JP4788990B2 - Method for producing sphingolipids from aquatic organisms - Google Patents

Description

The present invention relates to a method for efficiently producing sphingolipids from aquatic organisms, particularly gastropods and / or axpods (hereinafter sometimes referred to as “shellfish” or “shellfish”) and cephalopods. The sphingolipid obtained in the present invention can be widely used for food materials, cosmetic materials, pharmaceutical materials, research reagents and the like.

In recent years, it has been clarified that the supply of ceramide contained in keratin intercellular lipid has a strong effect on maintaining the health of the skin, and a large amount of supply as cosmetics is desired. Furthermore, since ceramide is involved in apoptosis, its use as a research reagent is being opened. Thus, although the demand for ceramide is rapidly expanding, sphingolipids containing ceramide as a skeletal structure are almost non-existent in nature, and the supply source is not other than bovine brain, so the supply does not remain, It could not be put into practical use for these applications (Non-patent Document 1). Recently, artificially chemically synthesized sphingolipids have been developed and are currently used as ceramide substitutes (Non-patent Document 2).
Recently, plant-derived ceramides have been found, although their concentration is low (Non-patent Document 3).

Published by Tokyo Chemical Co., Ltd. “Biochemical Dictionary” 2nd edition, page 702, Sphingomyelin (1990) “Oil Chemistry”, Vol. 43, No. 8, 656-658 (1994) J. et al. Oleo Sci. , Vol. 51, no. 5,347-354 (2002)

As described above, sphingolipids having ceramide as a basic skeleton have been increasing in demand in recent years, but only a very small amount exists in nature, and a new supply source has been demanded.
The present inventors extracted lipid components from aquatic organisms targeting sphingolipids and analyzed the lipids. Surprisingly, gastropods, axopoda, cephalopod soft bodies, such as pearl oysters, etc. It was found that a high content of sphingolipid was contained in the bivalve mollusc. The concentration of this sphingolipid reaches 10% or more in the crude lipid, and that of conventional sphingolipid (glycosylceramide) derived from plants such as wheat is about 0.7%. The headline and the method of collecting this were studied, and the present invention was completed.

That is, an object of the present invention is to provide a method for producing sphingolipid with high yield from a specific aquatic organism.
Moreover, the subject of this invention is recycling the aquatic organisms conventionally discarded, such as a soft body part of a pearl oyster, and it exists in contributing to environmental conservation.
Furthermore, the subject of this invention is providing the manufacturing method of the sphingolipid which can supply the sphingolipid which supply is desired in large quantities.

This invention is made | formed based on the said knowledge, Comprising: It relates to the method of manufacturing sphingolipid from the aquatic organism as follows. (1) A method for producing sphingolipids, wherein one or more soft body parts of aquatic organisms belonging to any of gastropods, axpods and cephalopods are used as a raw material and extracted with an organic solvent comprising ethanol or hydrous ethanol . (2) One or more types of aquatic organisms belonging to any of gastropods, axopoda and cephalopods are extracted with an organic solvent consisting of ethanol or hydrous ethanol , chromatographed, and fractionated with an organic solvent. And collecting a sphingolipid fraction. (3) One or more aquatic organisms belonging to any one of gastropods, axopoda and cephalopods are immersed and crushed in an organic solvent such as ethanol, hydrous ethanol, chloroform-methanol 2/1 mixture, A method for producing a sphingolipid, which comprises extracting with an organic solvent, concentrating, chromatography, fractionating with an organic solvent, and collecting a sphingolipid fraction. (4) ax legs such that scallop, oyster, mactra chinensis and mussels and cephalopods are Humboldt squid, neon flying squid, squid, okinawan squid, the is any molluscs are selected from the group consisting of squid (1) - The method according to any one of (3).

According to the method of the present invention, sphingolipids (ceramide aminoethyl phosphonate) can be obtained in high yield from aquatic organisms such as gastropods, axopoda and cephalopods. As a result, it is possible to produce a large amount of sphingolipid, which has been conventionally known to be present in a small amount in bovine brain, wheat and the like. The sphingolipid obtained by the method of the present invention contains a functional phospholipid even if it is a crude oil, and therefore can be widely used as a physiologically active substance or moisturizer in food materials, cosmetic materials, pharmaceutical materials, etc. It can also be used as a research reagent. Furthermore, when the soft body part of the pearl oyster after the pearl is collected is used as a raw material, resources of the pearl oyster soft body part, which has been rarely used in the past, can be reused.

Examples of gastropods in the present invention include snails such as abalone, sazae, tsubugai, etc., and examples of axpods include clams, clams, red scallops, oysters, scallops, scallops, lobsters, tiger oysters, rainbow trouts, squid butterflies, pearl oysters, turtles , Mussels and the like can be exemplified. In addition, the cephalopods include cuttlefish, cuttlefish, silly squid, blue cuttlefish, squid, bakery, ginkgo squid, cuttlefish, flying squid, squid, squid, squid, squid, octopus, sea lion, Octopuses such as willow octopus, mizudako, wamon octopus, chihiro octopus and shimada octopus. Also, most of these molluscs have a dietary experience.

  These aquatic creatures live in the ocean like abalone, clams, pearl oysters, mussels and squid and octopus, live in brackish water like swordfish, or in freshwater like squid butterflies There are creatures. The present invention can be used in the soft body part of any of these aquatic organisms, and not only one type but also two or more types can be used in combination. Among these aquatic organisms, octopuses and squids have a high sphingolipid content, and most of them can be used as raw materials. In addition, soft body parts such as pearl oysters, snails, and mussels have a high sphingolipid content and are used as preferable raw materials. In the case of shellfish, as the soft body part, any of the foot, mantle, cereal muscle, and other built-in muscles may be used. In particular, when sphingolipids are collected from the soft body after collecting pearls of pearl oysters by the method of the present invention, resources that have been discarded in the past can be recycled. Not only does it contribute, it is also clearly advantageous from the point of view of the content.

  Sphingolipid is also called sphingolipid and is a generic name for lipids having a long-chain base such as sphingosine. It is mainly composed of glycosphingolipids and sphingophospholipids and is widely distributed from lower animals to higher animals, but its content is extremely low as described above. The fatty acid is characterized by having an acid amide bond with an amino group at the C-2 position such as sphingosine, and is called ceramide. A sphingolipid has ceramide as a partial skeleton.

In the present invention, at least one of these gastropods, axopoda and cephalopods is extracted with an organic solvent, chromatographed, and fractionated with an organic solvent to obtain a sphingolipid fraction. Furthermore, the sphingolipid can be obtained in high yield by further purification as necessary. Extraction solvents include ethanol, hydrous ethanol, methanol, butanol, t-butanol, propyl alcohol, isopropyl alcohol, other alcohols, chloroform, dichloromethane, dichloroethane, trichloroethane, other halogenated hydrocarbons, acetone, ethyl acetate, ether Etc. are used. Carbon dioxide may be used as a supercritical solvent. These may be used alone or in combination with several kinds of solvents. In particular, hydrous ethanol and chloroform-methanol mixtures are preferred because they can dissolve sphingolipids at high concentrations.

  In addition, the chromatography treatment uses silica gel, alumina, florisil, etc. as the filler, and the HPLC treatment uses silica gel (normal phase), silica gel chemically bonded to C18, phenyl, CN, etc. (reverse phase) as the filler. Can be used.

In the production of sphingolipid, the soft body part of the aquatic organism is immersed in an organic solvent such as hydrous ethanol , crushed, the organic solvent is added to extract the sphingolipid, and concentrated to obtain crude total lipid. The crude total lipid is then chromatographed and fractionated using an organic solvent to give the sphingolipid. This compound may be identified by using thin-layer chromatography as long as the Rf value matches that of the standard sphingolipid. Moreover, it can carry out using a nuclear magnetic resonance spectrum (NMR).

  The inventor of the present invention analyzed lipids using a large number of aquatic organisms as raw materials, and found that gastropods, axopoda and cephalopods are rich in sphingolipids. An example of this analysis is as follows.

The pearl oysters used as the raw material of the present invention are 10 years old oyster oysters grown in Uwajima Bay, Ehime Prefecture, Ago Bay, Mie Prefecture, and Tsushima, Nagasaki Prefecture on the Japan Sea side, from 1998 to 2000. ~ 11 were collected and used.
The weight of the pearl oyster was 18-28 g in the summer season (Uwajima and Tsushima), which is near the egg-laying season, but 40-47 g in the winter season (Ago Bay), which is the growing season.
After immersing the soft body of these pearl oysters in chloroform-methanol 2/1 mixture, Folch
According to the method, crude lipids were extracted with the same solvent.

The lipid class was identified by silica gel column chromatography, TLC, NMR. The fatty acid composition was measured by gas chromatography (Hewlett Packard 5890 Series-II) and gas chromatography mass spectroscopy (Hewlett Packard G1800C MCD). The crude lipid was fractionated into each lipid class by silica gel column chromatography. The fractionated purified lipid was confirmed by comparing the Rf value of TLC with that of a standard product or by coloring with a phosphorus-molybdenum reagent or the like. Further, a part of the mixture was quantified at a ratio based on an NMR integration ratio.
Fatty acid-related compounds such as TAG and phospholipid were determined by gas chromatography analysis and gas chromatography mass spectroscopy after methyl esterification and DIMOX derivatives with a catalytic amount of hydrochloric acid-methanol by a conventional method. The results are shown in Table 1.

As shown in Table 1, the lipid content was low and was 2% or less (0.4 to 2.0%). The lipid class was mainly composed of triacylglycerols in neutral lipids, but also contained significant amounts of sterols.
Polar lipids contained phospholipid phosphatidylethanolamine, phosphatidylcholine, and the like, as well as ceramide aminoethylphosphonate (ceramide aminoethylphosphonic acid: sphingolipid), which is a sphingolipid, in a high content of about 10% on average. Therefore, in the present invention, sphingolipids contained in large quantities in aquatic organisms can be collected easily and with high yield using the above method.
Examples of the present invention are shown below, but the present invention should not be construed as being limited to these examples.

Akoya oyster (Pinctada fucata martensii: average shell length 5.7 ± 0.2cm, average shell width 5.8 ± 0.12cm, average body weight 18.6 ± 0.7g) cultured in Uwajima Bay, Ehime Prefecture 10 soft bodies (6.4-9.3g)
Were immersed in about 50 mL of a chloroform-methanol 2/1 mixed solution and crushed with a homogenizer. About 200 mL of saturated saline was added, extracted with 50 mL of dichloromethane three times, and concentrated under reduced pressure to obtain 84 to 213 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. In the dichloromethane / methanol (1/10) fraction, 3.8 to 16.8 mg (average yield: 11.9 ± 2.0%) of purified ceramide aminoethyl phosphate (sphingolipid) was obtained. The obtained ceramide was completely consistent with the standard sample by thin layer chromatography (Rf = 0.6).

  The soft body of 7 individuals collected from offshore Kisarazu, Chiba Prefecture (Mactra chinensis: average shell length 6.4 ± 0.9 cm, average shell width 4.7 ± 0.3 cm, average body weight 31.9 ± 1.8 g) The feet and mantle (1.8-5.1 g) ), The closed shell muscle (4.6 to 11.4 g), and the other internal (4.6 to 7.2 g) were separated and immersed in about 50 mL of a chloroform-methanol 2/1 mixture, and crushed with a homogenizer. About 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain 7.5 to 105.3 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. Purified ceramide aminoethylphosphonic acid (sphingolipid) in the dichloromethane / methanol (1/10) fraction was 0.7 to 6.3 mg (average yield: 15.8 ± 2.9%) in the foot and mantle, respectively, and 0 in the closed shell muscle. 0.4 to 1.1 mg (average yield: 7.7 ± 2.1%), and 3.8 to 13.8 mg (average yield: 9.3 ± 1.8%) were obtained with other built-in components. The obtained ceramide was completely consistent with the standard sample by thin layer chromatography (Rf = 0.6).

  Blue mussels (Mytilus galloprovincialis: average shell length 5.8 ± 0.9 cm, average shell width 3.2 ± 0.8 cm, average body weight 17.1 ± 1.3 g) collected along the coast of Yokohama, Kanagawa Prefecture. One mixture was immersed in about 50 mL and crushed with a homogenizer. About 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain 47.2 to 158.1 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. 4.0 to 15.9 mg (average yield: 9.9 ± 1.9%) of purified ceramide aminoethyl phosphate (sphingolipid) was obtained in the dichloromethane / methanol (1/10) fraction. The obtained ceramide was completely consistent with the standard sample by thin layer chromatography (Rf = 0.6).

  Immerse the soft body part (average 21.6 ± 1.2g) of oysters (Crassostrea gigas: average body weight 230.3 ± 8.2g) cultivated and collected in Yamada Bay, Iwate Prefecture into chloroform-methanol 2/1 mixture, about 50mL, The mixture was crushed with a homogenizer. About 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain 151.4 to 479.9 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. In the dichloromethane / methanol (1/10) fraction, 3.1 to 19.6 mg (average yield: 8.5 ± 0.8%) of purified ceramide aminoethyl phosphate (sphingolipid) was obtained. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  Ten soft body parts (Crassostrea nippona: average body weight 235.1 ± 7.9g) soft body (closed shell muscle: average 11.1 ± 0.6g and other soft body parts: average 11.5 ± 0.6g) cultured and collected near Jodogahama, Iwate Prefecture Was immersed in about 50 mL of a chloroform-methanol 2/1 mixture and crushed with a homogenizer. About 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain 56.8 to 199.6 mg of crude total lipid and 151.0 to 396.0 mg, respectively. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. In the dichloromethane / methanol (1/10) fraction, purified ceramide aminoethyl phosphate (sphingolipid) was 2.6 to 12.9 mg (average yield: 7.2 ± 3.3%) and 1.1 to 3.6 mg (average), respectively. Yield: 1.4 ± 0.3%). The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  3 soft body parts (0.63-1.59g) of black mussel (Pinctada margaritifera: body weight 1.6 ± 10.7g) bred and collected in Ishigakijima Sea of Okinawa Prefecture are immersed in about 50mL of chloroform-methanol 2/1 mixture, The mixture was crushed with a homogenizer. About 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain 6.2 to 13.4 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. 0.3 to 1.3 mg (average yield: 8.3 ± 0.5%) of purified ceramide aminoethyl phosphate (sphingolipid) was obtained in the dichloromethane / methanol (1/10) fraction. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  Soft-shelled mussels (Tridacna crocea: average body weight 84.7 ± 1.5g) collected in Shirahohama, Ishigaki Island, Okinawa Prefecture (mantle membrane 7.34 to 11.97g, closed shell muscle 2.50 ± 4.55g, other soft body parts 6.08 to 8.97 g) Each was immersed in about 50 mL of a chloroform-methanol 2/1 mixture and crushed with a homogenizer. Add about 200 mL of saturated saline, extract 3 times with 50 mL of dichloromethane, concentrate under reduced pressure, and add crude total lipid (mantle membrane 33.0-52.2 g, closed shell muscle 8.7-10.5 mg, other soft body parts 40.5-209.5 mg). Obtained. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. In the dichloromethane / methanol (1/10) fraction, 0.6 to 7.9 mg of purified ceramide aminoethyl phosphate (sphingolipid) (outer membrane, average yield: 5.7 ± 1.8%), 1.4 to 4. 1 mg (closed muscle, average yield: 5.0 ± 1.4%) and 0.4 to 6.3 mg (other soft body parts, average yield: 2.3 ± 1.5%) were obtained. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  Soft abalone (Haliotis discus hannai: average body weight 40.9 ± 7.8g) collected in Oshimari Island, Hokkaido Oshima Branch Office (soft body part of muscles 4.37-21.66g, midgut 2.64-6.38g, other soft body parts 1.43-4.85g) Each was immersed in about 50 mL of chloroform-methanol 2/1 mixed solution, and crushed with a homogenizer. Add about 200 mL of saturated saline, extract 3 times with 50 mL of dichloromethane, and concentrate under reduced pressure to obtain crude total lipid (muscle 34.3 to 176.5 mg, midgut 43.0 to 445.7 mg, other soft body parts 12.9 to 48.5 mg) It was. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. 4.7 to 24.7 mg of purified ceramide aminoethyl phosphate (sphingolipid) containing a small amount of phospholipid in the dichloromethane / methanol (1/10) fraction (muscle, average yield: 25.9 ± 5.4%), 4 0.1-12.3 mg (middle intestinal line, average yield: 15.0 ± 4.9%) and 5.9-28.4 mg (other soft body parts, average yield: 25.5 ± 5.4%) were obtained. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  About 50 mL of hydrous ethanol solution (ethanol containing 5-10% water) of 2 soft bodies (average 18.6 ± 2.6 g) of oysters (Crassostrea gigas: average weight 226.5 ± 3.6 g) cultivated and collected in Yamada Bay, Iwate Prefecture And crushed with a homogenizer. The crushed mixture was filtered and the filtrate was collected. About 100 mL of ethanol was divided into several portions and poured onto the solid on the filter paper to extract the lipids and added to the filtrate. Concentration under reduced pressure afforded 185.5-391.6 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. Extraction was performed using hydrous ethanol (ethanol containing 5% water) to obtain a fraction containing crude ceramide aminoethyl phosphate. Further, by purifying with dichloromethane / methanol, 5.6 to 7.8 mg (average yield: 6.7 ± 0.9%) of purified ceramide aminoethyl phosphate (sphingolipid) was obtained in the dichloromethane / methanol (1/10) fraction. )confirmed. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  Two soft-shelled oysters (Crassostrea gigas: average body weight 265.3 ± 0.1g) cultivated and collected in Yamada Bay, Iwate Prefecture, frozen at -40 ° C (under 5mmTorr) After freeze-drying and removing water (average dry weight, 3.34 ± 0.11 g), it was immersed in about 50 mL of an aqueous ethanol solution (ethanol containing 5 to 10% water) and crushed by a homogenizer. The crushed mixture was filtered and the filtrate was collected. About 100 mL of ethanol was divided into several portions to wash the solid on the filter paper and added to the filtrate. Concentration under reduced pressure afforded 294.8-329.7 mg of crude total lipid. Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. In the dichloromethane / methanol (1/10) fraction, 9.4 to 16.4 mg (average yield: 5.4 ± 1.5%) of purified ceramide aminoethyl phosphate (sphingolipid) was obtained. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  Onychoteuthis banksii japonica (average mantle length 28.8 ± 0.4 cm, average body weight 485.3 ± 16.6 g) collected from offshore Miyagi Prefecture in the North Pacific Ocean 5 individual soft body parts (29.5-55.0 g of mantle membrane, 9.5-28.0 liver) g, ovaries 8.5 to 13.5 g) were immersed in about 50 mL of a chloroform-methanol 2/1 mixture and crushed with a homogenizer. Approximately 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain crude total lipids (mantle membrane 326.8 to 430.3 mg, liver 3.75 to 8.88 g, ovary 95.3 to 148.7 mg), respectively. . Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. Purified ceramide aminoethyl phosphate (sphingolipid) in the dichloromethane / methanol (1/10) fraction was 12.0 to 36.8 mg (mantle membrane, average yield: 13.3 ± 0.9%) and 14.9 to 19. 1 mg (ovary, average yield: 20.6 ± 1.0%) was obtained. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

  Chihiro octopus (Benthoctopus profundorum: average mantle length 96.0 ± 10.1mm, average body weight 419.9 ± 59.8g) collected off the coast of Miyagi, North Pacific 4 soft body parts (28.7-70.4g part of mantle membrane, 12.7 ± 38.9g liver) Each part of the tentacles (12.6 to 31.6 g) was immersed in about 50 mL of a chloroform-methanol 2/1 mixed solution and crushed with a homogenizer. About 200 mL of saturated saline was added, extracted three times with 50 mL of dichloromethane, and concentrated under reduced pressure to obtain crude total lipids (mantle membrane 13.1 to 34.3 mg, liver 491.1 to 1247.8 mg, tentacle 51.2 to 73.5 mg), respectively. . Each crude total lipid was subjected to silica gel column chromatography and fractionated with an organic solvent. In the dichloromethane / methanol (1/10) fraction, purified ceramide aminoethyl phosphate (sphingolipid) was 0.6 to 1.5 mg (mantle membrane, average yield: 13.2 ± 1.4%), 9.6 to 37, respectively. 2 mg (liver, average yield: 4.9 ± 0.7%) and 10.8 to 13.0 mg (tentacles, average yield: 15.2 ± 0.7%) were obtained. The obtained ceramide was completely consistent with the sample by thin layer chromatography.

Industrial availability

As described above in detail, according to the present invention, sphingolipids (ceramide aminoethylphosphonate) can be produced in a large amount and in a high yield from aquatic organisms such as gastropods, axopoda, cephalopods and the like. Further, the sphingolipid obtained by the method of the present invention contains a functional phospholipid even if it is a crude oil, so it can be widely used as a physiologically active substance or a moisturizing agent in food materials, cosmetic materials, pharmaceutical materials, etc. It can also be used as a research reagent. Furthermore, when the soft body part of the pearl oyster after the pearl is collected is used as a raw material, the pearl oyster soft body part that has hardly been used in the past can be reused as a useful resource. As described above, the present invention is a useful invention that can be widely used in research fields such as foods, cosmetics, and pharmaceuticals and in production sites.

Claims (4)

Gastropods Onoashirui, and one or more soft body portion of one belonging aquatic organisms cephalopods as a raw material, ethanol or method for producing ceramide aminoethyl phosphonic acid and extraction with an organic solvent consisting of water-containing ethanol.
One or more soft body parts of aquatic organisms belonging to gastropods, axopoda, cephalopods are extracted with an organic solvent consisting of ethanol or hydrous ethanol, chromatographed, and fractionated with an organic solvent to ceramide amino method for producing ceramide aminoethylphosphonic acid, and collecting the fractions of ethyl phosphonic acid content.
One or more types of aquatic organisms belonging to any of gastropods, axopoda and cephalopods are immersed in an organic solvent composed of ethanol or hydrous ethanol and crushed and extracted with an organic solvent. method for producing ceramide aminoethylphosphonic acid to claim 1 or 2, wherein.
The soft body consists of mantles of scallops, pearl oysters, snails, mussels, clam muscles, midgut glands, feet, other internal organs, American giant squid, squid, squid, flying squid, squid liver, ovaries, tentacles, mantle 4. The method according to any one of claims 1 to 3, wherein the waste is any mollusk waste selected from the group.