only at (+ E + C). From this data, it was confirmed that the main band (FIG. 4) detected when the purified ganglioside GM2 was developed was indeed ganglioside GM2.

<1-6. Measurement of the percentage of ganglioside GM2 in camellia muscle>
The weight of sialic acid contained in the purified ganglioside GM2 obtained in 1-3 above was measured according to the method of Y. Uchida et al. (J. Biochem., 82, 1425-1433, 1977). From the measured sialic acid weight, the ganglioside GM2 weight was calculated based on the ratio of the molecular weight of sialic acid to the total molecular weight of ganglioside GM2. Furthermore, based on the weight of the muscle used for purification (wet weight), the proportion of ganglioside GM2 in the raw muscle of camellia was determined. As a result, the ratio was about 0.81 mg / g. This ratio was about 20 times as high as that of human muscle (Non-Patent Document 2).

  In addition, the ratio of ganglioside GM2 in fresh sea bream muscle was determined by the same method as described above using the crude glycosphingolipid extract obtained in 1-1 above, and it was about 1 mg / g. From this result, it was shown that the recovery rate of ganglioside GM2 in the above column purification steps 1-2 and 1-3 was as high as 81% (“0.81 mg / g” / “1 mg / g”). .

<1-7. ESI-MS analysis>
Purified ganglioside GM2 was analyzed by ESI-MS. The results and analysis conditions are shown in FIG.

As shown in FIG. 6, the main peak (1465.0) of purified ganglioside GM2 coincided with the molecular weight (1464.89) of ganglioside GM2 (C ₇₃ H ₁₃₀ N ₃ O ₂₆ ).

Example 2: Production of extract containing ganglioside GM2 from naruto ray and stingray muscle Purified ganglioside GM2 was obtained from naruto ray and stingray according to the same method as in Example 1. For all rays, the proportion of ganglioside GM2 in raw muscle (above 1-6) was similar to that of winged ray (above 1-6). In addition, the recovery rate of ganglioside GM2 in the column purification step (above 1-6) was also similar to that of Tsubakiray.

Comparative Example 1: Ganglioside GM2 extraction from bony fish muscle
According to the same method as in Example 1, ganglioside GM2 was extracted from bony fish muscle. However, ganglioside GM2 was not detected in the extract of bony fish muscle.

Claims (9)

A method for producing an extract containing ganglioside GM2, characterized by comprising steps (a) and (b):
(A) a step of extracting lipids from ray muscle, and (b) a step of neutralizing the saponified lipid extract obtained in step (a). The manufacturing method of Claim 1 whose extraction solvent in the said process (a) is a mixed solvent of chloroform and alcohol. Furthermore, step (c):
(C) The production method according to claim 1 or 2, comprising a step of fractionating the neutralized product obtained in step (b) by chromatography to obtain a ganglioside GM2-containing fraction. The production method according to claim 3, wherein the fractionation is performed by fractionation by anion exchange column chromatography, fractionation by silicic acid column chromatography, or a combination thereof. The manufacturing method according to claim 4, wherein the step (c) includes the steps (c3-1) and (c3-2):
(C3-1) The step of fractionating the neutralized product obtained in step (b) by anion exchange column chromatography to obtain a ganglioside GM2-containing fraction, and (c3-2) step (c3-1) The step of obtaining the ganglioside GM2-containing fraction by fractionating the ganglioside GM2-containing fraction obtained in) by silicic acid column chromatography. In fractionation by the anion exchange column chromatography, elution is performed using a solution containing ammonium acetate at a concentration of 5 to 15 mM, and a fraction eluted with the solution is obtained as a ganglioside GM2-containing fraction. The manufacturing method according to claim 4 or 5. In the fractionation by silicic acid column chromatography, elution is performed using a mixed solvent of chloroform / alcohol / water, and the fraction eluted with the solution is obtained as a ganglioside GM2-containing fraction. 6. The production method according to any one of 6 above. The production method according to any one of claims 4 to 7, wherein elution is performed by isocratic elution in the fractionation by silicic acid column chromatography. A ganglioside GM2-containing extract produced by the production method according to claim 1.

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