JP6169978B2 - Method for producing extract containing sphingoid base - Google Patents

Description

  The present invention relates to a method for producing an extract containing a sphingoid base, and more particularly to a method for producing an intestinal hormone secretion promoting component, and a functional food and a medicine containing the intestinal hormone secretion promoting component.

  It is known that when food is ingested, various hormones are secreted from the intestinal tract and exert various physiological functions. Among intestinal hormones, hormones having the ability to promote the release of insulin from pancreatic β cells are called incretins. As specific examples of incretin, a glucagon-like peptide (hereinafter referred to as GLP-1 (glucagon-like peptide 1)) and GIP (gastric inhibitory peptide) are known. Incretin is an important hormone that regulates tissue uptake of blood glucose through insulin secretion. Disorders of incretin secretion cause diabetes.

  Sugars, amino acids, peptides, lipids and fatty acids are known as food-derived components that promote incretin secretion from the intestinal tract. Both are used by the human body as nutrients. In addition, pharmaceuticals that control intestinal hormone secretion to promote insulin release and to treat diabetes have been developed. An approach of administering a GLP-1 derivative that is difficult to be degraded in the body and a degrading enzyme inhibitor (DPP4 inhibitor) that suppresses GLP-1 degradation in the body are attracting attention as next-generation diabetes drugs.

  Regarding the molecular mechanism of incretin secretion from the intestine, a signal enters the cell by binding of a fatty acid derived from food to the receptor protein (GPR120) present on the incretin-secreting cell of the intestine, and the incretin secretion is Be guided. Fatty acids having a strong action are long-chain unsaturated fatty acids. Among natural products, α-linolenic acid (αLA) and DHA are known to have the strongest incretin secretion action.

Sphingolipid is a general term for substances having the structure shown in FIG. 1, and is widely present in animals and plants. A long-chain base component (sphingoid bases) and a ceramide in which a fatty acid is bonded with an acid amide have a common structure, and a sugar or phosphoric acid and a base are further bonded thereto. It is classified into a sphingophospholipid in which a glycosphingolipid with a glycoside bond and a phosphate and a base are combined. Sphingolipids have been suggested to have skin beautifying effects and colorectal cancer prevention effects, and their food functions are attracting attention. Many of the animal long-chain base components (sphingoid bases) are sphingosine (2-amino-4-octadecene-1,3-diol), a long-chain amino alcohol with 18 carbons.

  Various structures as sphingoid bases other than sphingosine have been discovered from sphingolipids derived from animals and plants. Various biological activities have been reported for sphingolipids, ceramide as a component thereof, and sphingoid base as a component thereof itself. In particular, sphingosine-1-phosphate (S1P) in which sphingosine is phosphorylated is used as a ligand for S1P receptors existing on cell membranes, such as cell motility control, actin skeleton formation, cell proliferation, and adhesion bond formation. It is known to cause a cellular response.

  Since sphingolipids and their constituents have various physiological activities, the present inventors considered the possibility that they have an incretin secretion-promoting action, and further promoted the incretin secretion action of various sphingolipids and their constituents. It was investigated. There has been no report that sphingolipid, its component ceramide, and its component sphingoid base have an incretin secretion promoting action.

  Compounds with sphingoid structures such as sphingolipids and ceramides have been found to be partially degraded in the gastrointestinal tract. Therefore, when sphingolipid or ceramide is orally administered, it is considered that the degradation product (for example, sphingoid base) exhibits GLP-1 secretion promoting effect.

  However, sphingolipids are not easily degraded in the intestine, and sphingolipids or constituents thereof (for example, ceramide or sphingoid base) are difficult to be absorbed in the intestine. Since cells involved in GLP-1 secretion are present in the lower part of the small intestine or the upper part of the large intestine, the ingested sphingolipid or its constituent components are advantageous in that they reach the GLP-1 secreting cell that is the site of action. On the other hand, α-linolenic acid is mostly absorbed in the small intestine, and the amount that reaches the GLP-1 secreting cells is a part of the intake. Therefore, it can be expected that the sphingolipid or a component thereof exhibits the same GLP-1 secretion promoting effect as α-linolenic acid in a smaller amount than α-linolenic acid. A sphingolipid or a component thereof can be expected to have a higher GLP-1 secretion promoting effect than α-linolenic acid per calorie or per molecule (number of moles). In particular, since sphingolipids are not easily degraded in the intestinal tract, it is preferable to directly administer sphingoid base, which is a main body that acts on fatty acid receptors and promotes secretion of GLP-1, rather than administering sphingolipids or ceramides. It can be easily estimated that it is advantageous for promoting GLP-1 secretion. Because sphingoid bases are difficult to absorb in the intestinal tract, administration of sphingoid bases is less likely to be an excess of nutrients and induce obesity than administration of common lipids (eg, α-linolenic acid) It can be easily estimated that this is advantageous.

Diabetes Care. 2010, 33 (10): 2277-2284 Endocrinology. 2010, 151 (5): 1984-1989. Nature Medicine, 2005, 11 (1): 90-94. J Lipid Res. 2009, 50 Suppl: S91-96 Biochim Biophys Acta. 2009, 1791 (7): 692-696. Physiol Rev. 2007 Oct; 87 (4): 1409-1439. Biochim Biophys Acta. 1969 Jul 29; 187 (1): 113-121. J. et al. Lipid Res. 2011.51: 1761-1769

  From the clinical results of the next-generation diabetes drugs and the like, it has become clear that increasing the blood concentration of GLP-1 is effective for treating diabetes. On the other hand, GLP-1 is known to stimulate the satiety center and suppress appetite. The obesity-suppressing effect was clearly shown by the clinical results of GLP-1 derivatives. Therefore, if food is ingested, GLP-1 is released from the intestinal tract and the blood concentration of GLP-1 rises, so it can be said that ingesting food naturally also leads to prevention of diabetes.

  However, since food is also nutritious, excessive intake of food leads to obesity and eventually diabetes. It is well known that diabetes is induced as a lifestyle-related disease when an excessive amount of calories is taken and the patient becomes obese. Therefore, in order to increase the blood concentration of GLP-1, excessive intake of food is not appropriate as a method for preventing or treating diabetes.

  An object of the present invention is to provide a method for extracting from a food a component having a high ability to promote intestinal hormone secretion per calorie or per molecule (number of moles) or a component having an ability to promote intestinal hormone secretion and difficult to be absorbed from the intestinal tract. Therefore, it is intended to provide functional foods and medicines that can be used for the prevention or treatment of obesity or diabetes.

  The present inventors are present in food, have components that promote intestinal hormone secretion such as GLP-1 release activity per calorie or per molecule (number of moles), or have intestinal hormone secretion promoting ability and are absorbed from the intestinal tract. We thought that it would be possible to prevent or treat obesity or diabetes by taking out difficult ingredients and taking them. Based on this idea, a component having high intestinal hormone secretion promoting activity per calorie or molecule (number of moles), or a component having intestinal hormone secretion promoting activity and difficult to be absorbed from the intestinal tract was searched for in food ingredients. As a result of diligent efforts, it was discovered that sphingoid bases, which are components of sphingolipids widely present in animals and plants, have a high GLP-1 secretion promoting activity. Sphingoid bases are known to be difficult to absorb from the intestinal tract.

The present invention includes the following steps (1) to (3):
(1) a step of extracting a lipid containing sphingolipid from a natural product,
(2) a step of obtaining a glycosphingolipid by fractionating by chromatography from the extract obtained in step (1), and (3) an acid hydrolysis of the glycosphingolipid, and then a fatty acid methyl ester from the decomposition product. And a step of extracting and removing free fatty acids,
A method for producing an extract containing a sphingoid base is provided.

  The natural product is preferably sea cucumber or corn.

  The present invention also provides a functional food composition or a pharmaceutical composition containing an extract containing a sphingoid base.

  The extract is preferably a compound having a sphingoid base structure.

  The compound having a sphingoid base structure is preferably a sphingolipid.

  The compound having a sphingoid base structure is preferably ceramide.

  The composition of the present invention is suitable for preventing or treating diabetes, obesity, or metabolic syndrome or assisting in preventing or treating.

  The sphingoid base has a function of promoting GLP-1 secretion, for example.

で示される４−スフィンゲニン（ｄ１８：１４ｔ）、

で示される４，８−スフィンガジエニン（ｄ１８：２４ｔ，８ｔ（ｃ））、

で示される４−ヒドロキシ−８−スフィンゲニン（ｔ１８：１８ｔ（ｃ））、

で示される９−メチル−４，８−スフィンガジエニン（９−Ｍｅ−ｄ１８：２４ｔ，８ｔ）、

で示される４，８，１０−スフィンガトリエニン（ｄ１８：３４ｔ，８ｔ，１０ｔ）、及び、

で示される９−メチル−４，８，１０−スフィンガトリエニン（ｄ１９：３４ｔ，８ｔ，１０ｔ）
からなる群から選ばれる少なくとも一種であることが好ましい。The sphingoid base is

4-sphingenin (d18: 14t) represented by

4,8-sphingadienin represented by (d18: 24t, 8t (c)),

4-hydroxy-8-sphingenin (t18: 18t (c)) represented by

9-methyl-4,8-sphingadienin (9-Me-d18: 24t, 8t) represented by

4,8,10-sphingatrienin (d18: 34t, 8t, 10t) represented by:

9-methyl-4,8,10-sphingatrienin (d19: 34t, 8t, 10t)
It is preferably at least one selected from the group consisting of

  According to the method for producing an extract containing a sphingoid base of the present invention, a component having a high ability to promote secretion of intestinal hormone per calorie or a component having an ability to promote secretion of intestinal hormone from food and hardly absorbed from the intestinal tract is fractionated. Can be taken out. The extract can be provided as a functional food or medicine that can be used for the prevention or treatment of obesity and diabetes.

  The present inventors have established a method for extracting a component that promotes intestinal hormone secretion from the fat of a food component, and have attempted to provide a functional food or a pharmaceutical containing the separated component. By improving the purity of the target component, it is possible to avoid the risk of side effects that the impurities may have. Alternatively, by clarifying the structure and properties of the active body, it is possible to stably supply the target component.

It is a figure which shows the structure of a sphingolipid. It is a GC chromatogram of a corn-derived sphingoid base aldehyde derivative. It is a GC chromatogram of a sea cucumber-derived sphingoid base. It is a graph which shows the GLP-1 secretion promotion activity from STC-1 cells by a sea cucumber or corn-derived sphingoid base. It is a graph which shows the GLP-1 secretion promotion activity from STC-1 cell by sphingosine, sea cucumber, or a maize origin sphingoid base. It is a graph which shows the GLP-1 secretion promotion effect of the mouse portal vein by oral administration of sphingosine, sea cucumber, or a maize origin sphingoid base.

  The manufacturing method of the extract containing the sphingo base of this invention is demonstrated below. The raw material to be used is not particularly limited as long as it is a natural product containing sphingolipid. Examples of the natural product include animal origin such as sea cucumber, fish brain and cow brain, and plant origin such as corn, wheat and rice bran, preferably sea cucumber and / or corn. A total lipid containing sphingolipids is extracted by adding a lipid-soluble solvent (for example, a mixture of chloroform and methanol to a mixture ratio of chloroform: methanol = 2: 1) to the natural product. Decomposes glycerolipids in the extracted total lipids.

  Dissolve the total lipid in a solvent (eg methanol), add alkali (eg potassium hydroxide) to make it weakly alkaline with a final alkali concentration of 0.2-0.5 N and heat (eg 30-40 ° C., 0 Heat for 5 to 2 hours).

  After alkaline hydrolysis, chloroform and water are added so that the chloroform layer and the aqueous layer are separated. For example, twice the amount of chloroform and 40% of its water are added and stirred, and the separated chloroform layer is recovered.

  Next, the chloroform layer is washed with water. For example, half the amount of chloroform and half the amount of water are added and stirred, and then the chloroform layer is recovered. The chloroform layer contains an alkali-stable lipid fraction (crude sphingolipid fraction).

  The sphingolipid fraction is purified using various chromatographies. For example, silica gel column chromatography or high performance liquid chromatography equipped with a silica gel column is used.

  In silica gel column chromatography, for example, a silica gel suspended in chloroform is packed in a glass column, and the sphingolipid fraction dissolved in chloroform is applied to the column. For example, the eluate is eluted by gradually increasing the methanol ratio in a mixed ratio of chloroform: methanol. For example, elution is performed with an eluent having a high methanol ratio stepwise, such as chloroform: methanol = 95: 5, chloroform: methanol = 90: 10, chloroform: methanol = 80: 20, chloroform: methanol = 70: 30. Each eluate is used in an amount equivalent to 1.5 times the column volume.

  The eluate is collected by fractionating into 3 to 5 fractions for each eluate. The mixing ratio of chloroform and methanol may be changed stepwise or continuously.

  A part of the fraction is developed on a thin layer chromatography together with a sample (sphingomyelin, lactosylceramide, glucosylceramide, ceramide, cholesterol) to examine the components of the fraction. A fraction containing glycosphingolipid (glucosylceramide) is collected, and concentrated and dried with an evaporator and nitrogen gas to obtain a glycosphingolipid fraction.

  Hydrous methanolic 1N hydrochloric acid is added to the glycosphingolipid (glycosylceramide) fraction to cause acid hydrolysis. Acid hydrolysis at 75-85 ° C. for 10-20 hours. The decomposition product is washed 3 or 4 times with hexane to extract and remove fatty acid methyl esters and free fatty acids.

  4N KOH is added to the remaining methanol layer to adjust the pH to 9.7 or higher, approximately equal amount of water is added, and then sphingoid base is extracted three times with equal amount of diethyl ether. The collected diethyl ether is washed with an equal amount of water and then dried to nitrogen to obtain a sphingoid base.

  Methanol and 1/5 amount of 0.2M periodic acid aqueous solution are added to the prepared sphingoid base and reacted in the dark for 1 hour. Thereafter, the mixture is extracted three times with an approximately equal amount of hexane, and the collected hexane layer is washed with an equal amount of water and then concentrated with nitrogen gas.

  A part of the obtained aldehyde derivative solution is subjected to GC-MS. For GC-MS analysis, for example, GC-17A, QP5050A instrument (both Shimadzu, Kyoto, Japan) is used. DB-1 (0.25 mm ID × 30 m, 0.25 μm; Agilent Technologies, California, USA) is used as the column. The column temperature is 80 ° C. to 160 ° C. (temperature increase 2 ° C./min), 160 ° C. to 200 ° C. (temperature increase 1 ° C./min), and the interface and injector temperatures are 250 ° C.

The results of structural analysis of the sphingoid base component prepared from corn by GC-MS are shown in FIG. Their main components are as follows.

  Where both the cis form and the trans form exist at the 8-position of Compound 2 and Compound 3, this prepared sample contains many cis forms.

The results of structural analysis of the sphingoid base component prepared from sea cucumber by GC-MS are shown in FIG. Their main components are as follows.

  In addition to Compound 4, Compound 5, and Compound 6, there are many types of sphingoid bases such as those having 17 or 16 carbon chains.

  Functional foods containing GLP-1 secretion promoting substances or pharmaceuticals containing GLP-1 secretion promoting substances as active ingredients include the following uses.

  Examples of target diseases or symptoms associated with an increase in the concentration of GLP-1 include (1) antidiabetic drugs by promoting insulin secretion from pancreatic β cells (2) hyperglycemia and insulin resistance by promoting differentiation and proliferation of pancreatic β cells Diabetes preventive that prevents the transition from sex and obesity to diabetes. Or improvement of engraftment rate of transplanted cells at the time of β-cell transplantation; (3) Treatment with excessive gastric acid by inhibiting gastric acid secretion; (4) Treatment for diarrhea by inhibiting intestinal motility (5) Diseases caused by neuropathy while maintaining nerve plasticity and survival And (6) obesity prevention treatment by suppressing appetite, and the like. However, the target disease or symptom is not limited to the above, and all treatment and symptom improvement by increasing the concentration of GLP-1 are targeted.

  In the composition of the present invention, in addition to an extract containing a sphingoid base as an active ingredient, a pharmacologically or pharmaceutically acceptable carrier or auxiliary agent may be added within a range that does not inhibit the effect of the present invention. it can. Examples of such additives include excipients such as corn starch, crystalline cellulose, lactose; disintegrants such as starch, sodium alginate, gelatin, calcium carbonate, calcium citrate; methyl cellulose or salts thereof, ethyl cellulose, gum arabic, Binders such as gelatin; Lubricants such as talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil; xanthine derivatives, pH adjusters, cooling agents, suspending agents, thickeners, solubilizers, antioxidants , Coating agents, plasticizers, surfactants, water, alcohols, water-soluble polymers, sweeteners such as fructose, glucose, sorbitol, flavoring agents, acidulants such as citric acid, flavorings, colorants, vitamins, minerals And lipids.

  The shape of the composition of the present invention is not particularly limited, and for example, solid preparations such as powders, granules, capsules, pills, tablets, chewable tablets, drops, drinks, liquids, suspensions, emulsions. , Syrups and dry syrups.

  The usage of the composition of the present invention is not particularly limited. The pharmaceutical may be oral or parenteral (intravenous injection, intramuscular injection, subcutaneous administration, intraperitoneal administration, rectal administration, transdermal administration, etc.). The intake time of the composition of the present invention may be before or after a meal, and is preferably taken before or simultaneously with a meal.

  The intake amount of the composition of the present invention as a functional food can vary depending on the age, weight, and past disease (for example, obesity) of the subject.

  The pharmaceutical dosage of the composition of the present invention may vary depending on the age, weight and pre-existing disease (eg obesity) of the patient to whom it is administered.

  This invention also provides the foodstuff containing the said composition. By adding the composition of the present invention to foods, secretion of intestinal hormones, particularly GLP-1, can be promoted. Examples of food are not particularly limited, for example, side dishes such as salad, deep-fried tofu, tofu, konjac; soup; bread, cooked rice and noodles; cookies, muffins, cakes, chips, snacks, chocolate, jelly, pudding, chewing gum, Sweets such as candy; Dairy products such as yogurt and milk; Fish meat products such as ham, sausage and kamaboko; Beverages such as coffee, juice and sports drinks; and general processed foods such as seasonings such as dressings, soy sauce and sauces Can be mentioned.

[Example 1] Preparation of sphingoid base from corn or sea cucumber 1) Total lipid extraction Total lipid was extracted from 100 g of dried corn powder or sea cucumber powder by the Folch method using a chloroform-methanol (2: 1) mixture. . To the obtained total lipid, 100 mL of methanol and 10 mL of 4N KOH were added and incubated at 37 ° C. for 1 hour to decompose glycerolipid. To this, 200 mL of chloroform and 80 mL of water were added, and only the lower chloroform layer was collected with a separatory funnel. To the collected chloroform layer, 100 mL of methanol and 90 mL of water were further added and washed with water, and the lower layer was collected again with a separatory funnel. From this chloroform layer, an alkali-stable lipid fraction (crude sphingolipid fraction) was recovered.

2) Purification of glycosphingolipid by silicic acid column chromatography 46 g of silica gel 60 (0.063-0.200 mm for column chromatography, MERCK) was suspended in an appropriate amount of chloroform and a glass column (diameter 2 cm, length 25 cm). ). The above sample subjected to weak alkali decomposition treatment was dissolved in a small amount of chloroform and applied to a column. As an elution solvent, 50 mL of chloroform, 100 mL of chloroform-methanol (95: 5), 100 mL of the same (90:10), 100 mL of the same (80:20), 100 mL of the same (70:30), and 100 mL of methanol were used in this order. Each eluted fraction was collected.
Chloroform 50mL ・ ・ ・ Fraction 1
Chloroform-methanol (95: 5) 100 mL ... Fraction 2, 3
Chloroform-methanol (90:10) 100 mL ... Fraction 4, 5, 6, 7
Chloroform-methanol (80:20) 100 mL ... fractions 8, 9, 10, 11
Chloroform-methanol (70:30) 100 mL ... fractions 12, 13
Methanol 100mL ... Fraction 14,15

3) Thin layer chromatography of glycosphingolipid The sample and the sample were spotted on a silica gel thin layer plate (25HPTLC Plates 10 × 10 Silicagel 60, MERCK), and developed with a developing solvent (chloroform: methanol: water = 64: 16: 2). Then, a copper sulfate coloring reagent {2 mL of H ₂ SO ₄ , 20 mL of 33% acetic acid aqueous solution (6.6 mL acetic acid + 13.4 mL water), 20 mL H ₃ PO ₄ , 360 mL 0.5% CuSO ₄ aqueous solution (1. 8 g CuSO ₄ +360 mL water) was mixed} and the plate was dipped in it, dried with a dryer, and then heated with a hot plate to cause color development.

4) Purification of glycosphingolipid by silicic acid column chromatography Each fraction was collected and subjected to TLC together with samples (sphingomyelin, lactosylceramide, glucosylceramide, ceramide, cholesterol), and the glycosphingolipid (glucosylceramide) The fractions contained (fractions 11 to 14) were combined and concentrated and dried with an evaporator and nitrogen gas to obtain a glycosphingolipid fraction. The recovery rate of the glycosphingolipid fraction with respect to the dry powder was about 0.05% for corn and about 0.5% for sea cucumber.

5) Acid hydrolysis of glucosylceramide (Preparation of sphingoid base)
To 4 mg of corn or sea cucumber-derived glucosylceramide, add 1 mL of hydrous methanolic 1N hydrochloric acid (4.7 mL of concentrated hydrochloric acid and 4.7 mL of water and dilute to 50 mL with methanol), and in a screw cap tube at 80 ° C. for 16 hours. It was made to react with a block heater. After cooling to room temperature, it was washed with 1 mL of hexane three times to extract and remove fatty acid methyl esters and free fatty acids. 200 μL of 4N KOH was added to the remaining methanol layer, the pH was adjusted to 9.7 or higher, 1 mL of water was added, and the sphingoid base was extracted 3 times with 1 mL of diethyl ether. The collected 3 mL of diethyl ether was washed with an equal amount of water and then nitrogen-dried. The weight was measured and dissolved in chloroform / methanol (2: 1, v / v) to a concentration of 1 mg / mL. And stored at -30 ° C.

[Example 2] Estimation of structure of sphingoid base prepared from corn or sea cucumber The sphingoid base prepared in Example 1 was analyzed by GC-MS. Specifically, 100 μL of the sphingoid base solution obtained by acid hydrolysis was transferred to a glass tube and nitrogen-dried, and 1 mL of methanol and a 0.2 M periodic acid aqueous solution (428.0 mg of sodium periodate in water). 200 μL) was added and allowed to react in the dark for 1 hour. Thereafter, extraction was performed 3 times with 1 mL of hexane, and the collected hexane layer was washed with 3 mL of water and then concentrated with nitrogen gas. 1 μL of the obtained aldehyde derivative solution was subjected to GC-MS.

  For GC-MS analysis, GC-17A and QP5050A instrument (both Shimadzu, Kyoto, Japan) were used. The column used was CP-Sil 88 (0.25 mm ID × 50 m, 0.2 mm; Chrompak, Middelburg, The Netherlands). The column temperature was 80 ° C. to 160 ° C. (temperature increase 2 ° C./min), 160 ° C. to 200 ° C. (temperature increase 1 ° C./min), and the interface and injector temperatures were 250 ° C. The measurement results of the corn-derived sphingoid base are shown in FIG. The measurement results of the sea cucumber-derived sphingoid base are shown in FIG.

[Example 3] Measurement of GLP-1 secretion-promoting activity against STC-1 STC-1 of the following sample (cell line of hormone-secreting cells of the intestinal tract: Am. J. Pathol. 136: 1349-1363 (1990)) GLP-1 secretion promoting activity was measured. STC-1 cells were seeded at 1 × L in a 12-well plate at a concentration of 4 × 10 ⁵ cells / mL. After culturing for 48 hours, each sample (μmol, DMSO solution) dissolved in Dulbecco's Modified Eagle's Medium (DMEM (SIGMA, D6429)) (500 μL) was added. After stimulation for 1 hour, the amount of GLP-1 in the culture supernatant was measured by ELISA.

  Samples used were DMSO (control), α-linolenic acid 20 μM, sphingosine 10 μM and 40 μM, sea cucumber-derived sphingoid bases 10 and 40 μM, corn-derived sphingoid bases 10 and 40 μM. The concentration is the final concentration. For ELISA, Rat GLP-1 ELISA Kit wako (Wako Pure Chemical Industries, 29-59201) was used. FIG. 4 shows the GLP-1 release characteristics of each sample.

[Example 4] Measurement of GLP-1 secretion promoting activity against STC-1 STC-1 of the following sample (see cell line of hormone secreting cells of intestinal tract: Am. J. Pathol. 136: 1349-1363 (1990)) GLP-1 secretion promoting activity was measured. STC-1 cells were seeded at 1 × L in a 12-well plate at a concentration of 4 × 10 ⁵ cells / mL. After culturing for 48 hours, each sample (μmol, DMSO solution) dissolved in Dulbecco's Modified Eagle's Medium (DMEM (SIGMA, D6429)) (500 μL) was added. After stimulation for 1 hour, the amount of GLP-1 in the culture supernatant was measured by ELISA.

  Samples used were DMSO (control), α-linolenic acid 10 μM, 40 μM and 100 μM, sphingosine 4 μM, 10 μM and 40 μM, sea cucumber-derived sphingoid bases 4 μM, 10 μM and 40 μM, and corn-derived sphingoid base 4 μM, 10 μM and 40 μM. It is. The concentration is the final concentration. For ELISA, Rat GLP-1 ELISA Kit wako (Wako Pure Chemical Industries, 29-59201) was used. The results are shown in FIG.

  From the results of Example 3 and Example 4, regarding the GLP-1 secretion promoting activity against STC-1, sphingosine is comparable to α-linolenic acid, whereas sea cucumber-derived sphingoid base and corn-derived sphingoid base are α It can be seen that linolenic acid has an activity of promoting intestinal hormone secretion per molecule (number of moles).

[Example 5] Mouse administration test ICR mice, males 8 weeks old, were fasted for 18 hours and then orally administered with 1.5 mg / g body weight of glucose. Immediately thereafter, the following sample 27 mg / g body weight was suspended in 0.5% CMC and orally administered. For control, only 0.5% CMC solution was administered. The N number was 3 or 4. The sample used is α-linolenic acid, sphingosine, corn-derived sphingoid base, or sea cucumber-derived sphingoid base.

  One hour after administration of the sample, blood was collected from the portal vein to obtain plasma and stored frozen at −80 ° C. After thawing the stored plasma samples, the amount of GLP-1 was measured by ELISA. As the ELISA, Rat GLP-1 ELISA Kit wako (Wako Pure Chemical Industries, 29-59201) was used. The results are shown in FIG. For the significance test, Scheffe's multiple test was used. In the figure, * indicates that there is a significant difference compared to the control (P <0.05).

  Oral administration of sphingosine did not give a significant difference from the control in terms of plasma GLP-1 concentration. On the other hand, corn-derived sphingoid base and sea cucumber-derived sphingoid base significantly promoted GLP-1 secretion from the intestinal tract by oral administration. It was found that the sphingoid base has a GLP-1 secretion promoting effect that is almost the same as or stronger than α-linolenic acid.

  The extract containing the sphingoid base of the present invention can be used as an active ingredient of a medicine or functional food for the purpose of promoting intestinal hormone secretion.

Claims (2)

Containing sphingoid salt group, a GLP-1 secretagogue for functional food composition or a pharmaceutical composition,
The sphingoid base is

4-sphingenin (d18: 14t) represented by

4,8-sphingadienin represented by (d18: 24t, 8t (c)),

4-hydroxy-8-sphingenin (t18: 18t (c)) represented by

9-methyl-4,8-sphingadienin (9-Me-d18: 24t, 8t) represented by

4,8,10-sphingatrienin (d18: 34t, 8t, 10t) represented by:

9-methyl-4,8,10-sphingatrienin (d19: 34t, 8t, 10t)
The functional food composition or pharmaceutical composition for promoting GLP-1 secretion, which is at least one selected from the group consisting of: Diabetes, obesity, or metabolic syndrome of preventing or treating or preventing or GLP-1 secretagogue for functional food composition according to claim 1 for assisting a therapeutic or pharmaceutical compositions.

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