JP2024042331A - DPPIV inhibitor containing protein hydrolyzate derived from buckwheat - Google Patents

Abstract

The present invention provides a DPPIV inhibitor that is inexpensive and easily available. The present invention provides a DPPIV inhibitor that contains a hydrolysate of a protein derived from buckwheat.

Description

Application for application of Article 30, Paragraph 2 of the Patent Act Announced on April 25, 2020 on the website (http://www.jsfcs.org/2022/05/28-202251920.html) 2020 Presented at the 28th General Meeting/Academic Conference of the Japan Society of Food Chemistry on May 19th Presented at the 28th General Meeting/Academic Conference of the Japanese Society of Food Chemistry on May 20, 2020

The present invention relates to a DPPIV inhibitor comprising a hydrolyzate of proteins derived from buckwheat.

Diabetes is a disease characterized by high fasting blood sugar levels, ie, hyperglycemia. Diabetes is considered to be a lifestyle-related disease because its onset is related to daily factors such as the content of meals, overeating, obesity, and stress, in addition to genetic factors. From the perspective of improving quality of life, it is important to properly control blood sugar levels and prevent lifestyle-related diseases such as diabetes.

Hyperglycemia is induced by abnormalities in the regulation mechanism of blood sugar levels, and is also known as a pathological condition that causes other metabolically related diseases in the body. For example, diseases and pathological conditions that diabetics, characterized by hyperglycemia, may have include hypertension, arteriosclerosis, heart disease such as angina pectoris and myocardial infarction, stroke such as cerebral infarction and cerebral hemorrhage, decreased immunity, dyslipidemia, Examples include osteoporosis and periodontal disease. Complications are difficult to treat once they occur, so it is important to control blood sugar levels to prevent complications and delay their progression.

DPP (dipeptidyl peptidase) IV is a protease that exists in blood and recognizes the second proline or alanine from the N-terminus of a peptide to release a dipeptide from the N-terminus. By inhibiting DPPIV, it is possible to smooth the function of incretin hormones, and antidiabetic drugs that inhibit DPPIV have been approved. Incretin hormones are hormones consisting of two types of peptide hormones, and have the properties of promoting insulin secretion and suppressing glucagon secretion, and prevent hyperglycemic states by smoothing their functions. can be improved or improved.

In recent years, the functionality of food components made by hydrolyzing protein has attracted attention. Buckwheat is a crop grown in various countries including Japan, Russia, China, and Ukraine due to its high nutritional value. Although buckwheat is an inexpensive plant that is cultivated all over the world and is rich in protein, there are few reports on the functional evaluation of protein hydrolysates derived from buckwheat, and no studies have been conducted. .

Patent Document 1 discloses a technology related to a DPPIV inhibitor containing a hydrolyzate of protein derived from the milt of seafood.

International Publication No. 2014/199780

The problem to be solved by the present invention is to provide a DPPIV inhibitor that is inexpensive and easily available.

The present inventors have discovered that a protein hydrolyzate derived from buckwheat, which is inexpensive and grown all over the world, has a DPPIV inhibitory effect, and has completed the present invention.

The present invention relates to the following (1) to (9).
(1)
A DPPIV inhibitor comprising a protein hydrolyzate derived from buckwheat.
(2)
The DPPIV inhibitor according to (1), wherein the buckwheat is buckwheat.
(3)
The DPPIV inhibitor according to (1), wherein the hydrolyzate is a hydrolyzate using a protease.
(4)
The DPPIV inhibitor according to (1), wherein the protease is an endopeptidase and/or an exopeptidase.
(5)
According to (4), the proteolytic enzyme is one or more selected from the group consisting of alcalase, pandase, samoase, allase, neurase, proteax, peptidase R, esperase, neutrase, flavorzyme, and pepsin. DPPIV inhibitor of.
(6)
The DPPIV inhibitor according to (1), wherein the hydrolyzate is derived from a fraction eluted using a 10 to 50% alcohol solvent in reverse phase chromatography using an alcoholic organic solvent.
(7)
The DPPIV inhibitor according to (1), wherein the hydrolyzate is derived from a fraction eluted at an elution time of 50 to 60 minutes in molecular weight distribution analysis by gel filtration chromatography.
(8)
A food or drink containing the DPPIV inhibitor according to any one of (1) to (7).
(9)
A food/beverage product for inhibiting DPPIV, which contains a hydrolyzate of protein derived from buckwheat.

According to the present invention, it is possible to provide a DPPIV inhibitor that is inexpensive and easily available due to the DPPIV inhibitory effect of a hydrolyzate of proteins derived from buckwheat.

FIG. 1 shows a reverse phase HPLC chromatogram of the F3 fraction and the DPPIV activity inhibition rate of each fraction. FIG. 2 shows a gel filtration HPLC chromatogram of the R fraction and the DPPIV activity inhibition rate of each fraction. FIG. 3 shows the hydrophilic interaction HPLC chromatogram of the S fraction and the inhibition rate of DPPIV activity of each fraction. Figure 4 shows the LC-Q-TOF MS MS spectrum of the H fraction.

Embodiments of the present invention will be described below. The following embodiments are illustrative for explaining the present invention, and are not intended to limit the present invention only to the embodiments.

The present invention relates to a DPPIV inhibitor comprising a hydrolyzate of proteins derived from buckwheat.

1. Buckwheat In this embodiment, "buckwheat" means a plant belonging to the genus Fagopyrum. There are no particular restrictions on the varieties or harvest times of buckwheat, but examples include Natsuyoshi, Kitamitsuki, Nijiyutaka, Ruchiking, Cobalt Chikara, Gamma no Aya, Reranokaori, Sachiizumi, Haruno Ibuki, Natsumi, Soba Intermediate Mother Farm No. 1, Examples include Toyo Musume, Kitano Mashu, Hitachi Aki Soba, and Tartary Soba.

In the present embodiment, "protein derived from buckwheat" refers to proteins and peptides (including polypeptides, hereinafter the same) obtained from buckwheat. Buckwheat serving as a protein source is not particularly limited, but for example, buckwheat, which is known to contain protein, can be used.

For example, buckwheat may be powdered, or may be ground after roasting and hot water extraction. Examples of powdered buckwheat include buckwheat flour. Examples of products obtained by roasting buckwheat seeds and pulverizing them after extraction with hot water include ground buckwheat tea leaves.

Buckwheat flour may be produced by a person skilled in the art using a known method, or may be a commercially available product. Methods for producing buckwheat flour are not particularly limited, but include, for example, a method in which harvested buckwheat is dried, the pericarp (shell) is removed, and then powdered using a low-temperature milling device. The particle size and water content of buckwheat flour are not particularly limited as long as it can be subjected to protein hydrolysis. Commercial products of buckwheat flour are not particularly limited, but include, for example, buckwheat flour (Masudaya Foods Co., Ltd.), stone-ground buckwheat flour (Onishi Seifun), and the like.

The ground tea leaves of buckwheat tea are not particularly limited, but for example, the ground tea leaves remaining after preparing buckwheat tea by hot water extraction using commercially available roasted buckwheat seeds for buckwheat tea may be ground. It is available from etc. The degree of pulverization and the water content of the pulverized product are not particularly limited as long as it can be subjected to protein hydrolysis. Commercial products of buckwheat seeds roasted for buckwheat tea include, but are not particularly limited to, fragrant buckwheat tea (Naraya), domestic buckwheat tea (Izumo Cha Sandaiichi), and the like.

2. Chemical hydrolysis such as acid hydrolysis and alkaline hydrolysis, enzymatic hydrolysis, and the like are known as hydrolytic enzymatic hydrolysis. Although this embodiment will be described using an example of enzymatic hydrolysis, chemical hydrolysis may also be applied. In the present embodiment, the term "protein hydrolase" may be used to include proteases, proteinases, and peptidases, which are hydrolases that act on peptide bonds in proteins and peptides. The protein hydrolase is not particularly limited as long as it can hydrolyze proteins derived from buckwheat, but those that are also used as food additives are preferred.

In this embodiment, various hydrolases, crude products containing these various hydrolases, or disrupted bacterial cells containing these various hydrolases, etc., can be used as the proteolytic enzymes. Only one of these may be used, or two or more of them may be used in combination.

Examples of protein hydrolases include, but are not limited to, microbial protein hydrolases such as Alcalase, Flavorzyme, Panchidase, Samoase, Alloase, Neurase, Proteax, Peptidase R, Esperase, Neutrase, and Pepsin. It will be done. Among them, Alcalase and Flavorzyme are preferred.

Commercially available protein hydrolases include, but are not particularly limited to, Alcalase (registered trademark) 2.4 L FG (Novozymes Japan Co., Ltd.), Flavorzyme (registered trademark) (Novozymes Japan Co., Ltd.) , Panchidase (registered trademark) NP-2, Panchidase MP, Panchidase P (Yakult Pharmaceutical Co., Ltd.), Samoase (registered trademark) (Amano Enzyme Co., Ltd.), Aloase (registered trademark) XA-10, Aloase AP-10 , Aloase NP-10 (Yakult Pharmaceutical Co., Ltd.), Neurase (registered trademark) F 3 G (Amano Enzyme Co., Ltd.), Proteax (registered trademark) (Amano Enzyme Co., Ltd.), Peptidase R (Amano Enzyme Co., Ltd.) Examples of commercially available products include Esperase (registered trademark), Esperase (registered trademark) (Novozymes Japan Co., Ltd.), and Neutrase (registered trademark) (Novozymes Japan Co., Ltd.).

Only one type of protein hydrolase may be used, or two or more types may be used in combination. Although not intended to be limiting, it is preferred that the protein hydrolase has broad substrate specificity for the cleavage site from the viewpoint of uniformly hydrolyzing proteins. For example, serine proteases are known to have broad substrate specificity and are preferred. For example, alcalase, esperase, etc. are serine proteases. For example, hydrolytic enzymes that can randomly hydrolyze peptide bonds are preferred. For example, neutrase can randomly hydrolyze peptide bonds.

Protein hydrolases include endopeptidases (also called "endo-type proteases"), which hydrolyze non-terminal peptide bonds in proteins and peptides, and exopeptidases (also called "endo-type proteases"), which hydrolyze peptide bonds from the terminal ends of proteins and peptides. ) is known to exist. Examples of endopeptidase include, but are not limited to, alcalase, flavorzyme, pepsin, and the like. Examples of exopeptidase include, but are not limited to, Neutrase, Flavorzyme, Proteax, Peptidase R, and the like.

In this embodiment, as the protein hydrolase, either endopeptidase or exopeptidase may be used alone, or both may be used. Although not intended to be limiting, it is preferable to use both endopeptidase and exopeptidase from the viewpoint of uniformly hydrolyzing proteins and peptides. When using endopeptidase and exopeptidase as the protein hydrolase, the type, number and combination of endopeptidase and exopeptidase to be used are not particularly limited. For example, Alcalase may be used as the endopeptidase and Flavorzyme may be used as the exopeptidase, pepsin may be used as the endopeptidase, and Flavorzyme may be used as the exopeptidase, Alcalase may be used as the endopeptidase, and Proteax may be used as the exopeptidase. Alternatively, Alcalase may be used as the endopeptidase, and Peptidase R may be used as the exopeptidase. Among these, it is preferable to use Alcalase as the endopeptidase and Flavorzyme as the exopeptidase.

Hydrolysis conditions may be appropriately set by those skilled in the art depending on the protein hydrolase used. For example, those skilled in the art may appropriately set the temperature based on the instructions issued by the manufacturer of the protein hydrolase used.

3. Hydrolyzate In this embodiment, "hydrolyzate" refers to a product obtained by the action of a hydrolytic enzyme. In this embodiment, the protein hydrolyzate derived from buckwheat contains peptides.

The form of the hydrolyzate is not particularly limited, but it may be in a liquid form containing a solvent added during the hydrolysis process, or it may be in a powder form by being subjected to treatments such as freeze-drying. It may be in the form of

The content of hydrolysates in DPPIV inhibitors is not particularly limited and is not uniformly determined depending on the types and contents of other components contained therein, but is about 0.01% by mass to 90% by mass, for example, about 0.1% by mass to 70% by mass.

Components in the hydrolyzate are not particularly limited, but can be fractionated, for example, by separation and purification methods, molecular weight distribution analysis, and the like. Separation and purification methods and molecular weight distribution analysis can be carried out by appropriately known methods by those skilled in the art, such as separation using chromatography. Examples of chromatography include, but are not particularly limited to, reverse phase chromatography, normal phase chromatography such as hydrophilic interaction chromatography, gel filtration chromatography, and the like. As an analysis method, chromatography may be used alone or in combination of one or more types. Although not intended to be particularly limiting, an example of a case where one or more types of chromatography are used in combination as a separation and purification method is to confirm the DPPIV activity inhibition rate of each fraction obtained by chromatography, Alternatively, a fraction with a high activity rate may be collected and subjected to subsequent chromatography. For example, the hydrolyzate is subjected to reversed phase chromatography, the DPPIV inhibitory activity of each fraction obtained by reversed phase chromatography is confirmed, and active fractions with a high DPPIV activity inhibition rate are collected. Next, the collected active fractions are subjected to gel filtration chromatography, the DPPIV activity inhibition rate of each obtained fraction is confirmed, and the active fractions with a high DPPIV activity inhibition rate are collected. Next, it is also possible to subject the collected active fractions to hydrophilic interaction chromatography, check the DPPIV activity inhibition rate of each obtained fraction, and collect the active fraction with a high DPPIV activity inhibition rate. be.

Those skilled in the art may appropriately use known separation columns and devices for chromatography. Although not intended to be particularly limited, examples of separation columns include columns using silica gel, polymer, carbon, glass, titania, etc. as a filler, and columns with alkyl groups added to these fillers. Can be mentioned.

Examples of the solvent used in the mobile phase of reversed phase chromatography include buffer solutions and alcoholic organic solvents. Buffers used in the mobile phase are not particularly limited, and include, for example, ammonium bicarbonate buffer (eg, 150 mM), phosphate buffer, acetate buffer, formate buffer, carbonate buffer, and the like. The alcohol-based organic solvent used in the mobile phase is not particularly limited, but includes, for example, acetonitrile, methanol, a mixture of acetonitrile and methanol (eg, 10 to 50% alcohol concentration), and the like. The separation column used is not particularly limited, but includes, for example, a separation column filled with an octadecylsilylated silica gel filler (ODS column, C18 column, etc.).

The aqueous solution used as the mobile phase for gel filtration chromatography is not particularly limited, and examples thereof include ammonium bicarbonate buffer (e.g., 150 mM), acetonitrile, methanol, chloroform, a mixture of acetonitrile and methanol, and the like. The separation column used is not particularly limited, but includes, for example, a separation column packed with an agarose filler (Sperdex series).

The solvent used in the mobile phase of hydrophilic interaction chromatography is not particularly limited, and examples thereof include ammonia aqueous solution (e.g., 0.01%) and acetonitrile, but solvents that can be used in reversed phase chromatography may also be used. The separation column used is not particularly limited, but includes, for example, a separation column filled with an octadecylsilylated silica gel filler (ODS column, C18 column, etc.).

As a method for separation and purification, liquid chromatography, in which analysis is performed by linking a liquid chromatogram and a mass spectrometer, may be performed. Examples of such a liquid chromatography device include a liquid chromatograph quadrupole time-of-flight mass spectrometer (LC/Q-TOF-MS).

Conditions for separation and the like may be appropriately set by those skilled in the art depending on the separation column and chromatography apparatus used. For example, those skilled in the art may appropriately set the values based on the instructions issued by the manufacturers of the separation columns and chromatography devices used.

In this embodiment, the hydrolyzate may be derived from fractions obtained by various analyses. Deriving from fractions obtained by various analyzes including the above-mentioned chromatography is preferable because it is possible to increase the proportion of components having the desired effect in the hydrolyzate.

The fractions obtained by various analyzes may be, for example, specific fractions recovered in reverse phase chromatography or specific fractions recovered in gel filtration chromatography.

The specific fraction recovered in reversed phase chromatography may be, for example, a fraction eluted using a specified solvent in reversed phase chromatography. Examples of fractions eluted using a specified solvent in reversed phase chromatography include fractions eluted using a 10-50% alcohol solvent such as a mixture of acetonitrile and methanol in reversed phase chromatography. Can be mentioned. At this time, the separation column of this embodiment may be used, for example.

The specific fraction recovered in molecular weight distribution analysis by gel filtration chromatography may be, for example, an oligopeptide fraction or a fraction eluted at a designated elution time.

In this embodiment, the term "oligopeptide fraction" refers to a fraction containing peptides (including dipeptides, tripeptides, tetrapeptides, pentapeptides, etc.) consisting of 2 to 20 amino acids, and components other than peptides. may be included. The oligopeptide fraction may be a fraction containing peptides consisting of 2 to 20 amino acids, or may be a fraction containing peptides consisting of 2 to 18 amino acids, or may be a fraction containing peptides consisting of 2 to 16 amino acids. It may be a fraction containing a peptide consisting of 2 to 14 amino acids, or it may be a fraction containing a peptide consisting of 2 to 12 amino acids. , may be a fraction containing a peptide consisting of 2 to 10 amino acids, may be a fraction containing a peptide consisting of 2 to 8 amino acids, or may be a fraction containing a peptide consisting of 2 to 6 amino acids. It may be a fraction, it may be a fraction containing a peptide consisting of 2 to 5 amino acids, it may be a fraction containing a peptide consisting of 2 to 4 amino acids, and it may be a fraction containing a peptide consisting of 2 or 3 amino acids. It may be a fraction containing a peptide consisting of amino acids.

The specified elution time for a fraction eluted at a specified elution time in molecular weight distribution analysis by gel filtration chromatography includes, for example, the elution time in gel filtration chromatography using the mobile phase and separation column of this embodiment. Examples include elution time of 50 to 60 minutes, elution time of 54 to 60 minutes, and elution time of 50 to 56 minutes.

As described above, only one type of chromatography may be used as an analysis method, or one or more types may be used in combination. That is, for example, a specific fraction recovered in reverse phase chromatography may be further subjected to molecular weight distribution analysis by gel filtration chromatography, and the specific fraction may be recovered.

4. DPPIV inhibitory activity One of the mechanisms of glucose metabolism in the living body is the regulation of blood glucose concentration (blood glucose level). When a meal is ingested, carbohydrates, proteins, and fats are converted into glucose, peptides, amino acids, and fatty acids through the digestive process by various decomposition enzymes, and are absorbed in the digestive tract (intestinal tract). Glucose absorbed in the intestinal tract is released into the blood, temporarily increasing the glucose concentration in the blood. In order to do so, the cells of the pancreas sense the increase in blood glucose level and release insulin, a secretory hormone, into the blood. Insulin works to cause liver cells, fat cells, and muscle tissue to take up glucose in the blood, converting glucose into glycogen and storing it in the cells, thereby lowering blood glucose level. In addition, when blood glucose level drops during exercise, glycogen is converted into glucose and released into the blood. In this way, a blood glucose regulation mechanism that keeps blood glucose level constant is known. It is known that if the blood glucose regulation mechanism does not function properly, lifestyle-related diseases including obesity and diabetes develop.

It is known that incretin hormones secreted into the blood from the intestinal tract are involved in the regulation mechanism of blood sugar levels. Incretin hormones consist of two types of peptide hormones: glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). These peptides have the property of acting on pancreatic cells and promoting insulin secretion when blood sugar levels rise with meal intake. It also has the property of suppressing the secretion of glucagon, which increases blood sugar levels by converting glycogen into glucose and releasing it into the blood.

In addition to the pancreas, incretin hormones include GLP-1, which acts on the central nervous system and digestive tract, including the stomach, and has the property of suppressing appetite and inhibiting digestive tract motility, and GIP, which acts on fat cells and promotes the uptake of glucose from the blood.

On the other hand, it is also known that after incretin hormone secretion, it is rapidly degraded by the action of its degrading enzyme, DPPIV, and loses its activity. Therefore, by inhibiting DPPIV, it is possible to smooth the function of incretin hormones. By smoothing the function of incretin hormones, it is possible to make it easier to lower blood sugar levels that rise after meals, to moderate the rise in blood sugar levels after meals, and to prevent blood sugar levels from dropping too low when fasting. It will be done. In recent years, antidiabetic drugs having an action mechanism related to incretin hormones have been developed. For example, liraglutide and exenatide, which are GLP-1 passive agonists that exhibit insulin secretion effects only when blood sugar levels are high, and vildagliptin and alogliptin, which are DPPIV inhibitors, are known.

In this embodiment, "DPPIV inhibition" means inhibiting the activity of DPPIV. The inhibition rate of DPPIV activity can be appropriately examined by those skilled in the art using known methods. For example, those skilled in the art can examine the inhibition rate of DPPIV activity by adding a substrate for DPPIV (eg, Gly-Pro-MCA) and measuring the fluorescence wavelength using a fluorescence plate reader.

5. DPPIV Inhibitor The DPPIV inhibitor of the present invention can be used as part of foods and drinks, food additives, pharmaceuticals, quasi-drugs, feeds, and the like. Among these, although it is not intended to be particularly limited, it is preferable to take the form of an oral composition.

The form of the oral composition containing the DPPIV inhibitor of the present invention is not particularly limited, and examples thereof include tablets, granules, powders, drinks, capsules, and the like. The content of the DPPIV inhibitor in the oral composition is not uniformly defined depending on the form of the oral composition and the type and content of other components included, but it is 0.01% by mass to 90% by mass. %, for example, about 0.1% by mass to 70% by mass.

The form in which the DPPIV inhibitor of the present invention is ingested is not particularly limited, but includes, for example, ingestion and administration through general eating and drinking. Among these, it is preferable to take or administer it orally.

The guideline for the amount of the DPPIV inhibitor of the present invention to be taken is not particularly limited, and may be determined as appropriate by those skilled in the art.

6. Food and Drink The present invention also relates to food and drink containing the DPPIV inhibitor of the present invention.

In this embodiment, "food and drink" refers to general foods, foods for specified health uses, foods with nutritional function claims, foods with health claims such as foods with functional claims, foods for the sick, health foods (nutritional supplements, health supplements, It is used to include supplements, etc. (including foods labeled as nutrition-adjusted foods, etc.). The form of the food and drink is not particularly limited, but includes, for example, noodles such as soba, udon, and instant noodles; flours such as buckwheat flour and cake mix; confectionery such as candy, gum, chocolate, snacks, biscuits, and jelly; Processed fish and livestock foods such as breads, kamaboko, ham, and sausages, dairy products such as processed milk and fermented milk, processed oil and fat foods such as margarine, dressings, and mayonnaise, tea drinks, soft drinks, nutritional drinks, and fruit drinks. , beverages such as lactic acid drinks, and hot water-infused beverages such as buckwheat tea. The form of the supplement is not particularly limited, and examples thereof include tablets, granules, powders, drinks, capsules, and the like. Food and drink products may contain food additives such as preservatives, preservatives, fragrances, and pigments.

Foods and drinks containing the DPPIV inhibitor of the present invention may be produced by appropriately known methods by those skilled in the art, and may be ingested through ordinary eating and drinking.

Specific uses of the food and drink containing the DPPIV inhibitor of the present invention include, for example, to moderate the increase in blood sugar level after a meal, to slow down the absorption of sugar, to make it easier to lower the blood sugar level that has increased after a meal. Examples include preventing fasting blood sugar levels from falling too low, supporting fasting blood sugar levels to approach normal levels, and supporting blood sugar control. Foods and beverages containing the DPPIV inhibitor of the present invention are suitable for people who are concerned about blood sugar levels, suitable for people who are concerned about postprandial blood sugar levels, suitable for people with high blood sugar levels, and suitable for people who eat sugar-rich foods. It may also be provided as a food or drink with a label indicating that it is suitable for people who tend to eat food.

1. Preparation of hydrolyzed solution Buckwheat flour (Buckwheat flour (trade name), Masudaya Foods Co., Ltd.) was weighed out at 10.0 g on a scale, mixed with 0.1 M disodium hydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.1 M sodium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.), and dispersed in 150 mL of a mixture adjusted to pH 8.0. 500 μL of Alcalase (Alcalase 2.4 L FG, Novozymes Japan Co., Ltd.) and 10 mg of Flavorzyme (Flavorzyme 500 MG, Novozymes Japan Co., Ltd.) were added thereto, and the mixture was heated at 50 ° C., 450 rpm, and 17 hours with a hot stirrer to cause an enzyme reaction. The resulting reaction solution was centrifuged (4 ° C., 4,000 g, 10 minutes) and then subjected to suction filtration (filter paper used: No. 5A). The resulting filtrate was heated at 125 ° C., 450 rpm, and 1 hour with a hot stirrer to inactivate the enzyme. The reaction solution obtained was centrifuged (4°C, 4,000g, 10 minutes) and then subjected to suction filtration (filter paper used: No. 5A). The filtrate obtained was made up to 200mL with 0.1M phosphate buffer (pH 8.0) to obtain a hydrolyzate containing a hydrolyzate of protein derived from buckwheat. In addition, as an enzyme blank, a solution was prepared by adding the above-mentioned Alcalase and Flavorzyme to 0.1M phosphate buffer (pH 8.0). Specifically, 500μL of Alcalase and 10mg of Flavorzyme were added to 150mL of 0.1M phosphate buffer (pH 8.0) in the same manner as above, and the mixture was heated at 50°C, 450rpm, and 17 hours with a hot stirrer, centrifuged (4°C, 4,000g, 10 minutes), and then subjected to suction filtration. The filtrate was heated at 125°C and 450 rpm for 1 hour using a hot stirrer, centrifuged (4°C, 4,000 g, 10 minutes), and then suction filtered (filter paper No. 5A). The filtrate was diluted to 200 mL with 0.1 M phosphate buffer (pH 8.0) to prepare an enzyme blank.

2. Measurement of DPPIV inhibitory activity of hydrolyzate In 20 μL of the prepared hydrolyzate, 50 mM Tris-HCl buffer (pH 7.5) (Tris (Fuji Film Wako Pure Chemical Industries, Ltd.) was dissolved in pure water, and hydrochloric acid (Fuji Film Wako Pure Chemical Industries, Ltd.) was dissolved in pure water. DPPIV (human origin) was mixed with 150 μL of 0.3 mM Gly-Pro-MCA (Peptide Institute Co., Ltd.) and 20 μL of 0.3 mM Gly-Pro-MCA (Peptide Institute Co., Ltd.). Add 10 μL of (DPPIV (human), (recombinant) (product name), Enzo Life Science), and use a fluorescent plate reader (Infinite 200PRO M Nano (device name), Tecan Japan Co., Ltd.) (excitation wavelength 360 nm, fluorescence wavelength 465 nm). After heating at 37° C. for 45 minutes, measurements were taken. The DPPIV inhibition rate was calculated as a percentage from the fluorescence intensity when the hydrolyzate was added, setting the fluorescence intensity without the addition of the hydrolyzate as 100. As a control, DPPIV inhibitory activity was similarly measured for a test solution to which no hydrolase was added. Furthermore, the DPPIV inhibitory activity was similarly measured for the enzyme blank.

The results are shown in Table 1. The DPPIV inhibitory activity rate of the enzyme blank was below the lower limit of measurement.

3. Measurement of DPPIV inhibitory activity of hydrolyzate derived from each fraction of open column chromatography The prepared hydrolyzate was passed in its entirety to open column chromatography packed with COSMOSIL (registered trademark) 75C18-OPN (Nacalai Tesque Co., Ltd.). The filtered solution was washed with 100 mL of pure water, and the passed-through solution was collected as the F1 fraction. A solution of pure water and methanol was used as the eluent. The eluate with 20% methanol was collected as the F2 fraction, the eluate with 40% methanol was collected as the F3 fraction, and the eluate with 100% methanol was collected as the F4 fraction. Thereafter, the F1 fraction was freeze-dried. The F2 to F4 fractions were freeze-dried after methanol was distilled off under reduced pressure using a rotary evaporator (N-1300 (product name), Tokyo Rikakikai Co., Ltd.). The F1 to F4 fractions after freeze-drying were dissolved in pure water to a concentration of 10 mg/mL to prepare a hydrolysis solution containing hydrolysates derived from each of the F1 to F4 fractions. DPPIV inhibitory activity was calculated for the hydrolyzate derived from each of the F1 to F4 fractions by the same method as described in "2. Measurement of DPPIV inhibitory activity of hydrolyzate".

The results are shown in Table 2.

4. Measurement of DPPIV inhibitory activity of hydrolyzate derived from F3 fraction of open column chromatography The F3 fraction obtained by the method of "3." was subjected to reversed phase HPLC. The conditions are as follows.
Model: Alliance HPLC e2695 (Waters)
Column: Xselect (registered trademark) Peptide CSHTM C18 (4.6 x 150 mm)
Temperature: 40℃
Flow rate: 0.7 mL/min, 3.5 mL/tube Fraction A solution: 150 mM ammonium bicarbonate buffer B solution: Acetonitrile: Methanol = 1:1
Wavelength: 215nm

The results are shown in Figure 1. The fraction with a retention time of 15 to 20 minutes (R fraction) showed the highest DPPIV inhibitory activity rate of about 63%.

5. Measurement of DPPIV inhibitory activity rate of hydrolyzate derived from R fraction by reversed-phase HPLC The R fraction obtained by the method in "4." was collected and then concentrated. The concentration was carried out by heating at 50°C while spraying nitrogen gas using a spray-type test tube concentrator (MG-3100, Tokyo Rikakikai Co., Ltd.) to remove the organic solvent, and then by freeze-drying. The concentration was then subjected to gel filtration HPLC. The conditions were as follows.
Model: Alliance HPLC e2695 (Waters)
Column: Superdex™ 30 Increase 10/300 GL (10×300 mm)
Temperature: Room temperature (no heating)
Flow rate: 0.3 mL/min, 3.0 mL/tube fraction Solution A: 150 mM ammonium bicarbonate buffer Solution B: acetonitrile:methanol=1:1
Wavelength: 215 nm
Isocratic elution with A:B=80:20

The results are shown in Figure 2. The fraction with a passage time of 50 to 60 minutes (S fraction) showed the highest DPPIV inhibitory activity rate of about 49%.

Since it was speculated that rutin, a flavonoid contained in buckwheat, was involved as a DPPIV-inhibiting active ingredient, a standard rutin product (Rutin (product name), Nacalai Tesque Co., Ltd.) was subjected to gel filtration HPLC under the same conditions as above. When measuring at 350 nm, which is the wavelength used to measure flavonoids, and confirming the elution position of rutin, rutin was eluted at a much different flow time (105 to 115 minutes) than that of the S fraction, and this inhibitory activity effect was It was inferred that it was derived from a component different from rutin (see Figure 2).

6. Measurement of DPPIV inhibitory activity rate of hydrolysis solution derived from S fraction of gel filtration HPLC The S fraction was collected several times and dried under reduced pressure. For drying under reduced pressure, the organic solvent was removed by heating at 50°C while blowing nitrogen gas using a spray test tube concentrator (MG-3100, Tokyo Rikakikai Co., Ltd.), and then concentrated by freeze-drying. Ta. The dried product under reduced pressure was dissolved in a solution of 0.01% aqueous ammonia and acetonitrile = 1:9, centrifuged (4°C, 15,000g, 5 minutes), and the supernatant was subjected to hydrophilic interaction HPLC. Served. The conditions are as follows.
Model: Alliance HPLC e2695 (Waters)
Column: Inertsustain (registered trademark) Amide (4.6 x 150 mm)
Temperature: 40℃
Flow rate: 0.8 mL/min, 0.8 mL/tube Fraction A solution: 0.01% ammonia B solution: Acetonitrile Wavelength: 215 nm

The results are shown in Figure 3. The fraction with a retention time of 7 to 8 minutes (H fraction) showed the highest DPPIV inhibitory activity rate of about 65%.

7. Analysis of hydrolysis solution derived from H fraction of hydrophilic interaction HPLC The H fraction was derivatized with AccQ Tag (trademark) and subjected to LC-Q-TOF to estimate the peptide sequence in the H fraction. I did it. The conditions are as follows.
Model: Xevo G2-XS Qtof (Waters)
Column: Poroshell 120 EC-C18 (4.6 x 100 mm)
Temperature: 37℃
Flow rate: 0.3mL/min
Solution A: 0.1% formic acid Solution B: Acetonitrile containing 0.1% formic acid

The results are shown in Figure 4. Two types of candidate tripeptides, VPI and VPL, were estimated.

Since Ile and Leu have the same exact mass, we obtained a VPI synthetic product (synthesis was entrusted to Eurofin Genomics Co., Ltd.) and a VPL synthetic product (Diprotin B, BioVison) for VPI and VPL, respectively. When the IC ₅₀ value was calculated, the results shown in Table 6 were obtained.

9. Measurement of residual rate of DPPIV inhibitory activity after artificial digestion test A pepsin solution containing 0.1M hydrochloric acid (Pepsin 1:10000, from Porcine Stomach Mucosa, Fuji Film Wako Co., Ltd.) was added to VPI and heated at 37°C for 1 hour. and enzymatic reaction (VPI:pepsin=35:1). The obtained reaction solution was neutralized by adding 0.1M hydrochloric acid and an equivalent amount of sodium hydroxide and checking the pH to be approximately 7-8 using pH test paper, and then buffered with 0.1M phosphate buffer. A pancreatin solution containing liquid (pancreatin, Fuji Film Wako Co., Ltd.) was added and heated at 37° C. for 2 hours to cause an enzyme reaction (VPI: pancreatin = 25:1). The obtained reaction solution was heated at 100° C. for 10 minutes to inactivate the enzyme, and a test solution was obtained. The DPPIV inhibitory activity of the test solution was measured in the same manner as described in "2. Measurement of DPPIV inhibitory activity of hydrolysis solution."

The results are shown in Table 7.

Claims (9)

A DPPIV inhibitor comprising a protein hydrolyzate derived from buckwheat. The DPPIV inhibitor according to claim 1, wherein the buckwheat is buckwheat. The DPPIV inhibitor according to claim 1, wherein the hydrolyzate is a hydrolyzate using a protease. The DPPIV inhibitor according to claim 1, wherein the proteolytic enzyme is an endopeptidase and/or an exopeptidase. 5. The proteolytic enzyme is one or more selected from the group consisting of alcalase, panchidase, samoase, aloase, neurase, proteax, peptidase R, esperase, neutrase, flavorzyme, and pepsin. DPPIV inhibitor. The DPPIV inhibitor according to claim 1, wherein the hydrolyzate is derived from a fraction eluted using a 10 to 50% alcohol solvent in reverse phase chromatography using an alcoholic organic solvent. The DPPIV inhibitor according to claim 1, wherein the hydrolysate is derived from a fraction eluted at an elution time of 50 to 60 minutes in a molecular weight distribution analysis by gel filtration chromatography. A food or drink product comprising the DPPIV inhibitor according to any one of claims 1 to 7. A food/beverage product for inhibiting DPPIV, which contains a hydrolyzate of protein derived from buckwheat.