JP5634402B2 - Method for producing glucosylceramide-containing material - Google Patents

Description

  The present invention relates to a method for producing a glucosylceramide-containing material, and more particularly to a method for producing a glucosylceramide-containing material from a plant material.

  In recent years, plant-derived glucosylceramide, sterols and sterol glycosides have attracted attention as functional materials.

  Glucosylceramide is also called glucocerebroside and has a structure in which glucose is β-glycoside bonded to the hydroxy group at position 1 of ceramide. Ceramide has a structure in which a sphingoid base and a fatty acid are bonded with an acid amide. FIG. 1 shows the structure of glucosylceramide. Glucosylceramide is expected as a material having, for example, a skin moisturizing effect, an antiatopic effect, and an antitumor effect.

  It is known that sterol glycosides are hardly contained in animal tissues, while plant tissues contain various types. A specific example of a sterol glycoside is shown in FIG. Sterol glycosides are expected as a material that can obtain an antihyperlipidemic effect, an arteriosclerosis preventive effect, and the like by, for example, lowering the cholesterol concentration.

  However, glucosylceramide and sterol glycosides have similar polarities in the whole molecule because the polar groups are the same glucose. In addition, since sterols and ceramides in the fat-soluble part and β-glycoside bonds are common and stable to weak alkalis, it was difficult to separate them easily and with high purity.

  Plant-derived glucosylceramide is already commercially available from several sales companies (examples are shown in Table 1), but when the commercially available glucosylceramide material is analyzed by silicate thin layer chromatography (TLC), glucosyl It turns out that ceramide and a sterol glycoside are contained. Furthermore, since these Rf values (the value obtained by dividing the distance of movement of the spot by the distance of movement of the solvent) do not change much, it can be seen that TLC hardly separates (FIG. 3).

  Therefore, in order to obtain a glucosylceramide derived from a high-purity plant material separated from a sterol glycoside, a high-performance liquid chromatography (HPLC) is used after extracting a glucosylceramide-containing fraction from the plant material with a solvent. Therefore, it was necessary to perform separation in small amounts. For this reason, high-purity plant-derived glucosylceramide is very expensive, and a sufficient amount for the experiment cannot be easily obtained to confirm its functionality in animal experiments.

  Under such circumstances, a method for producing a large amount of high-purity glycosylceramide simply and inexpensively has been studied. For example, Patent Document 1 describes a method of obtaining a glycosylceramide-containing extract by hydrolyzing a plant raw material with alkaline ethanol and then subjecting it to an extraction step using a mixed solvent of hexane, acetone and water. However, in this method, an extract containing 12 to 50% by weight of glycosylceramide is usually obtained, and the sterol glycoside is not separated but remains as it is depending on the original raw material concentration. .

JP 2006-232699 A

  An object of the present invention is to provide a method for producing a large amount of a high-purity glucosylceramide-containing material easily and inexpensively from a plant raw material containing glucosylceramide and a sterol glycoside.

  The present inventors surprisingly, by subjecting a fraction derived from a plant raw material containing glucosylceramide and a sterol glycoside to alcohol precipitation, and recovering the supernatant, it is easy and inexpensive to produce a large amount of high purity. The present inventors have found that it is possible to produce a glucosylceramide-containing product, and have made further improvements to complete the present invention.

That is, this invention includes the manufacturing method of the glucosylceramide containing material of the following items 1-6.
Item 1.
A method for producing a glucosylceramide-containing product, comprising a step of subjecting a fraction derived from a plant raw material containing glucosylceramide and a sterol glycoside to alcohol precipitation, and collecting a supernatant.
Item 2.
Item 2. The method for producing a glucosylceramide-containing product according to Item 1, wherein the plant material-derived fraction is obtained by extraction from a plant material using alcohol or hydrous alcohol.
Item 3.
Item 3. The method for producing a glucosylceramide-containing product according to Item 2, wherein the plant material-derived fraction is obtained by extraction from a plant material using alcohol or hydrous alcohol and further lipid removal treatment.
Item 4.
Item 4. The method for producing a glucosylceramide-containing material according to Item 3, wherein the lipid removal treatment is performed by a step comprising hexane precipitation treatment, acetone precipitation treatment, hexane and acetone mixed solvent precipitation treatment, or a combination thereof.
Item 5.
Item 5. The method for producing a glucosylceramide-containing material according to any one of Items 1 to 4, wherein the alcohol precipitation treatment is ethanol and / or isopropanol precipitation treatment.
Item 6.
Any of paragraphs 1-5, wherein the plant material is at least one selected from the group consisting of wheat, soybeans, rice, cottonseed, rapeseed, corn, sugarcane, sesame, peanuts, rice cakes, eggplant, tangerine and sugar beet The manufacturing method of the glucosylceramide containing material of crab.

Furthermore, the present inventors are able to easily and inexpensively produce a large amount of high-purity sterol glycoside by subjecting a fraction derived from a plant raw material containing glucosylceramide and a sterol glycoside to alcohol precipitation, and collecting the precipitate. It has been found that it is also possible to produce body-containing materials. Therefore, this invention also includes the manufacturing method of the sterol glycoside containing material as described in the following term AF.
Term A.
A method for producing a sterol glycoside-containing product, comprising a step of subjecting a plant raw material-derived fraction containing glucosylceramide and a sterol glycoside to an alcohol precipitation treatment and collecting the precipitate.
Term B.
The method for producing a sterol glycoside-containing product according to Item A, wherein the plant raw material-derived fraction is obtained by extraction from a plant raw material using alcohol or hydrous alcohol.
Term C.
The method for producing a sterol glycoside-containing product according to Item B, wherein the plant raw material-derived fraction is obtained by extraction from a plant raw material using an alcohol or a hydrous alcohol and further a lipid removal treatment.
Term D.
The method for producing a sterol glycoside-containing product according to Item C, wherein the lipid removal treatment is performed by a step including hexane precipitation treatment, acetone precipitation treatment, hexane and acetone mixed solvent precipitation treatment, or a combination thereof.
Term E.
The method for producing a sterol glycoside-containing product according to any one of Items A to D, wherein the alcohol precipitation treatment is ethanol and / or isopropanol precipitation treatment.
Term F.
Any of paragraphs A to E, wherein the plant material is at least one selected from the group consisting of wheat, soybeans, rice, cottonseed, rapeseed, corn, sugarcane, sesame, peanuts, strawberries, eggplant, tangerine and sugar beet A method for producing a sterol glycoside-containing product according to claim 1.

  In addition, as apparent from these production methods, the glucosylceramide-containing product can be said to be a glucosylceramide-containing extract. Moreover, it can be said that the said sterol glycoside containing material sterol glycoside containing extract.

  According to the method for producing a glucosylceramide-containing material of the present invention, a large amount of high-purity glucosylceramide-containing material can be produced from a plant raw material simply and inexpensively. In addition, since a highly toxic solvent is not used in the production process, a safe and secure high-purity glucosylceramide can be provided. For this reason, for example, the amount of glucosylceramide necessary for testing and implementation as food raw materials, cosmetic raw materials, etc. can be provided at low cost.

A structural formula (left) of one example of glucosylceramide (sphingoid base is 4,8-unsaturated) and a schematic diagram (right) of the structure of main glucosylceramide are shown. The structure of a typical plant sterol glycoside is shown. The structural formula of sitosteryl glucoside as an example of a plant sterol glycoside is shown on the right, and the types of main side chains that bind to the steroid skeleton are shown on the left. The result of having analyzed the glucosylceramide raw material (commercial item) refine | purified from various plants by the silicic-acid thin layer chromatography (TLC) is shown. The glucosylceramide materials used are all those shown in Table 1, and the corn used in the fourth row from the top and the rice used in the eighth row from the top. The outline | summary of the process of the manufacturing method of the glucosylceramide containing material of this invention is shown. The description in dotted lines indicates an optional step. The chromatogram obtained by analyzing the glucosylceramide containing material manufactured by the manufacturing method of the glucosylceramide containing material of this invention by HPLC-ELSD is shown. The chromatogram obtained by analyzing the sterol glycoside containing material manufactured by the method which concerns on this invention by HPLC-ELSD is shown.

  Hereinafter, the present invention will be described in more detail.

  The present invention relates to a method for producing a glucosylceramide-containing product, comprising a step of subjecting a fraction derived from a plant raw material containing glucosylceramide and a sterol glycoside to alcohol precipitation treatment and collecting a supernatant.

  The fraction derived from a plant raw material containing glucosylceramide and sterol glycoside is not particularly limited as long as it is obtained from a plant raw material and contains glucosylceramide and sterol glycoside. Usually, when a fraction containing glucosylceramide is extracted from a plant raw material, a sterol glycoside is also contained in the fraction. Since these cannot be easily separated, it is more difficult to obtain a large amount of inexpensive high-purity glucosylceramide.

  The plant material used for obtaining the fraction derived from plant material is not particularly limited as long as glucosylceramide and sterol glycoside can be obtained. For example, wheat (wheat germ), soybean, rice (rice bran / rice germ) , Cottonseed, rapeseed, corn (corn germ), sugar cane, sesame, peanut, cocoon, eggplant, tangerine, sugar beet, etc. These edible parts can be used mainly, but when producing and refining oil from these plant raw materials, low cost resources such as oil extraction residue, degummed products generated in the oil refining process, or rice bran are costly. Is particularly preferable in terms of In addition, a plant raw material can be used 1 type or in combination of 2 or more types.

  As a preferable fraction derived from a plant raw material containing glucosylceramide and a sterol glycoside, a fraction obtained from the plant raw material by extraction with an organic solvent or a hydrous organic solvent, more preferably an alcohol or a hydrous alcohol, etc. Minutes.

  The alcohol used for the extraction is not particularly limited, and for example, a monohydric alcohol having 1 to 6 carbon atoms, preferably a monohydric alcohol having 1 to 4 carbon atoms can be used. Specifically, methanol, ethanol, propanol, butanol, Isopropanol, pentanol, hexanol and the like can be used. Of these, ethanol is preferred. In addition, alcohol can be used 1 type or in combination of 2 or more types.

  For extraction from plant materials, an organic solvent other than alcohol or water may be used instead of or in addition to alcohol. The organic solvent other than alcohol is not particularly limited as long as glucosylceramide can be extracted and has low toxicity. Toxic non-food solvents such as chloroform are not preferred. These organic solvents and water may be used separately from alcohol to perform each extraction step in order, or may be used for extraction after mixing with alcohol. Moreover, you may use, after mixing an organic solvent with water. A mixture of alcohol and water is hydrous alcohol. The water content of the hydrous alcohol is not particularly limited, but is preferably 50% (v / v%) or less, more preferably 30% (v / v%) or less. In addition, these alcohol, organic solvent, and water can be used 1 type or in mixture of 2 or more types.

  The specific extraction operation is not particularly limited, and can be performed by a conventional method. For example, it can be performed by an immersion method such as cold immersion or digestion, a percolation method, or the like. As a suitable example, for example, a method of adding an alcohol or hydrous alcohol to a plant raw material and allowing to stand or stir at room temperature for about 1 minute to 5 hours can be exemplified.

  The fraction obtained in this way and the solid obtained by concentrating and drying the fraction may be used as it is as a “plant-derived fraction containing glucosylceramide and sterol glycoside”. This may be further subjected to lipid removal treatment and used as “a fraction derived from a plant raw material containing glucosylceramide and a sterol glycoside”. Since the purity of the resulting glucosylceramide-containing product can be further increased by removing the lipid, it is preferable.

  A preferable lipid removal treatment method is not particularly limited, and examples thereof include hexane and / or acetone precipitation treatment. The said process can be performed by a conventional method. For example, in both hexane precipitation treatment and acetone precipitation treatment, an appropriate amount (for example, 5 to 10 mL for 1 g of sample) of hexane or acetone is added to the sample, and after stirring, centrifugal treatment or filtration under reduced pressure is performed to recover the precipitate or insoluble part. This can be done by performing the operation. Furthermore, you may perform the precipitation process (hexane / acetone precipitation process) using the mixed solvent of hexane and acetone. In this case, the mixing ratio of hexane and acetone can be appropriately set (for example, the volume ratio is hexane: acetone = 1: 1). The hexane precipitation process, the acetone precipitation process, and the hexane / acetone precipitation process may be repeated, or may be performed multiple times, either alone or in combination in any order. When combining, for example, it is preferable to combine each treatment 1 to 5 times, preferably 1 to 3 times. In addition, since the pigment | dye which cannot be removed only by acetone precipitation may become removable by using hexane, it is preferable to use a hexane precipitation process or a hexane / acetone precipitation process. As one aspect of a preferred lipid removal treatment, a treatment of repeating the acetone precipitation treatment 1 to 5 times and then repeating the hexane / acetone precipitation treatment 1 to 5 times can be mentioned. Moreover, as a sample, it is preferable to use what was concentrated and dried by drying under reduced pressure the fraction obtained by extracting from the above-mentioned plant raw material with an organic solvent or a water-containing organic solvent. Although not intended to be limited, it is considered that mainly phospholipids and glyceroglycolipids can be removed by hexane precipitation, and neutral lipids can be mainly removed by acetone precipitation.

  Moreover, instead of or in addition to the above-described hexane precipitation treatment and / or acetone precipitation treatment, an alkali hydrolysis treatment may be performed as the lipid removal treatment. Examples of the alkaline solution that can be used for the treatment include sodium hydroxide solution, and water, ethanol, and the like can be used as a solvent for the solution. A specific treatment method is not particularly limited, and can be performed by a conventional method.

  The alkali hydrolysis treatment may be performed before or after the hexane precipitation treatment and / or the acetone precipitation treatment, but is preferably performed before. In addition, it is thought that ester lipids, such as glycerophospholipid and glyceroglycolipid, are mainly decomposed | disassembled and removed from a polar lipid fraction by an alkaline hydrolysis process.

  The lipid-removed fraction thus obtained can also be preferably used as a “plant-derived fraction containing glucosylceramide and sterol glycoside”. It should be noted that most of the fraction derived from plant raw materials (90% by mass or more, preferably 95% by mass or more, more preferably 97%) is obtained by concentrating and drying the fraction obtained through the lipid removal treatment. (Mass% or more) consists of glucosylceramide and a sterol glycoside.

  The “plant-derived fraction containing glucosylceramide and sterol glycoside” obtained as described above is subjected to alcohol precipitation treatment to recover the supernatant, whereby glucosylceramide is separated and high-purity glucosylceramide Inclusions can be obtained. In addition, the method mentioned above is an example for obtaining a plant raw material origin fraction, and is not necessarily limited to this. Even if “a fraction derived from a plant raw material containing glucosylceramide and sterol glycoside” obtained by a known method (for example, the method described in Patent Document 1 above), this is subjected to an alcohol precipitation treatment to obtain a supernatant. By collecting, glucosylceramide is separated, and a high-purity glucosylceramide-containing material can be obtained.

  When using a plant raw material-derived fraction obtained through lipid removal treatment, most of the fraction is glucosylceramide and sterol glycoside. And usually 90% by mass or more, preferably 95% by mass or more, more preferably 97% by mass or more) sterol glycoside-containing product (precipitation) and high purity (in terms of dry mass, usually 90% by mass or more, preferably 95 mass% or more, more preferably 97 mass% or more) glucosylceramide-containing material (supernatant) can be obtained. Thus, this invention concerns not only the manufacturing method of a glucosylceramide containing material but the method of manufacturing a sterol glycoside containing material through the process mentioned above.

  Although it does not restrict | limit especially as alcohol used for an alcohol precipitation process, For example, a C1-C6 monohydric alcohol, Preferably a C1-C4 monohydric alcohol can be used, Specifically, ethanol and isopropanol are used. It can be preferably used. Alcohol can be used 1 type or in mixture of 2 or more types.

  The alcohol precipitation treatment can be performed by a conventional method. For example, an appropriate amount (for example, about 20 mL per 1 g of the concentrated dry-solid fraction) of alcohol is added to the plant raw material-derived fraction, and the mixture is stirred and then centrifuged or filtered under reduced pressure. The alcohol precipitation treatment may be repeated. Since glucosylceramide is contained in the supernatant after the alcohol precipitation treatment, when the alcohol precipitation treatment is repeated, the supernatant may be collected and combined for each treatment. In addition, since the sterol glycoside may be mixed into the supernatant when repeated many times, the alcohol precipitation treatment is usually performed 1 to 5 times, preferably 1 to 3 times, more preferably 1 to 2 times.

  The supernatant obtained by the treatment may be used as a glucosylceramide-containing material, or the supernatant may be concentrated and dried to obtain a solid (powder), which may be used as a glucosylceramide-containing material. The supernatant can be concentrated to dryness according to a conventional method. For example, the supernatant may be concentrated and dried with a rotary evaporator or the like, and washed as necessary.

  The glucosylceramide-containing product thus obtained can be preferably used for moisturizing the skin, inhibiting collagen degradation, inhibiting melanin production, relieving atopic symptoms, and the like, for example, by oral ingestion or percutaneous ingestion. In addition, glucosylceramide is also known to inhibit colon adenoma (polyp) onset, and can be used for this purpose. Furthermore, it can also be used for pharmacokinetics such as digestion and absorption of glucosylceramide that have not yet been elucidated, safety tests such as acute and chronic toxicity, and analysis of gene expression fluctuations due to intake of glucosylceramide. Furthermore, the glucosylceramide-containing product is considered to have particularly excellent lipid metabolism improving action (LDL-cholesterol lowering action, adiponectin secretion promoting action, etc.) and antihyperglycemic action.

  Therefore, the present invention also provides a moisturizer, a collagen degradation inhibitor, a melanin production inhibitor, an antiatopic agent, an antihyperlipidemic agent and an antihyperglycemic agent containing the glucosylceramide-containing product as an active ingredient. Furthermore, this invention also provides the cosmetics and food additive which contain the said glucosylceramide containing material and show | plays these effects. Furthermore, the present invention also provides a food containing the glucosylceramide-containing material, a moisturizer comprising the food, a collagen degradation inhibitor, a melanin production inhibitor, an antiatopic agent, an antihyperlipidemic agent, an antihyperglycemia. An agent is also provided.

  Hereinafter, the present invention will be specifically described, but the present invention is not limited to the following examples. In addition, the outline | summary of the process of the manufacturing method of the glucosylceramide containing material of this invention is shown in FIG.

8 L of ethanol was added to 4 kg of extracted corn germ containing glucosylceramide and sterol glycoside-containing fractions , stirred for 1 hour, and then filtered to recover the soluble part. And as a result of concentrating and drying this with a rotary evaporator, about 480 g of an extract was obtained. This was degummed according to a conventional method to obtain 80 g of a corn-derived crude glucosylceramide fraction (degummed product).

To 72 g (degummed product) of the crude glucosylceramide fraction derived from lipid-removed corn, 700 mL of acetone was added and stirred vigorously, and centrifuged to collect a precipitate. The acetone precipitation treatment was repeated 5 times. Furthermore, 350 mL of a hexane / acetone (volume ratio 1: 1) mixed solvent was added to the precipitate obtained by the acetone precipitation treatment, and the mixture was vigorously stirred and centrifuged to collect the precipitate. The hexane / acetone precipitation treatment was repeated three times.

  30 g of the precipitate obtained by the above treatment was used as a corn-derived glucosylceramide fraction.

Ethanol 600 mL (1 g / 20 mL) was added to 30 g of the ethanol-precipitated corn-derived glucosylceramide fraction, and the mixture was stirred and centrifuged to recover the supernatant. The ethanol precipitation treatment was repeated twice, and all the supernatants obtained were combined to obtain a glucosylceramide-containing fraction. In addition, the precipitation (sterol glycoside containing substance) obtained by the said ethanol precipitation process was also collect | recovered.

  The glucosylceramide-containing fraction was concentrated and dried with a rotary evaporator, and the resulting solid was dissolved in hexane and transferred to a tube, and further acetone precipitated with 4 volumes of acetone to obtain 18.4 g of precipitate. The precipitate was used as a glucosylceramide-containing material below.

Purity assay by evaporative light scattering detection-high performance liquid chromatography (HPLC-ELSD) 10 mg of the glucosylceramide-containing material obtained as described above was dissolved in 100 ml of chloroform / methanol (2: 1) solution, and 15 μl thereof was dissolved therein. And was analyzed by HPLC-ELSD to obtain a chromatogram. The obtained chromatogram is shown in FIG. The upper figure shows the result of analyzing a solution containing 1.5 μg of glucosylceramide-containing material, and the lower figure shows the result of analyzing a solution containing 10 μg of glucosylceramide-containing material. The conditions for the HPLC-ELSD analysis are as follows.

HPLC: Shimadzu LC-10A
Detector: Shimadzu ELSD-LT
Separation: 2 liquid gradient A liquid (eluent): hexane / isopropanol / acetic acid (82: 17: 1)
Liquid B (eluent): isopropanol / water / acetic acid / triethylamine (85: 14: 1: 0.2)

  The peak area of glucosylceramide with respect to the total peak area of the chromatogram was 98%. Therefore, it was confirmed that the obtained glucosylceramide-containing product had a glucosylceramide purity of 98%.

  Similarly, when the precipitate (sterol glycoside-containing product) obtained by the ethanol precipitation treatment was analyzed, the sterol of the sterol glycoside-containing product was determined from the peak area of the obtained chromatogram (FIG. 6). It was confirmed that the glycoside purity was 98%.

Claims (5)

The plant material from fractions containing glucosylceramide and sterol glycosides and processes alcohol precipitation, recovering the supernatant seen including,
The plant material-derived fraction is extracted from the plant material using alcohol or hydrous alcohol, and is further obtained by lipid removal treatment,
The alcohol precipitation treatment is ethanol and / or isopropanol precipitation treatment,
A method for producing a glucosylceramide-containing material. The method for producing a glucosylceramide-containing material according to claim 1 , wherein the lipid removal treatment is performed by a step including hexane precipitation treatment, acetone precipitation treatment, hexane and acetone mixed solvent precipitation treatment, or a combination thereof. Plant material is wheat, soybean, rice, cottonseed, rapeseed, corn, sugarcane, sesame, peanut, konjac potato, citrus, is at least one selected from the group consisting of oranges and sugar beet, to claim 1 or 2 The manufacturing method of the glucosylceramide containing material of description. The manufacturing method in any one of Claims 1-3 whose purity of the glucosylceramide containing material obtained is 90 mass% or more in conversion of dry mass. The manufacturing method in any one of Claims 1-4 which does not include the separation process by a high performance liquid chromatography.

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