JP7780965B2 - Peptides with dipeptidyl peptidase-IV inhibitory activity and uses thereof - Google Patents

Description

The present invention relates to a peptide having dipeptidyl peptidase-IV inhibitory activity. The present invention also relates to dipeptidyl peptidase-IV inhibitors, blood glucose level inhibitors, antidiabetic agents, and the like, which utilize the peptide.

In recent years, gastrointestinal hormones called incretins have been attracting attention in the treatment of type 2 diabetes. Incretins are hormones secreted from the gastrointestinal tract following meal intake and act on pancreatic beta cells to promote insulin secretion. They also inhibit the secretion of glucagon, which increases blood glucose levels, and protect and promote the proliferation of pancreatic beta cells. Incretins thus play an important role in lowering blood glucose levels, but they suffer from the problem of being rapidly degraded and inactivated in the blood by dipeptidyl peptidase-IV (hereinafter sometimes referred to as "DPP-IV"). Therefore, the use of DPP-IV inhibitors is expected as a treatment strategy for type 2 diabetes. Accordingly, various compounds with DPP-IV inhibitory activity have been discovered.

For example, Patent Document 1 describes that fluoropyrrolidone derivatives with a specific structure can be used as DPP-IV inhibitors of serine proteases such as DPP-IV. Furthermore, Patent Document 2 describes that proline derivatives with a specific structure can be used as DPP-IV inhibitors. However, the DPP-IV inhibitors reported in Patent Documents 1 and 2 are chemically synthesized products that have never been consumed as food, and have the disadvantage that they cannot be used on a daily basis as foods, etc.

On the other hand, several DPP-IV inhibitors utilizing naturally occurring ingredients have also been reported. For example, Patent Document 3 describes that the tripeptide Met-Lys-Pro contained in casein hydrolysate has DPP-IV inhibitory activity. Patent Document 4 describes that a specific peptide contained in the water-soluble fraction of cheese has DPP-IV inhibitory activity. Patent Document 5 describes that the tripeptide X-Pro-Gln obtained by hydrolyzing gluten with an enzyme derived from ginger rhizome has DPP-IV inhibitory activity. Patent Document 6 describes that tea leaf protein hydrolysates have DPP-IV inhibitory activity. As such, several DPP-IV inhibitors utilizing naturally occurring ingredients have been reported. However, from the perspective of increasing the variety of ingredients used as DPP-IV inhibitors, there is a need for the development of new DPP-IV inhibitors utilizing naturally occurring ingredients.

JP 2007-63286 A Special Publication No. 2007-537231 Japanese Patent Application Laid-Open No. 2016-136966 Japanese Patent Application Laid-Open No. 2014-1223 JP 2017-214334 A JP 2017-43618 A

The object of the present invention is to discover novel components with DPP-IV inhibitory activity and to provide DPP-IV inhibitors, blood glucose level suppressants, and antidiabetic agents.

The inventors conducted extensive research to solve the above-mentioned problems and discovered that the tetrapeptide Ile-Pro-Ala-Gly and the tripeptide Val-Ala-Tyr contained in almond-derived protein hydrolysates have DPP-IV inhibitory activity. The present invention was completed through further research based on this finding.

That is, the present invention provides the following aspects.
Item 1. A tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly.
Item 2. A tripeptide having the amino acid sequence Val-Ala-Tyr.
Item 3. A DPP-IV inhibitor comprising the tetrapeptide according to Item 1 and/or the tripeptide according to Item 2.
Item 4. The DPP-IV inhibitor according to Item 3, comprising an almond protein hydrolysate containing Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr as the tetrapeptide according to Item 1 and/or the tripeptide according to Item 2.
Item 5. A food or drink for inhibiting DPP-IV, comprising the DPP-IV inhibitor according to Item 3 or 4.
Item 6. A pharmaceutical for inhibiting DPP-IV, comprising the DPP-IV inhibitor according to Item 3 or 4.
Item 7. An agent for suppressing elevation of blood glucose level, comprising the tetrapeptide according to Item 1 and/or the tripeptide according to Item 2.
Item 8. The blood glucose level increase suppressant according to Item 7, comprising an almond protein hydrolysate containing Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr as the tetrapeptide according to Item 1 and/or the tripeptide according to Item 2.
Item 9. A food or drink for suppressing an increase in blood glucose level, comprising the blood glucose level increase suppressor according to Item 7 or 8.
Item 10. A pharmaceutical for suppressing an increase in blood glucose level, comprising the blood glucose level increase suppressor according to Item 7 or 8.
Item 11. An antidiabetic agent comprising the tetrapeptide according to Item 1 and/or the tripeptide according to Item 2.
Item 12. The antidiabetic agent according to Item 11, comprising an almond protein hydrolysate containing Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr as the tetrapeptide according to Item 1 and/or the tripeptide according to Item 2.
Item 13. An antidiabetic food or drink comprising the antidiabetic agent according to Item 11 or 12.
Item 14. An antidiabetic drug comprising the antidiabetic agent according to Item 11 or 12.

The present invention inhibits DPP-IV using peptides derived from almonds, a natural product, making it possible to provide a highly safe DPP-IV inhibitor, blood sugar level inhibitor, and antidiabetic agent that can be taken daily as food or beverages.

1 shows the results of HPLC analysis of fractions obtained by purifying almond-derived protein hydrolysate, confirming the presence of Ile-Pro-Ala-Gly and Val-Ala-Tyr. The upper chromatogram shows the fraction obtained by the purification process, and the lower chromatogram shows a chromatographic standard. FIG. 1 shows the results of measuring blood glucose levels over time after ingestion of an almond-derived protein hydrolysate containing Ile-Pro-Ala-Gly and Val-Ala-Tyr, followed by a glucose load (ingestion of an aqueous glucose solution).

1. Peptides The peptides of the present invention are peptides having DPP-IV inhibitory activity, specifically, a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly (SEQ ID NO: 1) and a tripeptide having the amino acid sequence Val-Ala-Tyr.

In the present invention, amino acids are represented using three-letter symbols, with Ile representing isoleucine, Pro representing proline, Ala representing alanine, Gly representing glycine, Val representing valine, and Tyr representing tyrosine. Furthermore, in the present invention, the symbols for tetrapeptides and tripeptides indicate that the left end is the N-terminus and the right end is the C-terminus. Specifically, Ile-Pro-Ala-Gly indicates that Ile, Pro, Ala, and Gly are arranged in this order from the N-terminus to the C-terminus, and Val-Ala-Tyr indicates that Val, Ala, and Tyr are arranged in this order from the N-terminus to the C-terminus.

The method for producing the peptides of the present invention is not particularly limited, as long as it produces a tetrapeptide and/or tripeptide having the amino acid sequence described above. Examples include methods such as hydrolyzing almond-derived proteins, biotechnology methods, and chemical synthesis methods. Of these, from the standpoint of safety, etc., the method of hydrolyzing almond-derived proteins is preferred.

The following describes a method for producing the peptide of the present invention by hydrolyzing almond-derived proteins.

Almonds are the seeds (sweet almonds) of the almond (Prunus amygdalus Batsch) of the Rosaceae family. Almond-derived proteins can be obtained by extracting almonds. Almonds used as a raw material for almond-derived proteins may be raw, roasted, or defatted almonds (e.g., defatted roasted almonds) generated during the almond oil production process. However, from the perspectives of reducing production costs and making effective use of unused resources, defatted almonds are preferred.

Protein extraction from almonds can be carried out according to conventional methods, for example, by crushing or powdering the almonds as necessary and then soaking them in an alkaline aqueous solution. The pH of the alkaline aqueous solution is, for example, 7 to 14, preferably 7.5 to 12, and more preferably 8 to 10. The alkaline aqueous solution is prepared by adding an alkaline agent such as sodium hydroxide or potassium hydroxide. The extraction temperature is, for example, 40 to 100°C, preferably 50 to 90°C, and more preferably 60 to 80°C. The extraction time is, for example, 0.5 hours or more, preferably 1 to 24 hours, and more preferably 2 to 12 hours.

The extract containing almond-derived protein thus obtained may be subjected to hydrolysis treatment after solid-liquid separation, concentration, etc., as necessary, or it may be subjected to hydrolysis treatment directly.

The method for hydrolyzing almond-derived proteins is not particularly limited, as long as Ile-Pro-Ala-Gly and/or Val-Ala-Tyr is produced. Any method, such as protease treatment, acid treatment, or alkali treatment, may be used, with protease treatment being preferred.

The type of protease used in the protease treatment is not particularly limited, as long as it is capable of producing Ile-Pro-Ala-Gly and/or Val-Ala-Tyr, but specific examples include endopeptidases, exopeptidases, and combinations thereof. The origin of the protease is also not particularly limited, and examples include plant-derived proteases such as papain, bromelain, and ficin; proteases derived from various microorganisms; and animal-derived proteases such as pepsin, (chymo)trypsin, and cathepsin. Commercially available proteases can be used. Commercially available proteases include, for example, Papain W-40, Thermoase PC10F, Neurolase F3G, Pancreatin F, Protin SD-AY10, Protin SD-NY10, Protease A "Amano" SD, Protease M "Amano" SD, Protease P "Amano" 3SD, Bromelain F, and Proteax (all manufactured by Amano Enzyme Co., Ltd.); Orientase AY, Orientase 10NL, Orientase 90N, and Orientase 10NL. Examples of such anti-inflammatory agents include tase OP, nucleisin, orientase 22BF, Bioprase SP-20FG, and Bioprase OP (manufactured by HI Biosciences, Inc.); Sumiteam LP-G, Sumiteam FL-G, Sumiteam CP, Sumiteam FP-G, Sumiteam MP, Sumiteam BR, and Sumiteam BNP (manufactured by Shin Nippon Chemical Industry Co., Ltd.); Aroase AP-10, Aroase XA-10, and Aroase NP-10 (manufactured by Yakult Pharmaceutical Co., Ltd.); Denateam CPO PEPRICH (manufactured by Nagase ChemteX Corporation); MaxiPro PSP (manufactured by DSM); purified papain (manufactured by Mitsubishi Chemical Foods Corporation); FormeaTL and FormeaCTL (manufactured by Novozymes).

Among proteases, from the viewpoint of efficiently producing Ile-Pro-Ala-Gly and Val-Ala-Tyr from almond-derived proteins, preferred are papain, protease derived from Geobacillus stearothermophilus, and chymotrypsin, and more preferred are Papain W-40 (papain, manufactured by Amano Enzyme Co., Ltd.), Thermoase PC10F (protease derived from Geobacillus stearothermophilus, manufactured by Amano Enzyme Co., Ltd.), and Formea CTL (chymotrypsin, manufactured by Novozymes).

These proteases may be used alone or in combination of two or more. In particular, the combined use of papain and Geobacillus stearothermophilus-derived protease, or the combined use of papain and chymotrypsin, makes it possible to efficiently produce the tetrapeptide Ile-Pro-Ala-Gly and the tripeptide Val-Ala-Tyr from almond-derived proteins.

Protease treatment can be carried out by allowing the protease and almond-derived protein to coexist in a solvent in which the protease reaction is possible. For example, the protease and almond-derived protein may be added to the solvent in any order, but from the perspective of reducing production costs, it is preferable to add the protease to the extract, concentrate, or diluted solution containing the almond-derived protein described above.

In the protease treatment, the amount of almond-derived protein added as a substrate is not particularly limited, but can be 10 to 300 g/L, preferably 25 to 200 g/L, and more preferably 50 to 100 g/L. The amount of protease added can be set appropriately depending on the type of enzyme used, but examples include a total amount of protease of 0.01 to 20 parts by mass, preferably 0.1 to 10 parts by mass, and more preferably 1 to 5 parts by mass per 100 parts by mass of almond-derived protein.

The pH during protease treatment may be within a range in which the protease used can act. For example, when using papain and/or a protease derived from Geobacillus stearothermophilus, the pH may be 7 to 14, preferably 7.5 to 12, and more preferably 8 to 10. Furthermore, when using papain and/or chymotrypsin, the pH may be 7 to 14, preferably 7.5 to 12, and more preferably 8 to 10.

The temperature conditions for protease treatment may be within a range in which the protease used can act. For example, when using papain and/or a protease derived from Geobacillus stearothermophilus, the temperature may be 30 to 70°C, preferably 40 to 60°C, and more preferably 45 to 55°C. Furthermore, when using papain and/or chymotrypsin, the temperature may be 30 to 70°C, preferably 40 to 60°C, and more preferably 45 to 55°C.

The treatment time for protease treatment can be appropriately set based on the amount of almond-derived protein used, the type and amount of enzyme, the pH and temperature during protease treatment, etc., and is, for example, 5 to 24 hours, preferably 10 to 22 hours, and more preferably 15 to 20 hours.

After protease treatment, if necessary, the protease may be inactivated, residue may be removed by solid-liquid separation, or the product may be concentrated or dried.

The almond protein hydrolysate thus obtained contains Ile-Pro-Ala-Gly and/or Val-Ala-Tyr and may be used as is as a peptide-containing product of the present invention. Alternatively, the obtained almond protein hydrolysate may be subjected to purification processes such as ultrafiltration, adsorption resin treatment, and chromatography, as necessary, to increase the purity of the tetrapeptide Ile-Pro-Ala-Gly and/or tripeptide Val-Ala-Tyr, or to produce a purified product of the tetrapeptide Ile-Pro-Ala-Gly and/or tripeptide Val-Ala-Tyr, which may then be used as a peptide-containing product of the present invention.

The uses of the peptides of the present invention are not particularly limited, but because the peptides of the present invention have DPP-IV inhibitory activity, they can be suitably used as DPP-IV inhibitors, blood glucose level inhibitors, antidiabetic agents, etc. These uses are described in detail in the section "2. DPP-IV inhibitors, blood glucose level inhibitors, and antidiabetic agents."

2. DPP-IV inhibitors, blood glucose level inhibitors, and antidiabetic agents The tetrapeptide Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr have DPP-IV inhibitory activity and can therefore be used as the active ingredient of a DPP-IV inhibitor.

Incretins also promote insulin secretion and inhibit the secretion of glucagon, which increases blood glucose levels. Inhibiting DPP-IV can inhibit the breakdown of incretins, so the tetrapeptide Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr can also be used as active ingredients in blood glucose level inhibitors or antidiabetic agents. Antidiabetic agents are used for the prevention or treatment of type 2 diabetes.

The DPP-IV inhibitor, blood glucose level increase suppressant, or antidiabetic agent of the present invention may contain at least one of the tetrapeptide Ile-Pro-Ala-Gly and the tripeptide Val-Ala-Tyr as an active ingredient, but it is preferable to contain both of these, as this will enable even more excellent DPP-IV inhibitory activity to be exhibited.

The tetrapeptide Ile-Pro-Ala-Gly and/or tripeptide Val-Ala-Tyr used as active ingredients in the DPP-IV inhibitor, blood glucose level inhibitor, or antidiabetic agent of the present invention may be in either a purified or unpurified state. For example, almond protein hydrolysate containing the tetrapeptide Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr can be used in an unpurified state or in a state in which the purity of these peptides has been increased.

The DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used by oral ingestion or administration. The intake or administration amount of the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is an amount effective for inhibiting DPP-IV in the body, and may be set appropriately depending on the product form used, etc. For example, the daily intake or administration amount for an adult is 1 mg to 500 mg, preferably 5 to 250 mg, and more preferably 10 to 100 mg, of the total amount of the tetrapeptide Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr.

The DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is incorporated into products that are required to have a DPP-IV inhibitory effect, a blood glucose level increase inhibitory effect, or an antidiabetic effect. Products that can incorporate the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention are not particularly limited, as long as they are orally ingested or administered, and examples include foods, beverages, pharmaceuticals, etc.

The dosage form of a product incorporating the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention may be solid, semi-solid, liquid, or the like, and is appropriately selected depending on the type and intended use of the product. Products incorporating the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention may contain food materials, food additives, nutritional ingredients, pharmaceutically acceptable base materials, pharmaceutically acceptable additives, pharmacological ingredients, etc., depending on the type, dosage form, etc., within the scope of the present invention, as long as the effects are not impaired.

When the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in the food and beverage field, the tetrapeptide Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr can be prepared into the desired form either as is or in combination with other food ingredients or additives, and provided as a DPP-IV inhibitory food and beverage, a blood glucose level increase inhibitor, or an antidiabetic food and beverage. Examples of such foods and beverages include general foods and beverages, as well as foods with health claims (including foods for specified health uses, foods with nutrient functions, supplements, etc.) and foods for patients. Foods for patients can be provided for patients who require DPP-IV inhibition, suppression of blood glucose level increase, or prevention or treatment of diabetes. The form of these foods and beverages is not particularly limited, but specific examples include confectioneries such as chocolate, cookies, biscuits, snacks, crackers, chewing gum, candy, caramels, mouth-refreshing sweets, rice crackers, etc.; Japanese sweets such as buns and dorayaki; energy bars or balanced nutritional foods kneaded with dried fruit, nuts, dietary fiber, etc.; frozen desserts such as ice cream, ice milk, lacto ice cream, and frozen desserts; desserts such as yogurt, pudding, and jelly; bakery products such as bread, cookies, biscuits, pizza dough, pie dough, ice cream cones, monaka wafer wrappers, and cream puff wrappers; Western sweets such as sponge cake, chiffon cake, castella, madeleines, financiers, pound cake, roll cake, and pancakes; noodles such as pasta, udon, buckwheat, hiyamugi, somen, Chinese soba, macaroni, rice vermicelli, and harusame; jams such as strawberry jam, apple jam, blueberry jam, and marmalade; curry, hayashi, stew, meat sauce, etc. sauce bases such as sashimi, white sauce, etc.; rice bowl bases for Chinese rice bowls, beef bowls, oyakodon, etc.; seasoning ingredients such as fried rice bases, mapo tofu bases, and kamameshi bases; freeze-dried foods such as miso soup and soups; sauces and condiments such as fruit sauce, Chinese bean paste, kabayaki sauce, mitarashi dango sauce, white sauce, dressing, Worcestershire sauce, meat sauce, and Thousand Island dressing; processed meat products such as ham, sausage, and roast pork; fish sausage, fish ham, fish paste, kamaboko, chikuwa, These include fish paste products such as hanpen (fish cakes) and satsuma-age (deep-fried fish cakes); cereals (processed grain products); supplements such as capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, and liposome preparations; and beverages such as soft drinks, milk drinks, lactic acid bacteria drinks, carbonated drinks, fruit juice drinks, vegetable drinks, vegetable and fruit drinks, powdered drinks, jelly drinks, coffee drinks, cocoa drinks, black tea drinks, green tea drinks, sports drinks, nutritional drinks, powdered drinks, energy drinks, non-alcoholic drinks, and alcoholic drinks.

Furthermore, when the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in the food and beverage field, the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention can be provided alone or in combination with other ingredients as a food additive for inhibiting DPP-IV, a food additive for inhibiting blood glucose level increase, or a food additive for antidiabetic purposes.

When the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in a food or beverage, the amount of the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent in the food or beverage may be appropriately determined within a range that satisfies the aforementioned intake amount depending on the type and form of the food or beverage, but examples of such ranges include a total amount of the tetrapeptide Ile-Pro-Ala-Gly and/or tripeptide Val-Ala-Tyr in the food or beverage of 0.0001 to 90% by mass, preferably 0.0005 to 75% by mass, and more preferably 0.001 to 50% by mass.

Furthermore, when the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in the pharmaceutical field, the tetrapeptide Ile-Pro-Ala-Gly and/or the tripeptide Val-Ala-Tyr can be prepared alone or in combination with other pharmacological ingredients, pharmaceutically acceptable bases, additives, etc. into a desired dosage form and provided as a DPP-IV inhibitor drug, blood glucose level increase inhibitor, or antidiabetic drug. The form of such pharmaceuticals is not particularly limited, but specific examples include oral pharmaceuticals such as drinks, tablets, pills, powders, fine granules, granules, capsules (including hard capsules and soft capsules), lozenges, chewable tablets, extracts (including soft extracts, dry extracts, etc.), jellies, syrups, and spirits.

When the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in a pharmaceutical product, the amount of the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent in the pharmaceutical product may be appropriately determined within a range that satisfies the aforementioned dosage amount depending on the type and dosage form of the pharmaceutical product. For example, the total amount of the tetrapeptide Ile-Pro-Ala-Gly and/or tripeptide Val-Ala-Tyr in the pharmaceutical product may be in the range of 0.0001 to 90% by mass, preferably 0.0005 to 75% by mass, and more preferably 0.001 to 50% by mass.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

Example 1: Preparation of almond protein hydrolysate 400 g of dry powder of defatted roasted almonds was added to 3600 g of water and thoroughly dispersed to prepare an almond-containing solution with a concentration of 10% by mass. Sodium hydroxide was added to this almond-containing solution to adjust the pH of the solution to 9.0, and the solution was heated and stirred at 70°C for 9 hours to obtain an almond protein extract.

Next, the almond protein extract obtained above was cooled to 50°C and readjusted to pH 9.0 by adding sodium hydroxide. Then, 4 g each of Papain W-40 (Amano Enzyme) and Thermoase PC10F (Amano Enzyme) (0.1% by mass based on the almond-containing liquid) were added, and the mixture was heated and stirred at 50°C for 18 hours to carry out the hydrolysis reaction. The enzymes were then inactivated by heating at 80°C for 30 minutes to terminate the hydrolysis reaction, and the mixture was cooled to room temperature. This hydrolyzed liquid was centrifuged (15,000 x g, 4°C, 10 minutes) to recover the supernatant, which was then suction filtered to remove the residue. The filtered aqueous solution was freeze-dried to obtain approximately 200 g of hydrolyzate (freeze-dried product).

Example 2: Identification of peptides with DPP-IV inhibitory activity
1. Separation of Peptides Having DPP-IV Inhibitory Activity The hydrolysate (lyophilized product) obtained in Example 1 was dissolved in ultrapure water to a concentration of 5% by mass to prepare a hydrolysate-containing aqueous solution. 500 μL of this hydrolysate-containing aqueous solution was injected into a solid-phase extraction cartridge (Sep-Pak Plus Short C18 Cartridge, manufactured by Waters), and then 3 mL of ultrapure water was injected using a syringe. The extruded solution was discarded without being recovered. Next, 1 mL of a 10% by volume aqueous methanol solution was injected into the solid-phase extraction cartridge, and the extruded solution was discarded without being recovered. Finally, 2 mL of a 10% by volume aqueous methanol solution was injected into the solid-phase extraction cartridge, and the extruded solution was recovered.

The recovered solution was concentrated and dried, then dissolved in 200 μL of ultrapure water. The solution passed through a 0.45 μm centrifugal filter (5,000 rpm/min) was used as a sample for peptide separation using reverse-phase HPLC under the following separation conditions 1 and 2.

<Separation conditions 1>
Column: Inertsil ODS-3 (GL Sciences)
Detection: UV 210 nm
Flow rate: 3mL/min
Eluent A: ultrapure water Eluent B: 50% aqueous acetonitrile solution Gradient: A linear gradient condition was set such that the proportion of eluent A increased from 100% to 50% of eluent B after 133 minutes.

<Separation conditions 2>
Column: LiChrospher 100 RP-18 (manufactured by MERCK)
Detection: UV 210 nm
Flow rate: 0.8mL/min
Eluent A: ultrapure water Eluent B: 50% aqueous acetonitrile solution Gradient: A linear gradient condition was set such that the proportion of eluent A was 100% and the proportion of eluent B became 100% after 20 minutes.

The DPP-IV inhibitory activity of the elution fractions obtained under separation condition 1 was measured using a DPP-IV drug discovery kit (BML-AK499, Enzo Life Sciences). A peptide with DPP-IV inhibitory activity was found in the elution fraction with a retention time of 80 minutes. The peptide separated under these conditions was designated "Peptide A."

Furthermore, the DPP-IV inhibitory activity of the elution fraction obtained under separation condition 2 was measured using a DPPIV drug discovery kit (BML-AK499, Enzo Life Sciences). A peptide with DPP-IV inhibitory activity was found in the elution fraction with a retention time of 12 minutes. The peptide separated under these conditions was designated "Peptide B."

2. Identification of the amino acid sequences of peptides A and B The amino acid sequences of peptides A and B, which were found to have DPP-IV inhibitory activity, were identified using a protein sequencer (PPSQ-53A, Shimadzu Corporation). As a result, it was found that peptide A is a tripeptide consisting of Val-Ala-Tyr, and peptide B is a tetrapeptide consisting of Ile-Pro-Ala-Gly.

3. Confirmation of DPP-IV inhibitory activity of tripeptide Val-Ala-Tyr and tetrapeptide Ile-Pro-Ala-Gly
3-1. Test Method The tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly were synthesized and their DPP-IV inhibitory activity was measured. The DPP-IV inhibitory activity was measured using a DPPIV drug discovery kit (BML-AK499, Enzo Life Sciences). The specific measurement conditions are as follows:

<Materials>
Buffer : 50mM Tris buffer (pH 7.5)
Substrate solution : H-Gly-Pro-pNA (10 mM in DMSO) The substrate solution included in the kit was diluted 50-fold with the buffer solution.
Enzyme solution : 40 μL of the enzyme solution included in the Recombinant Human DPPIV/CD26 kit was diluted with the buffer solution to a total volume of 1011.5 μL. This diluted solution was further diluted 2-fold with the buffer solution and used.
Sample solution : Synthesized Val-Ala-Tyr and Ile-Pro-Ala-Gly were used after being appropriately diluted stepwise with ultrapure water.

<Method>
The buffer solution, substrate solution, and sample solution were each added to a 96-well plate so that a total volume of 100 μL per well was determined according to Table 1. Next, this 96-well plate was inserted into a microplate reader (Infinite 200PRO, manufactured by TECAN) and pre-incubated at 37°C for 10 minutes. After pre-incubation, an enzyme solution was added to the microplate reader according to Table 1. Thereafter, the reaction was continued at 37°C for 60 minutes, and the increase in absorbance due to the cleavage of p-nitroaniline (pNA) from the substrate by DPP-IV was measured at 405 nm at 5-minute intervals.

The DPP-IV inhibition rate at each concentration of Val-Ala-Tyr and Ile-Pro-Ala-Gly was calculated according to the following formula, and IC ₅₀ (the concentration that inhibits 50% of DPP-IV activity) was determined.

3-2. Test results The results are shown in Table 2. As a result, it was confirmed that Val-Ala-Tyr and Ile-Pro-Ala-Gly have DPP-IV inhibitory activity, and that Ile-Pro-Ala-Gly has significantly higher DPP-IV inhibitory activity.

Example 3: Preparation of purified products of tripeptide Val-Ala-Tyr and tetrapeptide Ile-Pro-Ala-Gly. The hydrolysate obtained in Example 1 was subjected to a resin purification treatment to prepare purified products with increased purity of Val-Ala-Tyr and Ile-Pro-Ala-Gly. Specifically, the hydrolysate obtained in Example 1 was added to ultrapure water and mixed to prepare an aqueous solution. mL of this aqueous solution was loaded onto a column (50 mm inner diameter x 200 mm length, manufactured by BIORAD) packed with a resin (Prep C18, manufactured by Nihon Waters). The peptides were then eluted by dropwise addition of two aqueous ethanol solutions with different concentrations to the column, and the eluate was fractionated. Each fraction obtained was subjected to HPLC analysis under the following conditions using synthesized Val-Ala-Tyr and Ile-Pro-Ala-Gly as standards.

<HPLC conditions>
HPLC system: LC-2010 CHT (SHIMADZU)
Column: Waters Xterra C18 (4.6 x 150 mm, 5 μm)
Detection: UV 210 nm
Flow rate: 800μL/min
Eluent A: 0.1% acetic acid in water Eluent B: 50% ethanol in water containing 0.1% acetic acid Gradient:
Time (min) Solution A (%) Solution B (%)
0-4 96 4
4-20 60 40
Column temperature: 40°C
Injection volume: 5μL

As a result, a fraction containing both Val-Ala-Tyr and Ile-Pro-Ala-Gly was detected. The results of HPLC analysis of this fraction are shown in Figure 1. This fraction was also concentrated and lyophilized to prepare a purified product containing Val-Ala-Tyr and Ile-Pro-Ala-Gly, which was used in Example 4, described below.

Example 4: Confirmation of DPP-IV inhibitory activity The DPP-IV inhibitory activity of the hydrolysate obtained in Example 1 (containing 1.60 mg/g of Val-Ala-Tyr and 1.33 mg/g of Ile-Pro-Ala-Gly) and the purified product obtained in Example 4 (containing 5.10 mg/g of Val-Ala-Tyr and 4.30 mg/g of Ile-Pro-Ala-Gly) was measured. The DPP-IV inhibitory activity was measured using DPPIV-Glo ^™ Protease Assay (G8350, manufactured by Promega). The specific measurement conditions are as follows:

<Materials>
Buffer solution : 1M Tris-HCl (pH 8.0, manufactured by Nippon Gene Co., Ltd.) was diluted 100-fold with ultrapure water to obtain 10 mM Tris-HCl (pH 8.0).
Substrate solution : A 10 mM substrate solution was prepared by adding 25 μL of ultrapure water to the DPPIV-Glo ^™ Substrate powder provided with the kit.
Luciferin detection reagent : The luciferin detection reagent powder included in the kit was mixed with 10 mL of DPPIV-Glo ^™ Buffer.
Measurement reagent : 20 μL of the substrate solution was added to 10 mL of the luciferin detection reagent and mixed thoroughly to adjust the substrate concentration to 20 μM.
Enzyme solution : A 0.5 mg/mL solution of Dipeptidylpeptidase IV, human recombinant (manufactured by BioVision) was diluted 16,000 times with the buffer solution.
Sample solution (sample) : The hydrolysate obtained in Example 1 or the purified product obtained in Example 4 was prepared using ultrapure water to a concentration of 150 mg/mL. This aqueous solution was then mixed with an aqueous solution of dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical Industries, Ltd.) prepared to 10 vol% using ultrapure water in a ratio of 3:1 (so that the DMSO concentration was 2.5 vol%), and the mixture was used.
Sample solution (negative control) : Ultrapure water to which DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to make a concentration of 2.5 vol % was used.
Sample solution (positive control) : P32/98 (BML-PI142-0010, manufactured by Enzo Life Sciences) was added to 2.5 vol % DMSO to a concentration of 19.2 μM.

<Method>
5 μL of the measurement reagent, 1 μL of the sample solution, and 4 μL of the enzyme solution were added to each well of a 384-well plate and incubated at room temperature for 30 minutes. The 384-well plate was then inserted into a microplate reader (EnVision 2105, PerkinElmer) and the luminescence intensity was measured. Using medical statistical analysis software GraphPad Prism 9 (MDF), the luminescence intensity of the sample solution (negative control) was set to 100% and that of the sample solution (positive control) to 0%, and the IC ₅₀ (concentration required to inhibit 50% of DPP-IV activity) of the hydrolysate obtained in Example 1 and the purified product obtained in Example 4 was determined.

The results are shown in Table 3. The _IC50 of the purified product obtained in Example 4 was 0.6 times that of the hydrolysate obtained in Example 1. In other words, it was confirmed that Val-Ala-Tyr and Ile-Pro-Ala-Gly, which have DPP-IV inhibitory activity, were concentrated by purification using a resin, and thus exhibited higher DPP-IV inhibitory activity.

Example 5: Confirmation test of the effect of suppressing postprandial blood glucose elevation As described above, the tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly were found to have DPP-IV inhibitory activity in vitro. Therefore, a human test was conducted to confirm whether a composition containing Val-Ala-Tyr and Ile-Pro-Ala-Gly (the hydrolysate obtained in Example 1; containing 1.60 mg/g of Val-Ala-Tyr and 1.33 mg/g of Ile-Pro-Ala-Gly) has the effect of suppressing postprandial blood glucose elevation in the human body.

In accordance with the Declaration of Helsinki, healthy adults (3 men, 3 women; average age 35 years) were selected as subjects after a thorough explanation of the purpose, methods, and results of the study was provided, and they gave their consent. The study was conducted over two days. Furthermore, due to the need to measure fasting blood glucose levels during the study, subjects fasted from 10:00 PM the day before the study until the start of the study, and were only allowed to drink water until 1.5 hours before the start of the study, after which they were not allowed to drink water until the start of the study. There was no restriction on the dietary content of the day before the study. During the study, subjects were asked to limit exercise and activities that may affect blood glucose levels and to remain at rest.

The test meal was a beverage prepared by suspending the hydrolysate (lyophilized product) obtained in Example 1 in 100 mL of water to achieve an intake of 5 g. The placebo meal was prepared by suspending the dried powder of defatted roasted almonds, used as the raw material for the hydrolysate obtained in Example 1, in 100 mL of water to achieve a protein intake of 5 g. The reference meal for carbohydrate loading was a beverage prepared by dissolving glucose (D(+)-glucose, Fujifilm Wako Pure Chemical Industries, Ltd.) in 300 mL of water to achieve a carbohydrate intake of 50 g.

In this study, subjects ate a test meal or placebo meal, followed 30 minutes later by a reference meal. Blood samples were taken immediately before ingestion of the test meal or placebo meal (-30 minutes), immediately before ingestion of the reference meal (0 minutes), and 30, 60, 90, and 120 minutes after ingestion of the reference meal. Blood samples were taken by a doctor or nurse using an indwelling needle. Blood glucose levels were measured in the collected blood samples. Measurements were performed using a Labo Assay ^™ Glucose (Fujifilm Wako Pure Chemical Industries, Ltd.).

Figure 2 shows the time course of the average blood glucose levels of six subjects, from immediately before ingestion of the test meal or placebo meal to 120 minutes after ingestion of the reference meal. As a result, ingestion of the hydrolysate obtained in Example 1 was confirmed to have an effect of suppressing the rise in blood glucose levels immediately after glucose loading. In other words, it was revealed that the tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly have the effect of suppressing postprandial blood glucose elevation in the human body.

Claims (13)

A tetrapeptide with the amino acid sequence Ile-Pro-Ala-Gly. A dipeptidyl peptidase-IV inhibitor comprising a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and/or a tripeptide having the amino acid sequence Val-Ala-Tyr . A dipeptidyl peptidase-IV inhibitor comprising an almond protein hydrolysate containing a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and/or a tripeptide having the amino acid sequence Val-Ala-Tyr . A food or drink for inhibiting dipeptidyl peptidase-IV, comprising the dipeptidyl peptidase-IV inhibitor according to claim 2 or 3 . A pharmaceutical for inhibiting dipeptidyl peptidase-IV, comprising the dipeptidyl peptidase-IV inhibitor according to claim 2 or 3 . A blood glucose level increase inhibitor comprising the tetrapeptide according to claim 1. An agent for suppressing elevation of blood glucose level, comprising an almond protein hydrolysate containing a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and/or a tripeptide having the amino acid sequence Val-Ala-Tyr. A food or drink for suppressing an increase in blood glucose level, comprising the blood glucose level increase suppressor according to claim 6 or 7 . A pharmaceutical for suppressing an increase in blood glucose level, comprising the blood glucose level increase suppressant according to claim 6 or 7 . An antidiabetic agent comprising the tetrapeptide of claim 1. An antidiabetic agent comprising an almond protein hydrolysate containing a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and/or a tripeptide having the amino acid sequence Val-Ala-Tyr . An antidiabetic food or drink comprising the antidiabetic agent according to claim 10 or 11 . An antidiabetic pharmaceutical product comprising the antidiabetic agent according to claim 10 or 11 .