JP2011084546A - Method for manufacturing intestinal hormone secretion-modulating component, method of use, functional food, and medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide such a functional food and medicine from a fractional component in foods as can be used for the prevention or treatment of obesity, diabetes, or intestinal disease. <P>SOLUTION: The intestinal hormone secretion-modulating component is taken out from a denatured fatty acid by denaturing the fatty acid obtained from food and by fractionating the denatured fatty acid with the chromatography containing at least one time of HPLC. The fractionation may be performed by plurality times of HPLCs. The suitable fatty acid includes especially α-linolenic acid, unsaturated long chain fatty acid such as DHA, or natural oils and fats, for example, the fatty acid fractionated from perilla oil or fish oil. The fractionation with column chromatography gives the substances named SIHR and IIHR. SIHR serves for the prevention and betterment of diabetes without incurring obesity or the like even when taken in for a long period of time because of having a high intestinal hormone secretion modulation (accelerating or inhibiting) function per calorie. IIHR serves for the prevention and betterment of an intestinal disease or the like. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a production method and a use method of an intestinal hormone secretion regulating component, and a functional food and a medicine containing the intestinal hormone secretion regulating component.

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

  On the other hand, diseases caused by substance P (Subastance P) or neurokinin (Neurokinin) reacting with neurokinin receptor in the periphery include, for example, neurogenic inflammation of the intestinal tract (see Non-Patent Document 1), visceral pain / Hyperalgesia (see Non-Patent Document 2), diarrhea (See Non-Patent Document 3), inflammation of intestinal tract and pancreas (see Non-Patent Document 4), stress skin inflammation (see Non-Patent Document 5), neurological inflammation (Refer nonpatent literature 6), acute enteritis (refer nonpatent literature 7, 8), intestinal diverticulum disease (refer nonpatent literature 9), etc. are known.

  Intestinal hormones substance P, neurokinin and CCK, if excessively released, promote the onset and exacerbation of IBS (irritable bowel disease) and IBD (inflammatory bowel disease). It is a substance useful for the living body that works to promote intestinal immunity, protect the living body from pathogenic bacteria that enter the digestive tract, etc., and promote digestive activity by regulating secretion of the digestive fluid and intestinal motility. For patients who do not sufficiently secrete intestinal hormones such as substance P, neurokinin and CCK, therapies that promote intestinal hormone secretion are necessary.

  In addition, sugars, amino acids, peptides, lipids and fatty acids are known as ingredients in foods that promote intestinal hormone release 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.

Am J Physiol Gatrointesti Liver Physiol 279, G1298-G1306, 2000 Neuroscience, 98 (2), 345-352, 2000 Br. J. Pharmacol. 121 (3), 375-80) 1997 Am J Physiol 272, G785-93, 1997 Clin Exp Dermatol, 29, 644-648, 2004 Neurocience 125 (2), 449-459, 2004 Neuroscience, 145 (2), 699-707 2007 IBD (Eur J Pharmacol, 548 (1-3), 150-137, 2006 Dig Liver Dis 36 (5), 348-354,2004

  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 clinical results of GLP-1 derivatives clearly showed obesity-suppressing effects. 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, overdose of food leads to obesity and thus diabetes. As is well known, diabetes is induced as a lifestyle-related disease when the patient becomes obese due to excessive calories.
Therefore, excessive intake of food to increase the blood concentration of GLP-1 is not appropriate as a method for preventing or treating diabetes.

On the other hand, food intake promotes the release of various intestinal hormones in addition to GLP-1. Substance P, neurokinin, CCK and the like prevent invasion of microorganisms from the intestinal tract by promoting intestinal immunity and assist digestive activity. Those who have reduced ability to secrete intestinal hormones have a reduced function of protecting against invasion of microorganisms from the intestinal tract, and are at risk of infecting not only the intestine but also the whole body. They are also at risk for various diseases that stem from reduced digestive activity.
Intestinal hormone secretion-promoting substance administration is effective for people who have decreased intestinal hormone secretion capacity due to food intake, but it is natural that intestinal hormone secretion per calorie or molecule (number of moles) of normal food It is necessary to administer a highly capable substance.

  On the other hand, continued excessive secretion of intestinal hormones leads to the onset or exacerbation of intestinal diseases such as IBS and IBD. In order to prevent and treat intestinal diseases such as IBS and IBD, it is necessary to administer a substance that suppresses intestinal hormone secretion.

  An object of the present invention is to provide a functional food that can be used for the prevention or treatment of obesity, diabetes, or intestinal diseases by taking a means for extracting a component having a high function of regulating intestinal horn per calorie or molecule (number of moles) from food. To provide medicine.

If the present inventors can take out a component (secretory regulating component) having a high intestinal hormone regulating function such as GLP-1 releasing activity per calorie or per molecule (number of moles) present in food, We thought that it would be possible to prevent or treat obesity, diabetes or bowel disease by ingestion.
Based on this idea, as a result of diligent efforts, to extract ingredients with high intestinal hormone regulating function, such as ingredients with high intestinal hormone secretion promoting activity or intestinal hormone secretion inhibitory activity per calorie or molecule (number of moles) from food. Successful.
The present invention is based on the invention of Japanese Patent Application No. 2008-066909 filed by the present inventors, and is further developed. Therefore, the inventions in Japanese Patent Application No. 2008-066909 are assumed.

  That is, the method for producing an intestinal hormone secretion-regulating component of the present invention comprises modifying a fatty acid, fractionating the modified fatty acid by chromatography including at least one high performance liquid chromatography (HPLC), and intestinal hormone secretion from the modified fatty acid. This is a method of taking out the adjustment component.

More preferably, fatty acids include unsaturated long-chain fatty acids such as α-linolenic acid (αLA) and Docosahexanoic Acid (DHA), natural fats and oils such as shiso oil and fish oil, or fatty acid fractions prepared from natural fats and oils. Yes.
A known method can be employed as a method for modifying these. For example, it is heated at a high temperature (30 ° C. to 150 ° C.), stirred in air or oxygen, oxidized, or denatured by ultraviolet irradiation or the like. Alternatively, there is a method of modifying these in combination.

A method for preparing a fatty acid from natural fats and oils can be carried out by employing a well-known method. A typical method includes the following processing.
Saponify the original fat with alkali, fractionate the saponified product using a mixture of water and petroleum ether, and acid-treat the water-ethanol fraction to obtain a mixture of free fatty acid and glycerin as the water-ethanol fraction. And unsaponifiable matter is obtained as a petroleum ether fraction. Next, when the water-ethanol fraction is washed again with petroleum ether, fatty acids are fractionated on the petroleum ether side, and glycerin is fractionated on the remainder.

As the fractionation method, a solvent extraction method or various types of chromatography can be used, but at least one HPLC (high performance liquid chromatography) is always used in some cases. As described in each example described later, the present inventors have found that a substance can be specified or a substance can be specified with higher accuracy by using HPLC with high resolution.
For example, by HPLC using a reverse phase chromatograph of modified fatty acid, an eluate containing acetic acid having a volume ratio of methanol to water of 80-50: 20-50 and a volume ratio of 0.05% ± 0.01% is obtained. Elute and fractionate.
At that time, as described in Japanese Patent Application No. 2008-0669909, hexane, ether and acetic acid were used as developing solvents on a silica gel plate in a volume ratio of 60: 40: 1, and the Rf value was 0.10 to 0.10. Show spots in the region of 0.12, 0.13-0.15, 0.22-0.24, 0.23-0.25, 0.24-0.26, and 0.25-0.27 Fraction the fraction.

Also, as a fractionation method, chromatography using a silica gel column (for example, silica gel cartridge Sep-Pak), HPLC equipped with various columns (for example, reverse phase column, silica gel column, etc.), or other chromatographies) be able to. For example, the denatured lipid is charged into a silica gel cartridge (Sep-Pak Plus long, Waters, WT020520) and eluted with a stepwise change in the solvent hexane: ether ratio. When a silica gel cartridge is used as a pretreatment for HPLC, for example, the cartridge is washed with a mixed solution of hexane: ether = 100: 1 to 50 and then eluted with a mixed solution of hexane: ether = 0 to 50: 100. By this operation, unmodified α-linolenic acid can be removed, which helps to improve separation performance in the next purification step, HPLC, or to prevent column deterioration.
In addition, the intestinal hormone secretion regulating component can be roughly separated by chromatography using a silica gel cartridge. For example, as an eluent, the ratio of hexane: ether is
(1) 100: 5
(2) 90:10
(3) 80:20
(4) 70:30
(5) 60:40
(6) 50:50
(7) 40:60
(8) 30:70
(9) 20:80
(10) 10:90
(11) 0: 100
Elute stepwise into the mixed solution. Select the eluate to be used for washing and the eluate to be used for elution according to the target substance for regulating intestinal hormone secretion. For example, the eluate (2) is washed and the target substance is eluted with the eluent (6). When an intestinal hormone secretion regulating substance is used as a functional food or pharmaceutical ingredient, the purpose may be achieved with a partially purified product, and partial purification with a silica gel column is effective.
Details of the method using the silica gel cartridge are as described in Japanese Patent Application No. 2008-0669909.

  In the fractionation, the Rf value is 0.10 in thin layer chromatography (TLC) (using a silica gel plate and a developing solvent in which hexane, ether and acetic acid are mixed at a volume ratio of 60: 40: 1). Spots in the regions of ~ 0.12, 0.13 to 0.15, 0.22 to 0.24, 0.23 to 0.25, 0.24 to 0.26, and 0.25 to 0.27 By fractionating the fractions shown, a substance having a high GLP-1 (Glucagon-like peptide-1) secretion promoting activity per calorie or molecule (number of moles) is taken out. In the present specification, this secretory promoting component is referred to as SIHR (Stimulators of intestinal hormone release). SIHR is a substance itself having GLP-1 release promoting activity.

  When fractionating, HPLC (liquid delivery device (SHIMADZU, LC-6AD), column oven (SHIMADZU, CTO-10A), detector (SHIMADZU, SPD-M20A))) reverse phase column (TOSOH, TSKgel) ODS-80Ts, 4.6mmID × 250mm), column oven temperature 40 ° C ± 5 ° C, fractionation, HPLC, reverse phase column (TOSOH, TSKgel ODS-80Ts, 4.6mmID × 250mm) column oven temperature 40 ° C ± Fractionation was performed at 5 ° C., and the volume ratio of methanol to water was 64-66: 36-34, and the flow rate was 0.9- Elute at 1.1 ml / min, fractions with elution times of 11 to 12.5 minutes, 18.75 to 20.25 minutes, 26.5 to 28 minutes, 60 to 62 minutes, and 62 to 64 minutes Thus, SIHR that functions as an agonist can be obtained.

  In HPLC (liquid delivery device (SHIMADZU, LC-6AD), column oven (SHIMADZU, CTO-10A), detector (SHIMADZU, SPD-M20A)), reverse phase column (TOSOH, TSKgel ODS-80Ts, 4.6mmID) × 250 mm), fractionation under conditions of column oven temperature 40 ° C. ± 5 ° C., and acetic acid having a volume ratio of methanol to water of 49 to 51:51 to 49 and a volume ratio of 0.05% ± 0.01%. Elution at a flow rate of 0.9 to 1.1 ml / min with the eluate contained, and fractionating fractions with an elution time of 48.25 to 50.25 minutes and 50.25 to 52.5 minutes Can be obtained.

When fractionating, HPLC (liquid delivery device (SHIMADZU, LC-6AD), column oven (SHIMADZU, CTO-10A), detector (SHIMADZU, SPD-M20A))) reverse phase column (TOSOH, TSKgel) ODS-80Ts, 4.6mmID × 250mm), fractionation under conditions of column oven temperature 40 ° C ± 5 ° C, volume ratio of methanol to water is 69-71: 31-29, volume ratio 0.05% ± 0 Elution with an eluent containing 0.01% acetic acid at a flow rate of 0.9-1.1 ml / min, fractions with elution times of 0-5 min, 35-40 min, 45-50 min, and 65-70 min Is obtained as a secretion inhibitory component of intestinal hormones such as GLP-1, CCK, substance P and neurokinins (for example, neurokinin A).
It is known that excessive secretion or continuous secretion of intestinal hormones causes diseases, and excessive secretion of CCK, substance P and neurokinin A is harmful to the human body. In the present specification, a food ingredient that suppresses secretion of intestinal hormones is referred to as IIHR (Inhibitor of Intestinal Hormone-release). Substance P, neurokinin or CCK is known to be related to the onset and exacerbation of diseases in intestinal diseases such as IBS (irritable bowel disease syndrome) and IBD (inflammatory bowel disease), and IIHR is related to IBS, IBD. It is useful as a medicine for preventing or treating isotonic diseases or a functional food.

By performing any one of the above three types of HPLC as the first HPLC and further performing any one of the above three types of HPLC as the second HPLC for the obtained fraction, it is more complicated. SIHR and IIHR can be obtained with the product removed.
After the second HPLC, the third, fourth,..., And many other HPLCs may be performed.

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 increased concentrations of GLP-1 include (1) anti-diabetic 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 of hyperacidity by suppressing gastric acid secretion; (4) Treatment of diarrhea by suppressing intestinal motility (5) Disease 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 treatments and symptom improvements associated with increased GLP-1 concentration are targeted.

Functional foods containing a CCK secretion inhibitory substance or pharmaceuticals containing a CCK secretion inhibitory ingredient as an active ingredient include the following uses.
As a target disease or symptom associated with an increase in the CCK concentration, for example, (1) CCK promotes pancreatitis, and therefore, by reducing the CCK concentration, pancreatitis can be treated. (2) Since CCK promotes secretion of gastric acid, suppression of CCK concentration enables treatment of gastric hyperacidity. (3) Since CCK causes behavioral disturbances due to anxiety and stress at the center, suppression of CCK concentration enables treatment of behavioral disorders and use as a tranquilizer. (4) Since CCK antagonizes an analgesic such as morphine, the effect of the analgesic can be enhanced by lowering the CCK concentration. (5) Suppression of CCK concentration makes it possible to treat diarrhea such as infectious diarrhea. However, the target disease or symptom is not limited to the above, and any treatment or symptom improvement of a disease associated with an increase in CCK concentration can be targeted.

Functional foods containing substance S or substance that inhibits secretion of neuronine, or pharmaceuticals containing substance substance that inhibits secretion of substance P or neurokinin as an active ingredient include the following uses.
Examples of target diseases associated with increased concentrations of substance P and neurokinin in the periphery include, for example, intestinal nerve inflammation (see Non-Patent Document 1), visceral pain / hyperalgesia (see Non-Patent Document 2), diarrhea (Non-Patent Document 2) Inflammation of the intestinal tract and pancreas (see Non-Patent Document 4), stress skin inflammation (see Non-Patent Document 5), neurogenic inflammation (see Non-Patent Document 6), acute enteritis (Non-Patent Document 7, 8), intestinal diverticulum disease (see Non-Patent Document 9), and the like. IIHR (Substance P / Neurokinin secretion inhibitor) makes it possible to treat or improve these diseases or symptoms. However, the target disease or symptom is not limited to the above, and any treatment or symptom improvement of a disease associated with increased concentration of substance P or neurokinin can be targeted.

According to the method for producing an intestinal hormone secretion-regulating component of the present invention, a functional food or a medicine, the intestinal hormone-regulating function-promoting component per calorie is fractionated and taken out from food to provide for prevention or treatment of obesity and diabetes. Functional foods and medicines can be provided.

Established a manufacturing method for ingredients that promote or suppress intestinal hormone secretion from fats in food ingredients,
We tried to provide functional foods and medicines containing the separated components. 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.

FIG. 4 is a diagram showing a first HPLC chromatograph in Example 3. It is a figure which shows the GLP-1 release characteristic of the fraction fractionated by the 1st HPL in Example 6, and each fraction. FIG. 6 is a diagram showing a first HPLC chromatograph in Example 7. It is a figure which shows the GLP-1 secretion inhibitory activity of the fraction fractionated by the 1st HPLC in Example 7. It is a figure which shows the CCK secretion inhibitory activity of the fraction fractionated by the 1st HPLC in Example 7. It is a figure which shows the substance P secretion inhibitory activity of the fraction fractionated by the 1st HPLC in Example 7. It is a figure which shows the chromatogram of the 1st HPLC in Example 8, and the fraction fractionated by the short time side. It is a figure which shows the chromatogram of the 1st HPLC in Example 8, and the fraction fractionated by the long time side. It is a figure which shows the chromatogram of the 2nd HPLC with respect to the fraction (1) fractionated by the 1st HPLC, and the fraction fractionated. It is a figure which shows the intestinal hormone release characteristic of the fraction fractionated by the second HPLC as a table | surface with respect to the fraction (1) fractionated by the first HPLC. It is a figure which shows the intestinal hormone release characteristic of the fraction fractionated by the second HPLC as a table | surface with respect to the fraction (5) fractionated by the first HPLC. It is a figure which shows the intestinal hormone release characteristic of the fraction fractionated by the 2nd HPLC as a table | surface with respect to the fraction (9) fractionated by the 1st HPLC. It is a figure which shows the chromatogram of the 2nd HPLC with respect to the fraction (5) fractionated by the 1st time HPLC, and the fraction fractionated. It is a figure which shows the chromatogram of the 2nd HPLC with respect to the fraction (9) fractionated by 1st HPLC, and the fraction fractionated. It is a figure which shows the chromatogram of the 2nd HPLC with respect to the fraction (13) fractionated by the 1st HPLC, and the fraction fractionated. It is a figure which shows the intestinal hormone release characteristic of the fraction fractionated by the 2nd HPLC fraction with respect to the fraction (13) fractionated by the 1st HPLC as a table | surface. It is a figure which shows the intestinal hormone release characteristic of the fraction fractionated by the 2nd HPLC fraction with respect to the fraction (14) fractionated by the 1st HPLC as a table | surface. It is a figure which shows the chromatogram of the 2nd HPLC with respect to the fraction (14) fractionated by the 1st HPLC, and the fraction fractionated. It is a flowchart which shows the flow of the preparation method of SIHR and IIHR demonstrated in each Example. It is a figure which shows the TLC measurement result about each fraction obtained as a result of the 2nd HPLC. It is a figure which tabulates the TLC * Rf value of an intestinal hormone secretion regulating substance, and the elution time in HPLC. It is a figure which shows the mass spectrum by MS analysis of fraction SIHR1-1. It is a figure which shows the mass spectrum by MS analysis of fraction SIHR1-2. It is a figure which shows the NMR analysis result of SIHR1-1. It is a figure which shows the NMR analysis result of SIHR1-2. (A), (b) is a figure which shows the molecular structure of SIHR1-1 and SIHR1-2 in order. It is a figure which shows the mass spectrum by MS analysis of fraction SIHR2-1. It is a figure which shows the mass spectrum by MS analysis of fraction SIHR2-2. It is a figure which shows the mass spectrum by MS analysis of fraction SIHR3-1. It is a figure which shows the mass spectrum by MS analysis of fraction SIHR3-2. It is a figure which shows the structure of monohydroxy alpha-linolenic acid estimated, and a fragment structure. It is a figure which shows as a table | surface the specific activity of each fraction SIHR prepared by 2nd HPLC. It is a figure which shows the ratio of the hexane: ether of the solvent using a silica gel cartridge as a table | surface. It is a figure which shows the result of the TLC measurement about the modified | denatured (alpha) LA fraction by a silica gel cartridge.

This embodiment is based on the embodiment in Japanese Patent Application No. 2008-066909 describing the inventions of the present inventors, and the following examples were performed in addition to the examples. Therefore, the description in each example in Japanese Patent Application No. 2008-066909 is also applied to this application as it is.
Example 1
As a fatty acid, 475 μl of α-linolenic acid (αLA (Sigma, L2376)) is placed in a tube (2 ml capacity), rotated in a 60 ° C. constant temperature bath for 6 days (7 rpm), mixed with air, and denatured α-linolenic acid Obtained. The modified α-linolenic acid was stored at −30 ° C. until the next purification operation.

-Example 2-
200 μl of the modified α-linolenic acid prepared in Example 1 was dissolved in 3 ml of a hexane: ether = 95: 5 mixture and charged on a silica gel cartridge (Sep-Pak Plus long, Waters, WT020520). After washing the cartridge with 40 ml of the same solvent, the eluate eluted with ether was dried under a stream of nitrogen. The residue was dissolved in 200 μl of dimethyl sulfoxide (DMSO (Sigma, D5879)) and used as a sample for the next purification operation.
Similar operations were repeated to stock samples. Samples were stored at −30 ° C. until the next operation.
The purification operation using this silica gel cartridge is mainly intended to remove unmodified α-linolenic acid, and has the effect of improving the component separation effect in the HPLC column used in the next purification process and extending the column life.

Example 3
The sample obtained in Example 2 was subjected to HPLC fractionation using a liquid delivery device (SHIMADZU, LC-6AD), a column oven (SHIMADZU, CTO-10A), and a detector (SHIMADZU, SPD-M20A). It was. That is, reverse phase column (TOSOH, TSKgel ODS-80Ts, 4.6mmID × 250mm), column oven temperature 40 ° C, flow rate 1ml / min, mobile phase buffered with methanol: H2O = 65: 35 mixture (containing acetic acid 0.05%) The column was charged with 75 μl of the sample obtained in Example 2, and eluted with a methanol: H 2 O = 65: 35 mixture (containing 0.05% acetic acid).
FIG. 1 is a diagram showing a first-time HPLC chromatograph in Example 3. FIG.

Example 4
The elution time was 10 to 30 minutes, and 55 to 75 minutes were collected at 2-minute intervals. The same operation was performed twice, and the fat-soluble component was recovered from the eluate for three times by the Folch method. The Folch method is a method for extracting a fat-soluble component and is performed by the following operation.
The amount of each solvent is calculated from the solvent composition, and the final composition is adjusted to chloroform: methanol: H2O = 2: 1: 0.9 in the centrifuge tube. After stirring with a test tube mixer, the mixture is centrifuged at 2400 rpm, 4 ° C. for 10 minutes, and the lower layer (chloroform layer) separated into two layers is collected. Chloroform in the same amount as the recovered solvent is added to the centrifuge tube, and similarly stirred and centrifuged to recover the lower layer again. The collected solution is dried under a nitrogen gas stream and dissolved in a mixed solution (4 ml) of chloroform: methanol = 2: 1. Samples were stored at −30 ° C. until the next operation.

-Example 5
2 ml was sampled from the sample of Example 4, dried by nitrogen stream, dissolved in 5 μl of DMSO, and used as a sample for activity measurement. Samples were stored at −30 ° C. until the next operation.

-Example 6
The GLP-1 secretion-promoting activity from STC-1 of the sample of Example 5 (cell line of hormone-secreting cells in the intestinal tract: Am. J. Pathol. 136: 1349-1363 (1990)) was measured. STC-1 cells are seeded at a concentration of 4 × 10 5 cells / ml in a 12-well plate, cultured for 48 hours, and each fraction (2 μl) is dissolved in Dulbecco's Modified Eagle's Medium (DMEM (SIGMA, D6429)) (500 μl). After the stimulation, the amount of GLP-1 in the culture supernatant was measured by ELISA. As ELISA, Rat GLP-1 ELISA Kit wako (Wako Pure Chemical Industries, 29-59201) was used.
FIG. 2 is a diagram showing the fractions fractionated by the first HPLC in Example 6 and the GLP-1 release characteristics of each fraction.

-Example 7-
In order to measure the activity of suppressing GLP-1 secretion from STC-1, the sample obtained in Example 2 was subjected to HPLC fractionation. The apparatus conditions were the same as in Example 3, and the mobile phase was buffered with a methanol: H 2 O = 70: 30 mixture (containing 0.05% acetic acid), and this column was charged with 75 μl of the sample obtained in Example 2, and methanol was added. : H2O = 70:30 mixture (containing 0.05% acetic acid) (flow rate 1 ml / min). Fractionation was performed at intervals of 5 minutes between 0 and 80 minutes.
3 is a diagram showing a chromatograph of fractions fractionated by the first HPLC in Example 7. FIG.

The fat-soluble component was recovered from these eluates by the Folch method, and the solution was dried under a nitrogen gas stream and dissolved in 25 μl of DMSO.
Then, as in Example 6, STC-1 cells were seeded in a 12-well plate, cultured for 48 hours, 50 μM of αLA was added, and each fraction was added simultaneously with stimulation. After stimulation for 1 hour, GLP-1 of the culture supernatant was added. , CCK, and substance P concentrations were measured by ELISA. CCK and substance P ELISA include Cholecytokinin Octapeptide (CCK) (26-30) (Human, Rat, Mouse) (Non-Sulfated) EIA Kit (PHOENIX PHARMACEUTICALS, INC., EK-069-04), ParameterTM Substance P Assay (R & D Systems, KGE007) was used.
FIG. 4 is a diagram showing the GLP-1 secretion inhibitory activity of the fraction fractionated by the first HPLC in Example 7.
FIG. 5 is a diagram showing the CCK secretion inhibitory activity of the fraction fractionated by the first HPLC in Example 7.
FIG. 6 is a diagram showing the substance P secretion inhibitory activity of the fraction fractionated by the first HPLC in Example 7.

-Example 8-
75 μl of the sample prepared in Example 2 was subjected to HPLC under the same HPLC conditions as in Example 3, and fractions for the following times were collected.
Fraction 1 (11 to 12.5 minutes)
Fraction 5 (18.75-20.25 minutes)
Fraction 9 (26.5-28 minutes)
Fraction 13 (60-62 minutes)
Fraction 14 (62-64 minutes)
Similar HPLC fractionation was performed 4 times, and a total of 5 fat-soluble components were separately collected by the Folch method. FIG. 7 is a diagram showing the chromatogram of the first HPLC in Example 8 and the fraction fractionated on the short time side.
FIG. 8 shows the chromatogram of the first HPLC in Example 8 and the fraction fractionated on the long time side.
These samples are designated as the first HPLC fractions 1, 5, 9, 13, and 14, respectively. The recovered solution was dried under a nitrogen gas stream and dissolved in DMSO (50 μl). Samples were stored at −30 ° C. until the next operation.

-Example 9-
The first HPLC elution fraction 1 prepared in Example 8 (referred to as fraction (1)) was further subjected to HPLC fractionation. Here, the conditions for the first HPLC were changed, and a reverse phase column (the same column as in Example 3) was used and buffered with a methanol: H 2 O = 50: 50 mixture (containing 0.05% acetic acid). Fraction (1) was charged by 20 μl and eluted with a methanol: H 2 O = 50: 50 mixture (containing 0.05% acetic acid) (flow rate 1 ml / min). In the fractionation, a main peak and a region including the front and back thereof were collected.
As a result, as shown in FIG. 9, SIHR1-1 pre-fraction (46.25 to 48.25 min), SIHR1-1 fraction (48.25 to 50.25 min), SIHR1-2 fraction (50.25 to 52.5 min), SIHR1-2 The rear fraction (52.5-54.5 minutes) was collected.
As can be seen from FIG. 9, the fraction (1) that appeared to be one component in the first HPLC was divided into two components, SIHR1-1 fraction (48.25-50.25 min) and SIHR1-2 fraction (50.25-52.5 min). divided.
For the fractionated fraction, lipid components were collected by the Folch method, dissolved in 2 ml of a mixture of chloroform: methanol = 2: 1, and stored at −30 ° C. until the next operation.

-Example 10-
1.2 ml (60% portion) of the fraction fractionated in Example 9 was dried to dryness in a nitrogen stream, dissolved in 4 μl of DMSO, and 2 μl of GLP-1, CCK, and substance P secretion promoting activity. It was measured.
The measurement of GLP-1 secretion promoting activity was performed in the same manner as in Example 6.
FIG. 10 is a table showing the intestinal hormone release characteristics of the fraction fractionated by the second HPLC with respect to the fraction (1) fractionated by the first HPLC.

-Example 11-
The first HPLC elution fraction 5 prepared in Example 8 (referred to as fraction (5)) was further subjected to HPLC fractionation. Under the same conditions as the first HPLC, a reverse phase column (the same column as in Example 3) was used and buffered with a methanol: H2O = 65: 35 mixture (containing 0.05% acetic acid). Fraction (5) 20 μl was charged and eluted with methanol: H 2 O = 65: 35 mixture (containing 0.05% acetic acid) (flow rate 1 ml / min). The fractionation times were (16.75 to 18.75 minutes), (18.75 to 20.25 minutes), and (20.25 to 22.5 minutes), and HPLC fractionation was performed on 20 μl of fraction (5). This operation is the second HPLC fraction. For the fractionated fraction, the lipid component was collected by the Folch method, dissolved in a mixture of chloroform: methanol = 2: 1 (2 ml), and stored at −30 ° C. until the next operation.
As shown in FIG. 13, three fractions of SIHR2-1 pre-fraction (16.75-18.75 min), SIHR2-1 fraction (18.75-20.25 min), and SIHR2-1 post-fraction (20.25-22.5 min) It was sorted.

-Example 12-
1.2 ml (60%) of the fraction fractionated in Example 11 was dried to dryness in a nitrogen stream, dissolved in 4 μl of DMSO, and 2 μl of GLP-1, CCK, and substance P secretion promoting activity. It was measured. The measurement of GLP-1 secretion promoting activity was performed in the same manner as in Example 6.
FIG. 11 is a table showing the intestinal hormone release characteristics of the fraction fractionated by the second HPLC fraction relative to the fraction (5) fractionated by the first HPLC.

-Example 13-
The first HPLC elution fraction 9 (referred to as fraction (9)) prepared in Example 8 was further subjected to HPLC fractionation. Under the same conditions as in the first HPLC, a reverse phase column (the same column as in Example 3) was used and buffered with methanol: H 2 O = 65: 35 (containing 0.05% acetic acid). Fraction (9) was charged with 20 μl and eluted with methanol: H 2 O = 65: 35 (containing 0.05% acetic acid) (flow rate 1 ml / min). The fractionation times were (24.5 to 26.5 minutes), (26.5 to 28 minutes), and (28 to 30 minutes), and 20 μl of fraction (9) was subjected to HPLC fractionation. This operation is the second HPLC fraction. For fractionated fractions, lipid components were collected by the Folch method, dissolved in a chloroform: methanol = 2: 1 mixture (2 ml), and stored at −30 ° C. until the next operation.
As shown in FIG. 14, three fractions of SIHR2-2 pre-fraction (24.5-26.5 minutes), SIHR2-2 fraction (26.5-28 minutes), and SIHR2-2 post-fraction (28-30 minutes) It was sorted.

-Example 14-
In addition, 1.2 ml (60%) of the fractionated fraction of Example 13 was solidified under a nitrogen stream, dissolved in 4 μl of DMSO, and 2 μl of GLP-1, CCK, and substance P secretion promoting activity. It was measured. The measurement of GLP-1 secretion promoting activity was performed in the same manner as in Example 6.
FIG. 12 is a table showing intestinal hormone release characteristics of the fraction fractionated in the second HPLC fraction relative to the fraction (9) fractionated in the first HPLC.

-Example 15-
The first HPLC elution fraction 13 (referred to as fraction (13)) prepared in Example 8 was further subjected to HPLC fractionation. Under the same conditions as in the first HPLC, a reverse phase column (the same column as in Example 3) was used and buffered with a methanol: H2O = 65: 35 mixture (containing 0.05% acetic acid). Fraction (13) (20 μl) was charged and eluted with methanol: H 2 O = 65: 35 mixture (containing 0.05% acetic acid) (flow rate 1 ml / min). Fraction times are (58-60 minutes), (60-62 minutes), (62-64 minutes). This operation is the second HPLC fraction. For the fractionated fraction, lipid components were collected by the Folch method, dissolved in a chloroform: methanol = 2: 1 mixture (2 ml), and stored at −30 ° C. until the next operation.
As shown in FIG. 15, three fractions of SIHR3-1 pre-fraction (58-60 minutes), SIHR3-1 fraction (60-62 minutes), and SIHR3-1 post-fraction (62-64 minutes) It was sorted.

-Example 16-
1.2 ml (60% portion) of the sample of Example 15 was dried under a nitrogen stream, dissolved in 4 μl of DMSO, and 2 μl of GLP-1, CCK, and substance P secretion promoting activity were measured. The measurement of GLP-1 secretion promoting activity was performed in the same manner as in Example 6.
FIG. 16 is a table showing intestinal hormone release characteristics of the fraction fractionated by the second HPLC fraction relative to the fraction (13) fractionated by the first HPLC.

-Example 17-
The first HPLC elution fraction 14 (referred to as fraction (14)) prepared in Example 8 was further subjected to HPLC fractionation. Under the same conditions as in the first HPLC, a reverse phase column (the same column as in Example 3) was used and buffered with a methanol: H2O = 65: 35 mixture (containing 0.05% acetic acid). Fraction (14) was charged with 20 μl and eluted with methanol: H 2 O = 65: 35 mixture (containing 0.05% acetic acid) (flow rate 1 ml / min). Fraction times are (60-62 minutes), (62-64 minutes), (64-65.75 minutes). HPLC fractionation was performed on 20 μl of fraction (14). This operation is the second HPLC fraction. For the fractionated fraction, lipid components were collected by the Folch method, dissolved in a mixed gastric fluid (2 ml) of chloroform: methanol = 2: 1, and stored at −30 ° C. until the next operation.
As shown in FIG. 18, three fractions of SIHR3-2 pre-fraction (60-62 min), SIHR3-2 fraction (62-64 min), and SIHR3-2 post-fraction (64-65.75 min) It was sorted.

-Example 18-
1.2 ml (60% portion) of the sample of Example 17 was dried to dryness in a nitrogen stream, dissolved in 4 μl of DMSO, and 2 μl of GLP-1, CCK, and substance P secretion promoting activity were measured. The measurement of GLP-1 secretion promoting activity was performed in the same manner as in Example 6.
FIG. 17 is a table showing the intestinal hormone release characteristics of the fraction fractionated by the second HPLC fraction relative to the fraction (14) fractionated by the first HPLC.

-Example 19-
FIG. 19 is a flowchart showing the flow of the SIHR and IIHR preparation methods described in the above examples.
As shown in FIG. 19, SIHR fraction 1 was subjected to the second HPLC under conditions different from the first HPLC, and SIHR fractions 2 to 5 were subjected to the second HPLC under the same conditions as the first HPLC. Yes. However, for the SIHR fractions 2 to 5, the second HPLC can be performed under conditions different from the first HPLC. Similarly to SIHR, IIHR fractions 1 to 4 can be subjected to the second HPLC under the same conditions as the first, or the second HPLC under conditions different from the first HPLC.

-Example 20-
Of the prepared samples (2 ml) obtained in Examples 9, 11, 13, 15 and 17, 0.8 ml (40% portion) of the remaining portion excluding the portion used for the measurement of GLP-1, CCK and substance P secretion promoting activity ) In a nitrogen stream and dissolved in a chloroform: methanol = 2: 1 mixture (32 μl), of which 16 μl (20% portion) was dissolved in TLC (carrier: silica gel, Whatman, Thin Layer Chromatography Plate, Partisil (R) K6F Silica Gel 60 Å with Fluorescent Indicator Size: 10x20cm Layer Thickness 250 μm, catalogue No. 4861-720) and developed with solvent: (hexane: ether: acetic acid = 60: 40: 1 mixture) ( At room temperature, the presence of organic carbon was measured by carbonization with copper sulfate.
FIG. 20 is a diagram showing TLC measurement results for each fraction obtained as a result of the second HPLC.
FIG. 21 is a table showing TLC / Rf values of intestinal hormone secretion regulators and elution times in HPLC.
Each SIHR and IIHR can be identified from the data of FIGS. IIHR is as described in Japanese Patent Application No. 2008-066909.

-Example 21-
Mass spectrometry (MS) was performed on 1 μl of each sample prepared in Examples 10, 12, 14, 16, and 18. The HPLC section uses a reverse phase column (TOSOH, TSKgel super ODS (2.0 mm ID x 50 mm)), the mass spectrometer uses LCMS-IT-TOF (SHIMADZU), conditions (negative mode, CDL temperature 200 ° C, heat The analysis was performed at a block temperature of 200 ° C., a nebulizer gas of 1.5 L / min, a CID energy of 100%, and a collision gas of 100%.
FIG. 22 is a diagram showing a mass spectrum by MS analysis of the fraction SIHR1-1. FIG. 23 is a diagram showing a mass spectrum by MS analysis of the fraction SIHR1-2.
For fraction SIHR1-1 and fraction SIHR1-2, mass spectrum 223 (M-H) was confirmed at the main peak. This shows that the molecular weight of the main component of fraction SIHR1-1 and fraction SIHR1-2 is 224.

Next, Example 2 (Sep-pak fraction of denatured αLA), Example 8 (first HPLC fraction), and Example 9 (second HPLC fraction of SIHR1) were repeated, and SIHR1-1, 1- Each 2 was prepared at 400 nmol. Samples were stored at −30 ° C. until the next operation.
Next, NMR analysis of SIHR1-1 and 1-2 prepared by the above procedure was performed. The 1H NMR spectrum was measured with JEOL ECA500 (500 MHz) (JEOL). Chemical shift (δ) is expressed in ppm relative to trimethylsilane. Coupling constant (J value) is indicated in Hz. Multiplicity is indicated by abbreviations of singlet: s, doublet: d, and multiplet: m.
FIG. 24 shows the NMR analysis results of SIHR1-1. 1H NMR (500 MHz, CDCl3): δ = 1.30-1.39 (m, 6H), 1.41-1.50 (m, 2H) 1.60-1.69 (m, 2H), 2.30-2.40 (m, 4H), 5.97-6.04 (m, 1H), 6.15 (dd, 1H, J = 8.0, 14.9 Hz), 6.24-6.31 (m, 1H), 7.45 (dd, 1H, J = 11.5, 14.9 Hz), 9.62 ppm (d, 1H) , J = 8.0 Hz). The conditions for collecting the data in the figure are Original Points Count: 16.384, Actual Points Count: 16.384, Acquisition Times 1.7459 (sec), and Spectrum Width: 18.763 (ppm).
FIG. 26 (a) is a diagram showing the molecular structure of SIHR1-1 derived from the above results.
FIG. 25 shows the NMR analysis results of SIHR1-2, 1H NMR (500 MHz, CDCl3): δ = 1.29-1.38 (m, 6H), 1.41-1.50 (m, 2H) 1.57-1.69 (m, 2H), 2.18-2.25 (m, 2H), 2.29-2.24 (m, 2H), 6.09 (dd, 1H, J = 8.0, 15.4 Hz), 6.21-6.38 (m, 2H), 7.08 (dd, 1H, J = 9.7, 15.4 Hz), 9.54 ppm (d, 1H) , J = 8.0 Hz). The conditions for collecting the data in the figure are Original Points Count: 16.384, Actual Points Count: 16.384, Acquisition Times 1.7459 (sec), and Spectrum Width: 18.763 (ppm).
FIG. 26B is a diagram showing the molecular structure of SIHR1-2 derived from the above results.

FIG. 27 is a diagram showing a mass spectrum by MS analysis of the fraction SIHR2-1. FIG. 28 is a diagram showing a mass spectrum by MS analysis of fraction SIHR2-2.
Mass spectrum 309 (M-H) was confirmed in the main peaks of fraction SIGR2-1 and fraction SIHR2-2. From this, it was found that the molecular weight of the main component of fraction SIHR2-1 and fraction SIHR2-2 was 310. The structure may be a molecule in which α-linolenic acid is hydroxylated in two places.
FIG. 29 is a diagram showing a mass spectrum by MS analysis of the fraction SIHR3-1. FIG. 30 is a diagram showing a mass spectrum by MS analysis of the fraction SIHR3-2.
As shown in FIGS. 29 and 30, mass spectrum 293 (M−H) was confirmed at main peaks of fraction SIHR3-1 and fraction SIHR3-2. As a result, the molecular weight of the main component of fraction SIHR3-1 and fraction SIHR3-2 is 294, and the structure is presumed to be a monohydroxy structure formed by oxidation of any double bond of α-linolenic acid. It was done.
Furthermore, when MS / MS measurement was performed, in the fraction SIHR3-1, a molecular weight of 171 (MH) and a molecular weight of 235 (MH) were confirmed as fragment ions (see FIG. 29). In fraction SIHR3-2, a molecular weight of 195 (MH) and a molecular weight of 211 (MH) were confirmed as fragment ions (see FIG. 30).

  FIG. 31 is a diagram showing the structure and fragment structure of monohydroxy α-linolenic acid deduced from the above results. As for the position of auto-oxidation of linolenic acid, there are 4 patterns (8 patterns including cis and trans) that are oxidized at 9th, 12th, 13th and 16th positions (non-patent document: Prog Lipid Res. , 23 (4), 197-221, 1984), the possibility of fragmentation also occurs as shown in FIG. The molecular weight of 171 (MH) and molecular weight of 235 (MH) were confirmed as fragment ions in SIHR3-1 because it was α-linolenic acid mono-hydroxylated at the 9th or 16th position as the main component. Estimated. In SIHR3-2, the molecular weight of 195 (MH) and molecular weight of 211 (MH) were confirmed as fragment ions because it was α-linolenic acid that was monohydroxylated at position 12 or position 13 as the main component. Estimated.

-Example 22-
In this example, the specific activity of the SIHR fraction prepared in Examples 9, 11, 13, 15, and 17 was measured.
The specific activity was calculated by the following two methods.
1) After TLC results obtained in Example 20 were photographed with LAS-4000mini (FUJIFILM), the spots of each sample were quantified with Multi Gauge Ver3.0 (FUJIFILM). At the same time, the numerical value of the developed α-linolenic acid was used as a control. Coloring is carbonized with copper sulfate, and all substances that contain hydrocarbons develop color, so the color intensity is proportional to the number of carbons contained. The specific activity of each SIHR fraction was determined from the TLC value and the results of the intestinal hormone secretion-promoting activity of Examples 10, 12, 14, 16, and 18.
2) NEFA C-Test Wako (Wako Pure Chemical Industries, 279-75401), which quantifies the number of moles of free fatty acids by reaction of free fatty acids with carboxyl groups, was used. The intestinal hormone secretion-promoting activity prepared in Examples 9, 11, 13, 15 and 17 and the remaining 16 μl (20% portion) of each sample used for TLC in Example 20 were dried to dryness under a nitrogen stream. It was dissolved in H2O (5 μl) containing% Triton X-100 (SIGMA, T-8787), and 4 μl thereof was quantified with NEFA C-Test Wako. The specific activity was determined from this quantitative value and the results of the intestinal hormone secretion-promoting activity of Examples 10, 12, 14, 16, and 18.
FIG. 32 is a table showing the specific activity of each fraction SIHR prepared by the second HPLC.

-Example 23-
In this purification method, a silica gel cartridge (Sep-Pak Plus long, Waters, WT020520) was used as a pretreatment for HPLC. The main purpose is to remove native α-linolenic acid to improve the separation ability of the sample in HPLC and to prevent column deterioration. However, depending on the purpose, a crude product of SIHR or IIHR may be used. This silica gel cartridge can be used for the purpose of preparing a crude product.
200 μl of the modified α-linolenic acid prepared in Example 1 was dissolved in a hexane: ether = 10: 0.5 mixture (3 ml) and charged into Sep-Pak plus long (Waters). The hexane: ether ratio was changed and eluted stepwise.
FIG. 33 is a table showing the hexane: ether ratio of the solvent using a silica gel cartridge.

Each elution fraction was dried under a nitrogen stream, dissolved in a chloroform: methanol = 2: 1 mixed solution (200 μl), and TLC was performed on 4 μl thereof. TLC conditions are the same as in Example 19.
FIG. 34 is a diagram showing the results of TLC measurement for the modified αLA fraction using a silica gel cartridge.
In order to obtain the fraction SIHR1-1 and the fraction SIHR1-2 crude purified product, after treatment with any one of the eluates 1 to 5, elution is performed with the eluate 6 or the eluate 7.
In order to obtain the fraction SIHR2-1 and the fraction SIHR2-2 crude purified product, after treatment with any one of the eluates 1 to 10, elution is performed with the eluate 11 or the eluate 12.
In order to obtain the fraction SIHR3-1 and the fraction SIHR3-2 crude purified product, after treatment with any one of the eluates 1 to 6, elution is carried out with the eluate 7 or eluate 8. By performing the above operations, each roughly purified product can be prepared.

-Functional foods and pharmaceutical functions-
SIHR1-1, SIHR1-2, SIHR2-2, SIHR2-2, SIHR3-13 and SIHR3-2 promote the release of GLP-1 from the intestinal tract, which is effective in the prevention and treatment of diabetes and obesity. Fatty acids perform this function in a normal diet, but these substances have a GLP-1 release activity per calorie or per molecule of 2-50 times higher than fatty acids. Therefore, it is possible to provide an effective diabetes or obesity preventive or functional food or functional food that has lower calories and exhibits high GLP-1 releasing activity compared to fatty acid administration.

  In addition, the release of substance P and CCK causes inflammation and causes pain. It is thought to cause onset or exacerbation of enteritis such as IBS and IBD. IIHR1, IIHR2, IIHR3, and IIHR4 suppress the release of substance P and CCK. Therefore, these components can be used to provide preventive / therapeutic drugs or functional foods that suppress the onset or exacerbation of enteritis such as IBS and IBD. . Since the reaction to fatty acids is hypersensitive in the environment where IBS and IBD develop and exacerbate, suppressing this can provide an important drug or functional food for IBS and IBD.

  The intestinal hormone secretion regulator of the present invention can be used not only as a medicine but also as a functional food.

Claims (13)

A step (a) of modifying a fatty acid;
(B) a step of fractionating the modified fatty acid modified in the step (a) by chromatography including at least one high performance liquid chromatography to extract an intestinal hormone secretion regulating component from the modified fatty acid;
A method for producing an intestinal hormone secretion regulating component, comprising: In the method for producing an intestinal hormone secretion regulator according to claim 1,
In the step (b),
The modified fatty acid is fractionated by chromatography using a silica gel column,
Subsequently, after performing fractionation using the liquid feeding device of the high performance liquid chromatography device, the column oven, and the detector,
Elution with an eluate containing acetic acid with a volume ratio of methanol to water of 80-50: 20-50 and a volume ratio of 0.05% ± 0.01%,
On a silica gel plate, hexane, ether and acetic acid were used as developing solvents at a volume ratio of 60: 40: 1, and the Rf values were 0.10 to 0.12, 0.13 to 0.15, 0.22. Fractionating the fractions showing spots in the regions of ~ 0.24, 0.23-0.25, 0.24-0.26, and 0.25-0.27,
A method for producing an intestinal hormone secretion regulator. In the method for producing an intestinal hormone secretion regulator according to claim 1 or 2,
In the step (b),
The modified fatty acid is fractionated by high performance liquid chromatography under conditions of reverse phase column and column oven temperature of 40 ° C. ± 5 ° C.,
Elution at a flow rate of 0.9 to 1.1 ml / min with an eluent containing acetic acid with a volume ratio of methanol to water of 69 to 71:31 to 29 and a volume ratio of 0.05% ± 0.01%,
Fraction fractions with elution times of 0-5 minutes, 35-40 minutes, 45-50 minutes, and 65-70 minutes,
A method for producing an intestinal hormone secretion regulator. In the method for producing an intestinal hormone secretion regulator according to claim 1 or 2,
In the step (b),
The modified fatty acid is fractionated by high performance liquid chromatography under conditions of reverse phase column and column oven temperature of 40 ° C. ± 5 ° C.,
Elution at a flow rate of 0.9 to 1.1 ml / min with an eluent containing acetic acid having a volume ratio of methanol to water of 64 to 66:36 to 34 and a volume ratio of 0.05% ± 0.01%,
Fractions with elution times of 11 to 12.5 minutes, 18.75 to 20.25 minutes, 26.5 to 28 minutes, 60 to 62 minutes, and 62 to 64 minutes,
A method for producing an intestinal hormone secretion regulator. In the method for producing an intestinal hormone secretion regulator according to claim 1 or 2,
In the step (b),
The modified fatty acid is fractionated by high-performance liquid chromatography under conditions of a reverse phase column and a column oven temperature of 40 ° C. ± 5 ° C., respectively.
Elution at a flow rate of 0.9 to 1.1 ml / min with an eluent containing acetic acid with a volume ratio of methanol to water of 49 to 51:51 to 49 and a volume ratio of 0.05% ± 0.01%,
Fraction fractions with elution times of 48.25 to 50.25 minutes, 50.25 to 52.5 minutes,
A method for producing an intestinal hormone secretion regulator. In any one of claims 3-5,
The step (b) according to any one of claims 3 to 5 is performed as the first high performance liquid chromatography, and the obtained fraction is further used as the second high performance liquid chromatography. Performing step (b) according to any one of 5;
A method for producing an intestinal hormone secretion regulator. In the method for producing an intestinal hormone secretion regulator according to any one of claims 1 to 6,
In the step (a), a method for producing an intestinal hormone secretion regulator, wherein one substance selected from unsaturated long-chain fatty acids, natural fats and oils, and a fatty acid fraction prepared from the natural fats and oils is used as the fatty acids. In the method for producing an intestinal hormone secretion regulator according to claim 7,
The unsaturated long chain fatty acid is α-linolenic acid or DHA,
The method for producing an intestinal hormone secretion regulator, wherein the natural fat is shiso oil or fish oil.   A functional food containing an intestinal hormone secretion regulating component extracted by the method for producing an intestinal hormone secretion regulating agent according to any one of claims 1 to 8.   The pharmaceutical which contains the intestinal hormone secretion adjustment component taken out by the manufacturing method of the intestinal hormone secretion regulator as described in any one of Claims 1-8 as an active ingredient.   A method of using an intestinal hormone secretion regulator that promotes the release of GLP-1 from the intestinal tract by administering the functional food according to claim 9 or the pharmaceutical according to claim 10.   A method for using an intestinal hormone secretion regulator, which suppresses the release of substance P from the intestinal tract by administering the functional food according to claim 9 or the pharmaceutical according to claim 10.   A method for using an intestinal hormone secretion regulator, which suppresses the release of CCK from the intestinal tract by administering the functional food according to claim 9 or the pharmaceutical according to claim 10.

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