JP5877560B2 - Dipeptidyl peptidase-IV inhibitor - Google Patents

Description

  The present invention relates to a dipeptidyl peptidase-IV inhibitor and the like.

  Dipeptidyl peptidase-IV (hereinafter referred to as “DPP-4”) is a multifunctional transmembrane glycoprotein having N-terminal dipeptidase activity. It is present on most mammalian cells and is present in various tissues such as the liver, kidney, small intestine, salivary gland, blood cells and plasma, so it is assumed that it has a broad role in vivo and is created. It is an enzyme that has recently attracted attention as a drug target.

  Dipeptidyl peptidase-IV is known to be a degrading enzyme of incretin glucagon-like peptide 1 (hereinafter referred to as “GLP-1”) and glucose-dependent insulinotropic polypeptide (hereinafter referred to as “GIP”). ing. This GLP-1 is released after meals, glucose-induced stimulation of insulin biosynthesis and secretion, glucagon secretion suppression, regulation of gene expression, nutritional effects on sputum cells, suppression of food intake, And multifaceted action including slowing of gastric emptying. And inhibition of dipeptidyl peptidase-IV suppresses the degradation of GLP-1 and GIP of incretin and increases their concentration in the blood. As a result, it is known that insulin secretion is promoted and the blood glucose level is lowered. The promotion of insulin secretion by incretin is an operating condition that the blood glucose level is high. For this reason, it can be said that inhibition of dipeptidyl peptidase-IV in type 2 diabetes due to a decrease in insulin secretion has a low risk of the side effect of hypoglycemia caused by conventional insulin secretagogues.

  As described above, dipeptidyl peptidase-IV inhibitors having an action of inhibiting dipeptidyl peptidase-IV activity are expected to be used as antidiabetic agents. However, dipeptidyl peptidase-IV inhibitors are still a new genre of therapeutic agents compared to insulin secretion inhibitors, glucose absorption inhibitors, etc., and many new substances having dipeptidyl peptidase-IV inhibitory activity can be found. It is desired. Furthermore, the first step in the treatment of diabetes is diet therapy and exercise therapy, and when the blood glucose level cannot be controlled even now, pharmaceuticals for treating diabetes are currently used. For these reasons, if it is possible to provide a substance having dipeptidyl peptidase-IV inhibitory activity as a supplement or food additive, it is considered that it can widely contribute to the improvement of blood glucose level.

For example, Patent Document 1 discloses that a bicyclic pyrimidine is used as a dipeptidyl peptidase-IV inhibitor for treating or preventing diabetes.
In addition, for example, in Patent Document 2, casein is subjected to alkaline decomposition by adjusting pH and temperature, and then various peptides such as proteases are further adjusted to separate and purify various peptides from hydrolyzed products. Furthermore, it has been disclosed that a dipeptidyl peptidase-IV inhibitory action has been found among them. In addition, there are disclosures about food-derived dipeptidyl peptidase-IV inhibitors (see, for example, Patent Documents 3 to 5).

Special table 2008-527011 US Patent Application Publication No. 2008/0075904 JP 2011-144167 A JP 2007-039424 A JP 2008-280291 A

Thus, although dipeptidyl peptidase-IV inhibitors have been disclosed in the past, for example, in Patent Document 1, an organic synthetic compound requires a complicated synthesis process, and future verification is necessary for safety. Any dipeptidyl peptidase-IV inhibitor should be examined, and the current situation is that the search for dipeptidyl peptidase-IV inhibitors is not sufficient.
Therefore, this invention is providing the outstanding dipeptidyl peptidase-IV inhibitor.

  As a result of intensive studies to solve the above problems, the present inventor has a dipeptidyl peptidase-IV inhibitory action in a milk protein hydrolyzate obtained by hydrolyzing milk protein with a specific enzyme. Dipeptide VA (SEQ ID NO: 1), dipeptide IA (SEQ ID NO: 2), dipeptide LA (SEQ ID NO: 3) (hereinafter also referred to as “dipeptide of the present disclosure” or “dipeptide”) have been found to complete the present invention. It was.

Moreover, since dipeptide VA (SEQ ID NO: 1), dipeptide IA (SEQ ID NO: 2), and dipeptide LA (SEQ ID NO: 3) are components derived from food, it is considered to be highly safe, so these dipeptides are supplements and food additives. It is also possible to provide as. Therefore, it is considered that the dipeptide of the present disclosure can widely contribute to the improvement of blood glucose level. In addition, since the dipeptides of the present disclosure include peptides having an angiotensin converting enzyme inhibitory action, the dipeptides of the present disclosure are considered to be able to widely contribute to the improvement of hypertension.
From the above, it can be said that the dipeptide of the present disclosure is an effective substance with more added value.

That is, the present invention contains a peptide comprising the amino acid sequence of the following (a) as an active ingredient, a dipeptidyl peptidase -IV inhibitors (except for aspects of the food).
Also, increase in blood glucose level inhibitor comprising a peptide consisting of the amino acid sequence of the following (a) as an active ingredient, also antidiabetic agents vascular endothelial disorder inhibitor (excluding aspects of food).
(A) Val-Ala (VA: SEQ ID NO: 1 )

  The present invention provides a dipeptidyl peptidase-IV inhibitor, a blood glucose level increase inhibitor, a hyperglycemia improving agent, a vascular endothelial disorder inhibitor, etc. can do.

It is a figure which shows the result of MS / MS analysis of dipeptide VA of this indication. It is a figure which shows the result of MS / MS analysis of dipeptide IA of this indication. It is a figure which shows the result of MS / MS analysis of dipeptide LA of this indication.

The dipeptide of the present disclosure is an amino acid sequence represented by a peptide consisting of Val-Ala (SEQ ID NO: 1), an amino acid sequence represented by a peptide consisting of Ile-Ala (SEQ ID NO: 2), or Leu-Ala (SEQ ID NO: 3). It has an amino acid sequence represented by a peptide consisting of
Here, in the present disclosure, Ala (A) is an L-alanine residue, Val (V) is an L-valine residue, Ile (I) is an L-isoleucine residue, and Leu (L) is an L-leucine residue. Indicates.
One or more selected from dipeptide Val-Ala (VA: SEQ ID NO: 1), dipeptide Ile-Ala (IA: SEQ ID NO: 2) and dipeptide Leu-Ala (LA: SEQ ID NO: 3) as active ingredients Can be used.
Dipeptide VA, dipeptide IA and dipeptide LA are dipeptides having a dipeptidyl peptidase-IV inhibitory action. Dipeptide VA and dipeptide IA are dipeptides having an angiotensin converting enzyme inhibitory action.

  The dipeptide of the present disclosure may be a salt of this peptide. Examples of the salts include alkali metals such as potassium and sodium; alkaline earth metals such as calcium and magnesium, and one or more of them may be used as appropriate.

About the manufacturing method of the dipeptide of this indication, it can carry out according to the method as described in WO2003 / 044044 pamphlet [reference literature 1] etc., for example, Although the following methods are illustrated, There is no particular limitation.
For example, a protein or peptide containing one or more amino acid sequences selected from the amino acid sequences represented by SEQ ID NO: 1 (VA), SEQ ID NO: 2 (IA), and SEQ ID NO: 3 (LA) is hydrolyzed. A method obtained by separating and purifying from the obtained degradation product; synthesizing the dipeptide of the present disclosure by a chemical peptide synthesis method, and then separating and purifying the dipeptide of the present disclosure from the obtained synthetic product. And a method of extracting from a plant, animal or microorganism that produces the dipeptide of the present disclosure and a peptide containing the same, and separating and purifying from the obtained extract.

The dipeptide of the present disclosure can be produced by, for example, appropriately hydrolyzing a protein such as casein or whey with an acid alkali or an enzyme.
A method for obtaining the dipeptide by hydrolyzing a raw material protein with a hydrolase is exemplified.
First, before hydrolyzing a raw material protein with an enzyme, the protein is dissolved, dispersed or suspended in water.
The raw material protein is not particularly limited as long as it is a protein containing the dipeptide of the present disclosure and can produce the dipeptide of the present disclosure when appropriately digested with a hydrolase. Examples of the protein include those derived from animals and microorganisms, and casein that is available in large quantities is preferable.
At this time, the treatment method varies depending on the properties of the raw protein, but if the raw protein is soluble, the raw protein may be dispersed and dissolved in water or warm water, and if it is poorly soluble, the protein will be dissolved in hot water. May be homogenized by mixing and stirring.

Then, an alkaline agent or an acid agent may be added to the solution containing the protein to adjust the pH. This pH is preferably adjusted to the optimum pH of the hydrolase used or in the vicinity thereof.
It does not specifically limit as said alkali agent or acid agent, What is necessary is just to use what is accept | permitted by a foodstuff or a pharmaceutical. Examples of the alkali agent include hydroxides such as sodium hydroxide and calcium hydroxide; carbonates such as potassium carbonate, and the like, and these may be alkali metal salts or alkaline earth metal salts. Examples of the acid agent include inorganic acids such as hydrochloric acid and phosphoric acid; organic acids such as citric acid, acetic acid, and formic acid. Of these, one or more may be used as appropriate.
In addition, it is desirable from the viewpoint of preventing deterioration due to contamination by various bacteria that the protein-containing solution is heat-sterilized at 70 to 90 ° C. for 15 seconds to 10 minutes.

Next, a predetermined amount of hydrolase is added to the solution containing the protein and reacted at a temperature of about 10 to 85 ° C. for 0.1 to 48 hours to obtain a hydrolyzate.
At this time, after adding the hydrolase, the solution is maintained at an appropriate temperature according to the type of the enzyme, for example, 30 to 60 ° C., preferably 45 to 55 ° C., to start protein hydrolysis. Is desirable.
The hydrolysis reaction time may be continued until the desired decomposition rate is reached while monitoring the decomposition rate of the enzyme reaction. In order to obtain the dipeptide of the present disclosure, the degradation rate of the raw material protein is preferably 20 to 30%.
The hydrolase reaction is stopped, for example, by deactivation of the enzyme in the hydrolyzate, and can be performed by a heat deactivation treatment by a conventional method. The heating temperature and holding time of the heat deactivation treatment can be set as appropriate under conditions that allow sufficient deactivation in consideration of the thermal stability of the enzyme used. For example, the heating temperature and the holding time can be set within a temperature range of 80 to 130 ° C. for 30 minutes. It can be performed with a holding time of ˜2 seconds.

  The hydrolase is not particularly limited, but is preferably an enzyme that can hydrolyze the raw protein to produce the dipeptide of the present disclosure. Specifically, the enzyme that can be produced is preferably an endopeptidase.

Examples of the endopeptidase include those derived from microorganisms and animals. Specific examples include proteases derived from bacteria belonging to the genus Bacillus and proteases derived from animal pancreas. A commercially available protease can be used. Examples of commercially available proteases include proteases derived from Bacillus bacteria such as biolase sp-20 (manufactured by Nagase Seikagaku Corporation) and protease N (manufactured by Amano Enzyme); PTN 6.0S (manufactured by Novozymes Japan), and the like. Animal pancreatic proteases and the like can be exemplified as preferable ones.
For example, when using a protease derived from a genus Bacillus, it is desirable to add it at a rate of 100 to 5000 active units per gram of protein. In addition, when using a protease derived from animal pancreas, it is desirable to add it at a rate of 3000 to 8000 active units per gram of protein.
You may use the hydrolase used in this indication individually or in combination of 2 or more types. When using 2 or more types of enzymes, you may perform each enzyme reaction simultaneously or separately.
In the present disclosure, it is preferable to use biorase sp-20, protease N, and PTN 6.0S in combination, and it is particularly preferable to use these three enzymes in combination.

The method for calculating the decomposition rate of the raw material protein was determined by measuring the total amount of nitrogen in the sample by the Kjeldahl method (edited by the Japan Food Industry Association, “Food Analysis Method”, page 102, Korin Co., Ltd., 1984). Measure the amount of formol nitrogen in the sample by the titration method (Matsuda et al., “Food Engineering Experiments”, Volume 1, Page 547, Yokendo, 1970), and calculate the decomposition rate from these measurements using the following formula To do.
Decomposition rate (%) = (formol nitrogen amount / total nitrogen amount) × 100

It is preferable to isolate or purify the dipeptide of the present disclosure from the hydrolyzate.
Purification of the dipeptide of the present disclosure is usually performed in the same manner as used for the purification of oligopeptides, such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography, etc. It can be carried out by appropriately combining various methods such as chromatography, solvent precipitation, salting out and distribution between two liquid phases.

  When isolating or purifying the dipeptide of the present disclosure, the fraction containing the target substance can be determined using dipeptidyl peptidase-IV inhibitory action and / or angiotensin converting enzyme inhibitory action described below as an index. Moreover, the active ingredient of those fractions can be identified by mass spectrometry.

  In addition, the proteolysate obtained by hydrolase contains at least one selected from VA (SEQ ID NO: 1), IA (SEQ ID NO: 2) and LA (SEQ ID NO: 3). preferable.

The dipeptide of the present disclosure can also be produced by chemical synthesis.
The chemical synthesis of the dipeptide of the present disclosure can be performed by a liquid phase method or a solid phase method which is usually used for the synthesis of oligopeptides. The synthesized peptide is deprotected as necessary, and unreacted reagents and by-products can be removed to isolate the dipeptide of the present disclosure.
Such peptide synthesis can be performed using a commercially available peptide synthesizer. The fact that the target peptide has been obtained can be confirmed by using dipeptidyl peptidase-IV inhibitory action and / or angiotensin converting enzyme inhibitory action described below as an index.

The dipeptide of the present disclosure has a dipeptidyl peptidase-IV inhibitory action, as shown in Examples below.
Various diseases and symptoms may occur when dipeptidyl peptidase-IV degrades a compound involved in physiological functions in a living body. For this reason, when dipeptidyl peptidase-IV is inhibited, the lifetime of a compound involved in physiological functions in vivo that has been degraded by dipeptidyl peptidase-IV is extended, resulting in dipeptidyl peptidase-IV. Prevention, amelioration, or treatment of the disease or symptom that occurs.
Examples of the compound involved in the physiological function include GLP-1 and GIP. And by inhibiting the degradation of GLP-1, glucose-induced stimulation of insulin biosynthesis and secretion, glucagon secretion suppression, regulation of gene expression, nutritional effects on sputum cells, suppression of food intake, and gastric emptying Each action such as slowing down can be achieved. This can contribute to normalization of the elevated blood glucose level and improvement / adjustment of hunger and body weight.

Further, it is known that the function of vascular endothelial cells is lowered or the endothelial cells of blood vessels are damaged by the action of dipeptidyl peptidase-IV. This decrease in blood vessel endothelial function or damage causes vascular disorders such as vasoconstriction, arteriosclerosis, and thrombus formation due to increased vascular tension, and these causes cause organ blood flow disorders, organ dysfunction, and diabetes. Complications will be induced.
In recent years, many effects of improving endothelial cell function by administration of dipeptidyl peptidase-IV inhibitors have been reported (for example, see References 2 to 5 [Reference 2] Endocrine Journal 2011, 58 (1), 69-73. [Reference 3] J Am Coll Cardiol. 2012, 59 (3), 265-76; [Reference 4] Diabetes Care. 2011, 34 (9), 2072-7; [Reference 5] Cardiovascular Diabetology 2011, 10 (85) (http://www.cardiab.com/content/10/1/85)).
These effects are considered to be mediated by the protective action of blood vessels by incretin in addition to the improvement by simply lowering the blood glucose level. When blood sugar decreases and blood vessel suppleness is lost due to hyperglycemia, the blood pressure rises, and the increased blood pressure further damages the blood vessels, causing adverse effects on organs such as the heart, kidney, and brain appear. In order to break this vicious circle, a combination of dipeptidyl peptidase-IV inhibitor and angiotensin converting enzyme inhibitor has been tried, and the inhibitor of dipeptidyl peptidase-IV also plays an important role in the treatment of cardiovascular system It is considered a thing.

That is, since the dipeptide of the present disclosure has a dipeptidyl peptidase-IV inhibitory action, it suppresses blood sugar levels, suppresses hyperglycemia, suppresses vascular endothelial function, suppresses vascular endothelial damage, suppresses vascular disorders, and vascular endothelium. It shows cell protective effect and appetite suppressive effect. In addition, it is considered that the dipeptide of the present disclosure can suppress appetite and protect vascular endothelial cells. Note that “suppressing the increase in blood glucose level” here means including a decrease in blood glucose level, and particularly means “being able to lower a blood glucose level that is higher than a normal value or higher than necessary”. The determination of the normal value of the blood glucose level may be based on the 2010 diagnostic criteria of the Japan Diabetes Society.
Furthermore, by inhibiting dipeptidyl peptidase-IV, it is considered possible to prevent, ameliorate, or treat diseases and symptoms caused by dipeptidyl peptidase-IV. Examples of various diseases and symptoms caused by dipeptidyl peptidase-IV of the present disclosure include hyperglycemia, diabetes, diabetic complications, vascular endothelial disorder, vascular disorder and the like. The various diseases caused by dipeptidyl peptidase-IV may be various diseases mediated by dipeptidyl peptidase-IV.
Furthermore, various diseases caused by hyperglycemia, diabetes and hyperglycemia conditions include, for example, diabetic microangiopathy (eg retinopathy, nephropathy, neuropathy, etc.) and macrovascular complications (eg angina) Ischemic heart disease such as symptom / myocardial infarction, cerebral infarction, obstructive arteriosclerosis, gangrene, etc.).

The fact that the dipeptidyl peptidase-IV inhibitor can be used as a prophylactic or therapeutic agent for the various diseases is as disclosed in the aforementioned Patent Documents 1 to 5 and Reference Documents 2 to 5, It goes without saying that the disclosed dipeptide can also be implemented as a prophylactic or therapeutic agent for the various diseases.
Therefore, the dipeptide of the present disclosure may be used for dipeptidyl peptidase-IV inhibition, suppression of increase in blood glucose level, improvement of hyperglycemia, suppression of vascular endothelial damage, anti-diabetes and the like as described above. In order to produce these various preparations, which can be used for various preparations intended for the above-mentioned use, such as peptidyl peptidase-IV inhibitors, blood glucose level increase inhibitors, vascular endothelial disorder inhibitors and antidiabetic agents. Can be used.

In addition, some of the dipeptides of the present disclosure have an angiotensin converting enzyme (hereinafter, also referred to as “ACE”) inhibitory action as shown in Examples below.
Here, the angiotensin converting enzyme is an enzyme that acts on angiotensin I generated from angiotensinogen by cleavage with renin, liberates two C-terminal amino acids, and converts them into angiotensin II.
Angiotensin converting enzyme generates angiotensin II having a strong pressor action and also has an action of inactivating bradykinin having a hypotensive action. For this reason, angiotensin converting enzyme inhibitors have already been used as therapeutic agents for hypertension, and captopril and renibase have been used as pharmaceuticals. However, as side effects, captopril and renibase may have excessive antihypertensive effects and impaired renal function. For this reason, high safety is desired even if it is provided as a supplement or food additive, and many searches for substances having an angiotensin converting enzyme inhibitory action have been reported (see References 1 and 6 to 8). [Reference 1] Pamphlet of International Publication No. 2003/044404; [Reference 6] Japanese Patent Laid-Open No. 6-40944; [Patent Document 7] Japanese Patent Laid-Open No. 2001-136995: [Reference 8] Japanese Patent Laid-Open No. 7 -101982).
For example, in References 6 and 7, it is known that Val-Pro-Pro and Ile-Pro-Pro obtained by degrading casein with lactic acid bacteria or a combination of proteinase and peptidase have angiotensin converting enzyme inhibitory action. Yes. In addition, as a tripeptide having an angiotensin converting enzyme inhibitory action, for example, Leu-Leu-Trp is known in Reference Document 8, and Met-Lys-Pro is known in Reference Document 1.

Inhibiting angiotensin-converting enzyme, in addition to blood pressure lowering action and bradykinin inactivating action, cardiac hypertrophy retraction action, peripheral vasodilation action, renal protection action, substance P degradation inhibition action, etc. are possible, and depending on the hypertension state It is considered that an inhibitory action on vascular endothelial injury, an inhibitory action on vascular injury, and the like are possible. It is also known that an angiotensin converting enzyme inhibitor is effective in improving essential hypertension.
Further, by inhibiting angiotensin converting enzyme, it is considered possible to prevent, improve or treat diseases and symptoms caused by angiotensin converting enzyme.
Examples of diseases and symptoms caused by angiotensin converting enzyme include hypertension, cardiac hypertrophy, and renal hypertrophy. The various diseases caused by angiotensin converting enzyme may be various diseases mediated by angiotensin converting enzyme.
Examples of various diseases and symptoms caused by hypertension or hypertension include cardiovascular diseases and vascular disorders such as cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, renal failure, visual impairment, and angioedema. .

That is, since the dipeptide of the present disclosure has an angiotensin converting enzyme inhibitory action, the blood pressure lowering action, the bradykinin inactivating action, the hypertensive symptom improving action, the vascular endothelial disorder inhibiting action caused by the hypertension state, and the vascular disorder inhibiting action. Etc. Furthermore, by inhibiting angiotensin converting enzyme, it is considered possible to prevent, ameliorate, or treat the diseases and symptoms caused by the above-mentioned angiotensin converting enzyme.
Note that the angiotensin converting enzyme inhibitor can be used as a preventive or therapeutic agent for the above-mentioned various diseases, as disclosed in Patent Documents 1 and 6 to 8, and the same applies to the dipeptides of the present disclosure. Needless to say, the present invention can also be implemented as a prophylactic or therapeutic agent for the various diseases.
Therefore, the dipeptide of the present disclosure may be used for angiotensin converting enzyme inhibition, blood pressure lowering, hypertension and the like, and uses as described above such as angiotensin converting enzyme inhibitor, blood pressure lowering agent and antihypertensive agent. It can be used for various preparations intended, and can be used to produce these various preparations.

In addition, the dipeptide of the present disclosure can be provided as a more useful compound when it has two effects of a blood glucose level increase inhibitory action and a blood pressure lowering action.
By the way, if the hyperglycemic state continues, the function of the endothelial cells of the blood vessels decreases and vascular endothelial damage is likely to occur, but high blood pressure that gives a strong load to the blood vessels increases the risk of causing vascular damage. Are known. Factors (NO and PGI2) that relax vascular smooth muscle are produced from vascular endothelial cells. And when the function of vascular endothelial cells decreases due to hyperglycemia, their releasing ability also decreases. As a result, diabetics have high blood pressure. Furthermore, high blood pressure falls into a vicious circle that places a load on vascular endothelial cells.

A person who is diabetic or a spare group thereof (the spare group is a person who has not yet been diabetic but has a high blood glucose level) needs to pay attention to hypertension. However, in general, it cannot be said that blood pressure can be lowered even if the blood sugar level is lowered with a hyperglycemic drug, whereas blood sugar can be lowered even if the blood pressure is lowered with a blood pressure lowering drug. I can't say that. For this reason, diabetic and hypertensive humans are often prescribed both hyperglycemia-improving drugs and antihypertensive drugs, and there is a concern of side effects due to the combination of drugs.
By having such an effect, it is possible to reduce the amount of drug used, and therefore, side effects can be expected to be reduced.

That is, the dipeptide of the present disclosure can be said to be very effective for improving blood vessels, suppressing vascular endothelial damage and preventing, improving or treating diabetes, and particularly effective for preventing, improving or treating vascular endothelial damage caused by hyperglycemic conditions. It is thought that.
Specific diseases and symptoms of the vascular endothelial disorder of the present disclosure include, for example, diabetic vascular disorder.

Accordingly, the dipeptide of the present disclosure and the above-mentioned various preparations containing this as an active ingredient (hereinafter referred to as “the dipeptidyl peptidase-IV inhibitor etc.”) are ingested or administered to animals including humans, and Prevent, improve and / or treat hyperglycemia, diabetes (especially type 2 diabetes), hypertension, and diabetic complications (eg, diabetic vascular endothelial disorder, nephropathy, hypertension, etc.), vascular disorders, etc. It can be used in a method for planning.
Further, the dipeptidyl peptidase-IV inhibitor, etc. is a pharmaceutical for humans or animals for the prevention, improvement and / or treatment of hyperglycemia, diabetes, vascular disorders, hypertension, etc. It can be used as an active ingredient of products, external preparations for skin, cosmetics and foods.
When blended in a pharmaceutical product, it can be an oral or parenteral agent. In addition, when blended in food, various diseases caused by the above-mentioned dipeptidyl peptidase-IV and / or various diseases caused by angiotensin converting enzyme, and various diseases caused by hyperglycemia and / or hypertension, etc. Functional foods based on the concept of physiology such as prevention, improvement or treatment, dipeptidyl peptidase-IV inhibition, blood sugar level increase suppression, hyperglycemia improvement, angiotensin converting enzyme inhibition and blood pressure lowering, food for the sick, food for specified health use It can be applied to.
The dipeptidyl peptidase-IV inhibitor or the like is a pharmaceutical or quasi-drug for human or animal use for the prevention, improvement and / or treatment of hyperglycemia, diabetes, hypertension, vascular disorders, etc. as described above. In addition, it can be used as an active ingredient for skin external preparations, cosmetics, foods and the like.

The dipeptidyl peptidase-IV inhibitor and the like of the present disclosure may be administered either orally or parenterally, but oral administration is desirable. Parenteral administration includes intravenous injection, rectal administration, inhalation and the like. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, acid agents, ointments and the like.
In formulation, in addition to milk protein hydrolyzate and the dipeptide of the present disclosure, components such as excipients, pH adjusters, colorants, and flavoring agents that are usually used for formulation can be used. It is also possible to use a drug having a dipeptidyl peptidase-IV inhibitory action and / or angiotensin converting enzyme inhibitory action, an antidiabetic drug, an antihyperglycemic drug, a blood pressure lowering drug, etc. .

Further, the dipeptide of the present disclosure is contained in a food as an active ingredient, and processed as a food having a dipeptidyl peptidase-IV action and / or an angiotensin converting enzyme inhibitory action as one aspect of the dipeptidyl peptidase-IV inhibitor and the like. It is also possible.
Examples of such foods include liquids, pastes, solids, powders, etc. In addition to tablet confectionery, liquid foods, feeds (including for pets), etc., for example, flour products, instant foods, processed agricultural products, Processed marine products, processed livestock products, milk / dairy products, fats and oils, basic seasonings, compound seasonings / foods, frozen foods, beverages, and other commercial products.

For example, examples of the flour product include bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread crumbs and the like. Examples of the instant foods include instant noodles, cup noodles, retort / cooked food, cooking canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, and other instant foods.
Examples of the processed agricultural products include canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (cereal processed products), and the like. Examples of the processed fishery products include canned fishery products, fish hams and sausages, fish paste products, marine delicacies, and tsukudani. Examples of the processed livestock products include canned livestock, pastes, livestock ham, sausages and the like.
Examples of the milk / dairy products include processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheese, ice creams, prepared powdered milks, creams, and other dairy products. Examples of the fats and oils include butter, margarines, and vegetable oils.
For example, the basic seasonings include soy sauce, miso, sauces, tomato processed seasonings, mirins, vinegars, etc., and the combined seasonings and foods include cooking mixes, curry ingredients, sauces, Examples include dressings, noodle soups, spices, and other complex seasonings.
Examples of the frozen food include raw material frozen food, semi-cooked frozen food, cooked frozen food, and the like.
Examples of the confectionery include caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, and other confectionery.
For example, the beverages include carbonated beverages, natural fruit juices, fruit juice beverages, soft drinks with fruit juices, fruit drinks, fruit drinks with fruits, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks Sports drinks, nutritional drinks, alcoholic drinks, other taste drinks, and the like.
For example, examples of commercially available foods other than the above include baby foods, sprinkles, and tea paste.

In the dipeptidyl peptidase-IV inhibitor and the like of the present disclosure, the amount of the dipeptide of the present disclosure is preferably at least 0.001% by mass with respect to the final composition of the formulation.
The dose of the dipeptide of the present disclosure varies depending on age, symptoms, etc., but is usually 0.001 to 3000 mg / day, preferably 0.01 to 30 mg / day, and is administered once to 3 times a day. May be.

  Specific examples and the like will be described below, but the present invention (the present disclosure) is not limited thereto.

Production Example 1: Production of dipeptide VA and dipeptide IA by enzymatic degradation of casein <1> Enzymatic degradation of casein 90 g of water was added to 10 g of commercially available casein (manufactured by New Zealand Daily Board), well dispersed, and sodium hydroxide was added. The pH of the solution was adjusted to 7.0 to completely dissolve casein to prepare an aqueous casein solution having a concentration of about 10%. The aqueous casein solution was sterilized by heating at 85 ° C. for 10 minutes, adjusted to 50 ° C., sodium hydroxide was added to adjust the pH to 9.5, and then biolase sp-20 (manufactured by Nagase Seikagaku Corporation) 10 , 000 active units (1,250 active units per gram of protein), protease N (manufactured by Amano Enzyme) 17,000 active units (2,000 active units per gram of protein), and PTN 6.0S (manufactured by Novozymes Japan) 60,000 activity units (7,000 activity units per gram of protein) were added to initiate the hydrolysis reaction. When the degradation rate of casein reached 24.5%, the enzyme reaction was stopped by heating at 80 ° C. for 7 minutes to inactivate the enzyme and cooled to 10 ° C. This hydrolyzed solution was ultrafiltered with an ultrafiltration membrane (manufactured by Asahi Kasei Co., Ltd.) having a fractional molecular weight of 10,000, concentrated and freeze-dried to obtain 8 g of a freeze-dried product.

<2> Separation of peptides by HPLC The casein hydrolyzate was separated by reversed-phase HPLC. The HPLC conditions are shown in the following HPLC condition 1.
[HPLC condition 1]
Column: Cadenza CD-C18
10 mmI. D. × 250mm (manufactured by Intact Corporation)
Detection: UV 215nm
Flow rate: 3 ml / min Eluent A: Aqueous solution containing 0.1% TFA Eluent B: Acetonitrile solution containing 0.1% TFA From the 98% eluent A ratio, 75% after 30 minutes, 50% after 40 minutes The hydrolyzate was separated under a gradient condition of 20% after 43 minutes, and the eluate was fractionated every 0.75 ml. When the dipeptidyl peptidase-IV inhibitory ability was measured for the eluted fraction, a strong inhibitory activity ability was observed in the fraction eluted at a retention time of 10.25 minutes.
Furthermore, in order to refine | purify, it refine | purified by HPLC of another condition. The conditions at this time are shown in the following HPLC condition 2. At this time, the eluents A and B of the condition 1 were changed to the eluents A and B of the condition 2, respectively, and the other conditions were the same as the condition 1.
[HPLC condition 2]
Column: Cadenza CD-C18
10 mmI. D. × 250mm (manufactured by Intact Corporation)
Detection: UV 215nm
Flow rate: 3 ml / min Eluent A: Aqueous solution containing 0.2% formic acid Eluent B: Acetonitrile solution containing 0.2% formic acid

Under the same gradient conditions, the hydrolyzate was separated, and the eluate was fractionated every 0.75 ml. When the dipeptidyl peptidase-IV inhibitory ability was measured for the eluted fraction, a strong inhibitory activity ability was observed in the fraction eluted at a retention time of 25.5 minutes. The 50% inhibitory concentration of enzyme activity in this fraction was 12 μg / ml.
The compound having the active peak was identified by a protein sequencer (PPSQ-23A) manufactured by Shimadzu Corporation. As a result, it was found to have a structure of Val-Ala (SEQ ID NO: 1). These amino acid residues were L-type.
Furthermore, as shown in FIG. 1, m / z = 72 as shown in FIG. 1 by MS / MS analysis using ThermoQuest Corporation mass spectrometer LTQ with a molecular weight of 188.1 and m / z = 189.1 (MH +) as a parent ion. Daughter ions such as 0.0 were detected.
Thus, it was revealed that the structure of the peptide having dipeptidyl peptidase-IV inhibitory activity was Val-Ala. Also, 50% of the synthetic peptide VA (SEQ ID NO: 1) obtained in Production Example 2 described below. The blocking concentration was 12.2 μg / ml, consistent with the value of the purified fraction.

  Further, the same operation was performed, and the <1> casein enzyme degradation product contained dipeptide IA (SEQ ID NO: 2) as a peptide having dipeptidyl peptidase-IV inhibitory activity in addition to dipeptide VA. Was confirmed. All of these amino acid residues were L-type.

Production Example 2: Chemical synthesis of dipeptide VA / IA / LA Using a peptide synthesizer (Model 433A, Applied Biosystems), Fmoc-Val (Peptide Institute, Inc.)), Fmoc-Ala-Wang-PEG Resin Tripeptide Val-Ala was synthesized by solid phase synthesis method using (Watanabe Chemical Co., Ltd.) as a raw material.
The operation was performed according to the manual of Applied Biosystems and then deprotected. This peptide was purified by the above HPLC condition 1.
The obtained dipeptide Val-Ala has a molecular weight (M) of 188.1 measured by mass spectrometry, and a spectrum in a purified fraction by MS / MS analysis using m / z = 189.1 (MH +) as a parent ion. A similar spectrum was obtained (see FIG. 1).

In addition, the dipeptide Ile-Ala was obtained by the solid-phase synthesis method in the same manner as described above, except that Fmoc-Val as the raw material was replaced with Fmoc-Ile. The obtained dipeptide Ile-Ala has a molecular weight (M) of 202.1 measured by mass spectrometry, and a spectrum in a purified fraction by MS / MS analysis using m / z = 203.1 (MH +) as a parent ion. A similar spectrum was obtained (see FIG. 2). Similarly, Leu-Ala was obtained (see FIG. 3).
In addition, Val-Pro-Pro (VPP: SEQ ID NO: 4), Ile-Pro-Pro (IPP: SEQ ID NO: 5), and Leu-Tyr (LY: SEQ ID NO: 6) were chemically synthesized.

Test Example 1: Dipeptidyl peptidase-IV inhibitory activity confirmation test for each peptide The dipeptidyl peptidase-IV inhibitory activity confirmation test for each peptide obtained in Production Example 2 shown in Table 1 was performed, and the results are shown in Table 1. .

Test Example 2: Angiotensin-converting enzyme inhibitory activity confirmation test of each peptide As a result of conducting angiotensin-converting enzyme inhibitory activity confirmation test of VA (SEQ ID NO: 1) and IA (SEQ ID NO: 2), 50% inhibition concentration is VA 52 μg / ml and IA was 91 μg / ml. Therefore, since VA and IA have both properties of dipeptidyl peptidase-IV inhibitory activity and angiotensin converting enzyme inhibitory activity, they are very effective for improving blood vessels, suppressing vascular endothelial damage and preventing, improving or treating diabetes. It can be said.

Dipeptide VA, dipeptide IA, and dipeptide LA have a dipeptidyl peptidase-IV inhibitory action, and among them, dipeptide VA and dipeptide IA have angiotensin converting enzyme inhibitory action.
Furthermore, it is considered that an enzyme degradation product of casein containing VA and / or IA can be used for foods, pharmaceuticals, etc. as a material or food material having a dipeptidyl peptidase-IV inhibitory action, an angiotensin converting enzyme inhibitory action or the like.
When a commercially available casein degradation product containing at least VA and / or IA was searched, VA and IA were found in Morinaga Milk Peptide MKP (manufactured by Morinaga Milk Industry Co., Ltd.).
Morinaga Milk Peptide MKP (manufactured by Morinaga Milk Industry Co., Ltd.) had a dipeptidyl peptidase-IV inhibition 50% inhibitory concentration of dipeptidyl peptidase-IV inhibitory activity: 84 μg / ml. Dipeptide VA and IA are thought to contribute to the dipeptidyl peptidase-IV inhibitory action of Morinaga Milk Peptide MKP (manufactured by Morinaga Milk Industry Co., Ltd.).

<Method for Measuring Dipeptidyl Peptidase-IV Inhibitory Activity>
Measurement of dipeptidyl peptidase (DPP-4) inhibition was performed according to the method of Kato et al. “Kato, T. et al. Biochem. Med. 19, p351, 1978”.
Specifically, the enzyme reaction was performed using Recombinant Human DPPIV / CD26 (R & D Systems, Inc.) as the enzyme (DPP-4) and H-Gly-Pro-AMC (Biomol GmbH) as the substrate.
To each well of a 96-well microplate (nunc 137101), water or an aqueous solution of each concentration of the test substance or HPLC fraction was added, and 20 μl of Tris-HCl (0.25 M, pH 8.0) was added. The total volume was adjusted to 80 μl. After stirring, the plate was warmed in a 37 ° C. incubator for about 10 minutes, 10 μl of DPP-4 solution and 10 μl of substrate solution were added (total volume 100 μl), and the reaction was started by stirring. A well to which water was added instead of the enzyme was used as a control.
The enzyme reaction was measured using a microplate reader (SH-9000, Corona Electric Co., Ltd.) under the condition where the internal temperature was maintained at 37 ° C. (5 minute interval, ex360 nm / em460 nm).
Vildagliptin (JS Research Chemicals Trading) was used as a positive target whose inhibitory activity was calculated from the fluorescence intensity value during a period in which the increase in fluorescence intensity over time was linear (within 30 minutes from the start of the reaction).

Inhibition rate (%) = 100% − [(Y−b) / (X−a)] × 100%
X: water + enzyme + substrate Y: test substance + enzyme + substrate a: water + substrate b: test substance + substrate <How to determine the concentration of IC ₅₀ >
The concentration of the test substance was diluted stepwise (10 to 2000 μg / ml), and the inhibition rate was determined. Based on the results, a relational expression between the logarithm (log ₁₀ ) of the addition concentration of the test substance and the inhibition rate was obtained. From this relational expression, IC ₅₀ was calculated by calculating back the concentration at which the inhibition rate of the enzyme was 50%.

<Method for measuring angiotensin converting enzyme inhibitory activity>
Measurement of angiotensin converting enzyme inhibition (ACE inhibition) was performed according to the method of Araujo et al. [Araujo, MC, et al., Biochemistry 39, 8519, 2000].
Enzyme reaction was performed using Angiotensin Converting Enzyme, from rabbit lung (SIGMA) as the enzyme (ACE) and Abz-FRK (Dnp) -P (Enzo Life Sciences International, Inc.) as the substrate (Araujo, MC, et al., Biochemistry 39, 8519, 2000).
To each well of a 96-well microplate (nunc 137101), add water or an aqueous solution of each concentration of the test substance or HPLC fraction, add 20 μl of Tris-HCl (0.25M, pH 8.0), and add the total amount. Prepared to 80 μl. After stirring, the plate was warmed in a 37 ° C. incubator for about 10 minutes, 10 μl of ACE solution and 10 μl of substrate solution were added (total volume 100 μl), and the reaction was started by stirring. A well to which water was added instead of the enzyme was used as a control.
The enzyme reaction was measured using a microplate reader (SH-9000, Corona Electric Co., Ltd.) under the condition where the internal temperature was maintained at 37 ° C. (5-minute interval, ex320 nm / em420 nm).
The inhibitory activity was calculated by the following formula from the fluorescence intensity value during a period in which the increase in fluorescence intensity with time was linear (within 30 minutes from the start of the reaction).
Inhibition rate (%) = 100% − [(Y−b) / (X−a)] × 100%
X: Water + Enzyme + Substrate Y: Test substance + Enzyme + Substrate a: Water + Substrate b: Test substance + Substrate <How to determine the concentration of IC ₅₀ >
The concentration of the test substance is diluted stepwise (10 to 2000 μg / ml) and the inhibition rate is determined. Based on the result, a relational expression between the logarithm (log ₁₀ ) of the addition concentration of the test substance and the inhibition rate was obtained. From this relational expression, IC ₅₀ was calculated by calculating back the concentration at which the inhibition rate of the enzyme was 50%.

  Since some of the disclosed dipeptides can be isolated from casein hydrolysates, they are highly safe and can be used in a wide range of fields such as pharmaceuticals, foods, topical skin preparations, and functional foods. is there.

Further, the present technology can also take the following configurations.
[1] As an active ingredient, one or more selected from peptides consisting of any of the following amino acid sequences (a) to (c):
A dipeptidyl peptidase-IV inhibitor, a blood sugar level increase inhibitor, an antidiabetic agent, a vascular endothelial disorder inhibitor, a blood vessel improving agent, an angiotensin converting enzyme inhibitor, a blood pressure lowering agent or an antihypertensive agent.
(A) Val-Ala (VA: SEQ ID NO: 1)
(B) Ile-Ala (IA: SEQ ID NO: 2)
(C) Leu-Ala (LA: SEQ ID NO: 3)

[2] For the production of dipeptidyl peptidase-IV inhibitor, blood glucose level increase inhibitor, antidiabetic agent, vascular endothelial disorder inhibitor, vascular ameliorating agent, angiotensin converting enzyme inhibitor, antihypertensive agent or antihypertensive agent Use of one or more selected from VA, IA and LA.
[3] Production of dipeptidyl peptidase-IV-inhibiting foods, blood glucose level-suppressing foods, anti-diabetic foods, vascular endothelial disorder-suppressing foods, vascular-improving foods, angiotensin-converting enzyme-inhibiting foods, and blood pressure-lowering foods Use of one or more selected from VA, IA, LA.

[4] One or two or more selected from VA, IA, LA, dipeptidyl peptidase-IV inhibitor, blood glucose level increase inhibitor, antidiabetic agent, vascular endothelial disorder inhibitor, blood vessel improving agent, angiotensin conversion Use as an enzyme inhibitor, antihypertensive agent or antihypertensive agent.
[5] diseases caused by dipeptidyl peptidase-IV, diseases caused by hyperglycemia, diabetes, diseases caused by vascular endothelial disorder or vascular injury, diseases caused by angiotensin converting enzyme, diseases caused by hypertension, One or more selected from VA, IA, and LA for prevention, amelioration or treatment as active ingredients, diseases caused by dipeptidyl peptidase-IV, diseases caused by hyperglycemia, diabetes, A method for preventing, ameliorating, or treating a vascular endothelial disorder or a disease caused by a vascular disorder, a disease caused by an angiotensin converting enzyme, or a disease caused by a hypertension state.

The disease and / or symptom caused by the dipeptidyl peptidase-IV is preferably selected from hyperglycemia, diabetes, diabetic complications, vascular endothelial disorder and vascular disorder.
The disease and / or symptom caused by the angiotensin converting enzyme is preferably selected from hypertension and cardiovascular disease.

Claims (3)

Dipeptidyl peptidase -IV inhibitor comprising a peptide consisting of the amino acid sequence of the following (a) as an active ingredient (except for aspects of the food).
(A) Val-Ala (SEQ ID NO: 1 ) Blood glucose increase inhibitor comprising a peptide consisting of the amino acid sequence of the following (a) as an active ingredient (except for aspects of the food).
(A) Val-Ala (SEQ ID NO: 1 ) Vascular endothelial dysfunction inhibitor containing a peptide comprising the amino acid sequence of the following (a) as an active ingredient (except for aspects of the food).
(A) Val-Ala (SEQ ID NO: 1 )

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