JPWO2013125622A1 - Dipeptidyl peptidase-IV inhibitor - Google Patents

Abstract

To provide an excellent dipeptidyl peptidase-IV inhibitor and the like. A dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent or a vascular endothelial disorder inhibitor containing a peptide consisting of Met-Lys-Pro as an active ingredient; A method for preventing, ameliorating, or treating a disease caused by a disease, a disease caused by a hyperglycemic state, diabetes, or a disease caused by vascular endothelial disorder or vascular disorder. The disease caused by the dipeptidyl peptidase-IV is preferably selected from hyperglycemia, diabetes, diabetic complications, vascular endothelial disorder and vascular disorder.

Description

  The present invention relates to a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, and a vascular endothelial disorder inhibitor.

  Dipeptidyl peptidase-IV (hereinafter also referred to as “dipeptidyl peptidase-4” or “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 and insulin resistance improvers, and have a new dipeptidyl peptidase-IV inhibitory action. It is desirable to find many substances. 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).
Further, for example, in Table 2 of Patent Document 6, isolated peptides obtained by isolating only one peptide from casein or whey hydrolyzate are IPI, LPL, KVLP, LPVPQK, VPLGTQ, VPYPQ, PLLQ, GPFP, LPVPQ, LPQYL, MPLW, YVPEPF, PQSVLS, PFP, LPVP, EMPFPK, LPLP, GPFPIIV, APFPE, HPIK and AFPPEVF are described, and dipeptidyl peptidase-IV inhibition test results of these isolated peptides are disclosed.

Special table 2008-527011 US Patent Application Publication No. 2008/0075904 JP 2011-144167 A JP 2007-039424 A JP 2008-280291 A International Publication 2006/068480 Pamphlet

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.
In addition, since peptides may have beneficial physiological activity, search for novel peptides and various physiological activity of peptides have been conducted. However, if the amino acids in the peptide increase or decrease or some of the amino acids are different, the physiological activity may be reduced or eliminated, and countless peptides even in the hydrolyzate of milk-derived proteins such as casein Therefore, it is difficult to find a new or known peptide having the desired physiological activity.
The present invention is to provide an excellent dipeptidyl peptidase-IV inhibitor.

  As a result of intensive studies to solve the above problems, the present inventor has found that Met- having a dipeptidyl peptidase-IV inhibitory action in a casein hydrolyzate obtained by hydrolyzing casein with a specific enzyme. A peptide having the sequence represented by Lys-Pro (SEQ ID NO: 1) (hereinafter also referred to as “peptide MKP”) was found, and the present invention was completed. Moreover, since this peptide MKP is a food-derived component, it can also be provided as a supplement or food additive. Therefore, it is considered that the peptide MKP of the present disclosure can contribute widely to the improvement of blood glucose level, and also has an ACE inhibitory action, and thus can be said to be an effective substance with more added value.

  That is, this invention is a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, or a vascular endothelial disorder inhibitor which contains the peptide which consists of Met-Lys-Pro as an active ingredient.

  Since the peptide MKP of the present invention has an excellent dipeptidyl peptidase-IV inhibitory action, the present invention can provide a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, a vascular endothelial disorder inhibitor, and the like.

The peptide of this indication has an amino acid sequence (sequence number 1) represented by Met-Lys-Pro. In the present disclosure, Met (M) represents an L-methionine residue, Lys (K) represents an L-lysine residue, and Pro (P) represents an L-proline residue.
The peptide 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.

Examples of the method for producing the peptide MKP of the present disclosure include the following methods as described in, for example, International Publication No. 2003/044044 pamphlet, but are not particularly limited thereto.
For example, a method of obtaining a protein or peptide containing the amino acid sequence represented by Met-Lys-Pro (SEQ ID NO: 1) by hydrolysis or the like, and separating and purifying from the obtained degradation product; A method of obtaining peptide MKP by separating and purifying peptide MKP from the obtained product after synthesis with peptide; Extract obtained from plants, animals and microorganisms that produce peptide MKP and peptides containing the peptide MKP And a method of separating and purifying from the above.

The peptide MKP of the present disclosure can be produced by, for example, appropriately hydrolyzing a protein such as casein with an acid alkali or an enzyme.
A method for obtaining the peptide MKP 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 MKP in its structure and can generate the peptide 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 sterilized by heating at 70 to 90 ° C. for about 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 peptide of the present disclosure, the degradation rate of the raw 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 for sufficient deactivation in consideration of the thermal stability of the enzyme used. It can be performed with a holding time of ˜2 seconds.

  The hydrolase is not particularly limited, but is preferably an enzyme capable of hydrolyzing the raw material protein to generate the peptide of the present disclosure, and specifically, the enzyme that can be generated 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 peptide MKP of the present disclosure from the hydrolyzate.
Purification of the peptide MKP of the present disclosure is usually performed in the same manner as used for 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 methods such as various types of chromatography, solvent precipitation, salting out, and partitioning between the two liquid phases.

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

The peptide MKP of the present disclosure can also be produced by chemical synthesis.
The chemical synthesis of the peptide MKP 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 peptide MKP of the present disclosure.
Such peptide synthesis can be performed using a commercially available peptide synthesizer. The fact that the target peptide was obtained can be confirmed by using the dipeptidyl peptidase-IV inhibitory action described later as an index.

The peptide MKP of the present disclosure has a dipeptidyl peptidase-IV inhibitory action as shown in Examples described later.
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 incretin glucagon-like peptide 1 (GLP-1) and glucose-dependent insulin secretion stimulating polypeptide (GIP). And by inhibiting the degradation of glucagon-like peptide 1 of this incretin, glucose-induced stimulation of insulin biosynthesis and secretion, glucagon secretion inhibition, regulation of gene expression, nutritional effects on sputum cells, suppression of food intake, In addition, it is possible to achieve various actions such as slowing of gastric emptying. Thereby, it is possible to contribute to normalization of the elevated blood glucose level and adjustment of hunger and 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, a large number of effects of improving endothelial cell function by administration of dipeptidyl peptidase-IV inhibitors have been reported (for example, see References 1 to 4 [Reference 1] Endocrine Journal 2011, 58 (1), 69-73. [Reference 2] J Am Coll Cardiol. 2012, 59 (3), 265-76; [reference 3] Diabetes Care. 2011, 34 (9), 2072-7; [reference 4] 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 vessel flexibility is lost due to injury of endothelial cells due to hyperglycemia, a vicious cycle in which blood pressure rises and the increased blood pressure further damages blood vessels appears as an adverse effect on organs such as the heart, kidney, and brain. In order to break this vicious cycle, a combination of a dipeptidyl peptidase-IV (DPP-4) inhibitor and an angiotensin converting enzyme (ACE inhibitor) has been tried, and an inhibitor of dipeptidyl peptidase-IV is used to treat the cardiovascular system. Are also considered to play an important role.

That is, since the peptide MKP of the present disclosure has a dipeptidyl peptidase-IV inhibitory action, it suppresses hyperglycemia, reduces blood sugar, suppresses vascular endothelial function, suppresses vascular endothelial damage, suppresses vascular damage, vascular endothelial cells Protective effect and appetite suppression effect. In addition, it is considered that the peptide MKP of the present disclosure is capable of suppressing appetite and protecting vascular endothelial cells.
Furthermore, by inhibiting dipeptidyl peptidase-IV with the peptide MKP of the present disclosure, it is considered possible to prevent, improve, or treat diseases and symptoms caused by dipeptidyl peptidase-IV. Therefore, the peptide MKP of the present disclosure can be used in a method for ingesting or administering to animals including humans to prevent, ameliorate, and / or treat diseases and symptoms caused by dipeptidyl peptidase-IV. it can.
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 1 to 4, It goes without saying that the disclosed peptide MKP can also be applied to similar diseases and symptoms because it has a dipeptidyl peptidase-IV inhibitory action.
Therefore, the peptide MKP of the present disclosure may be used for dipeptidyl peptidase-IV inhibition, hypoglycemia, vascular endothelial disorder suppression, anti-diabetes, etc. as described above, and dipeptidyl peptidase-IV inhibition It can be used in various preparations intended for use as described above, such as agents, hypoglycemic agents, vascular endothelial disorder inhibitors and antidiabetic agents, and can be used to produce these various preparations.

In addition, if the blood glucose level continues, the function of the endothelial cells of the blood vessels decreases and vascular endothelial damage tends 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.
For this reason, diabetic humans or their preparatory group (this preparatory group is a person who has not yet developed diabetes but has a high blood glucose level) needs to pay attention to hypertension. Humans are often prescribed hypoglycemic drugs and antihypertensive drugs, and there are also concerns about side effects due to the combination of drugs.

However, the peptide MKP of the present disclosure also has an angiotensin converting enzyme (hereinafter also referred to as “ACE”) inhibitory action and bradykinin inactivating action, as well as a blood pressure lowering action, as shown in Reference Example 1 in the Examples below. Currently, it has the strongest ACE inhibitory activity among peptides obtained from foods. (For example, refer to the pamphlet of International Publication No. 2003/044044).
Therefore, it can be said that the peptide MKP of the present disclosure is a very useful compound having two effects of a blood glucose lowering action and a blood pressure lowering action. Thereby, since it becomes possible to reduce the usage-amount of a chemical | medical agent, reduction of a side effect can be anticipated. That is, the peptide MKP of the present disclosure can be said to be very effective in improving blood vessels, suppressing vascular endothelial damage and preventing, improving or treating diabetes, and particularly in preventing, improving or treating vascular endothelial damage caused by hyperglycemic conditions. It is valid.
Specific diseases and symptoms of the vascular endothelial disorder of the present disclosure include, for example, diabetic vascular disorder.

Accordingly, the peptide MKP of the present disclosure can be ingested or administered to animals including humans to produce hyperglycemia, diabetes (particularly type 2 diabetes) and diabetic complications (eg, diabetic vascular endothelial disorder and nephropathy). , Hypertension, etc.), vascular endothelial disorder, vascular disorder and the like can be used in a method for preventing, improving and / or treating.
Further, the above-mentioned various preparations containing the peptide MKP of the present disclosure as an active ingredient are ingested or administered to animals including humans, and the above-mentioned hyperglycemia, diabetes (especially type 2 diabetes) and diabetic complications (for example, , Diabetic vascular endothelial disorder, nephropathy, hypertension, etc.), vascular endothelial disorder, vascular disorder and the like can be used in a method for preventing, improving and / or treating.
In addition, the peptide MKP of the present disclosure and the various preparations containing the peptide MKP as an active ingredient are for human or animal use for the prevention, improvement and / or treatment of hyperglycemia, diabetes, vascular disorders and the like as described above. It can be used as an active ingredient of pharmaceuticals, quasi-drugs, 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 foods, prevention, improvement or treatment of various diseases and symptoms caused by the above-mentioned dipeptidyl peptidase-IV and various diseases and symptoms caused by hyperglycemia, dipeptidyl peptidase-IV inhibition, It can be applied to functional foods based on the concept of physiological functions such as hypoglycemia, foods for the sick, and foods for specified health use.

The peptide MKP of the present disclosure and the above-described various preparations containing the peptide MKP as an active ingredient may be either oral administration or parenteral administration, 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 formulating, in addition to whey protein hydrolyzate and peptide MKP, components such as excipients, pH adjusters, colorants, corrigents and the like that are usually used for formulation can be used. It is also possible to use a known or future-found drug having DPP-4 inhibitory action, an antidiabetic drug, a hypoglycemic drug, a hypotensive drug, an ACE inhibitor and the like in combination.

Further, the peptide of the present disclosure is contained in a food as an active ingredient, and processed as a food having dipeptidyl peptidase-IV action as one embodiment of the peptide MKP of the present disclosure and the above-mentioned various preparations containing the peptide as an active ingredient. 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 peptide MKP of the present disclosure is preferably at least 0.001% by mass with respect to the final composition of the preparation.
The dose of the peptide MKP 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 divided into 1 to 3 times a day. It may be administered.

  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 MKP Peptide by Enzymatic Degradation of Casein <1> Enzymatic Degradation of Casein 900 g of water was added to 100 g of commercially available casein (manufactured by New Zealand Daily Board), dispersed well, and sodium hydroxide was added to adjust the pH of the solution. Was adjusted to 7.0, casein was completely dissolved, and an aqueous casein solution having a concentration of about 10% was prepared. 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 biopulase sp-20 (manufactured by Nagase Seikagaku Corporation) 100 , 800 active units (1,200 active units per gram of protein), protease N (manufactured by Amano Enzyme) 168,000 active units (2,000 active units per gram of protein), and PTN 6.0S (manufactured by Novozymes Japan) The hydrolysis reaction was initiated by adding 588,000 active units (7,000 active units per gram of protein). When the degradation rate of casein reached 24.1%, the enzyme reaction was stopped by heating at 80 ° C. for 6 minutes to deactivate the enzyme, and then cooled to 10 ° C. This hydrolyzed solution was ultrafiltered with an ultrafiltration membrane (manufactured by Asahi Kasei Co., Ltd.) having a molecular weight cutoff of 3,000, concentrated and freeze-dried to obtain 85 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: Capsule pack C18 (UG120, 5 μm) 20 mmI. D. × 250mm (Shiseido)
Detection: UV 215nm
Flow rate: 16 ml / min Eluent A: 1% acetonitrile aqueous solution containing 0.05% TFA Eluent B: 25% acetonitrile aqueous solution containing 0.05% TFA

The hydrolyzate was separated under a linear gradient condition such that the ratio of the eluent A was 100% and the ratio of the eluent B was 100% after 40 minutes. About the elution fraction, when the dipeptidyl peptidase-IV inhibitory ability was measured by the method mentioned later, the peptide having the dipeptidyl peptidase-IV inhibitory ability was eluted at a retention time of 22 minutes. Furthermore, in order to purify this peptide, it refine | purified further by HPLC.
The conditions at this time are shown in the following HPLC condition 2.
[HPLC condition 2]
Column: Capsule pack C18 (UG300, 5 μm) 2.0 mmI. D. × 250mm (Shiseido)
Detection: UV 215nm
Flow rate: 0.2 ml / min Eluent A: 1% acetonitrile aqueous solution containing 0.05% TFA Eluent B: 10% acetonitrile aqueous solution containing 0.05% TFA Elution rate of eluent A from 100% to 15 minutes later A strong dipeptidyl peptidase-IV inhibitory ability was observed at a peak at a retention time of 13 minutes under a linear gradient condition such that the ratio of solution B was 100%.

The compound having the above active peak was identified by a protein sequencer (Model-473A) manufactured by Applied Biosystem. As a result, it was found to have a structure of Met-Lys-Pro. Further, by mass spectrometry LCQ manufactured by Thermoquest, the molecular weight (M) was 374.2, and m / z = 260,215 by MS / MS analysis using m / z = 375.2 (MH +) as a precursor ion. , 129 and the like were detected.
Thus, it was revealed that the structure of the peptide having dipeptidyl peptidase-IV inhibitory ability was H-Met-Lys-Pro-OH. In addition, 42.5 mg of tripeptide Met-Lys-Pro was contained in 85 g of the lyophilized product.

Production Example 2: Chemical Synthesis of MKP Peptide Using a peptide synthesizer (Model 433A, Applied Biosystems), Fmoc-L-Met (Applied Biosystems), Fmoc-Lys (Boc) (Applied Biosystems), The tripeptide Met-Lys-Pro was synthesized by solid phase synthesis using Fmoc-Pro-TrtA-PEG Resin (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 tripeptide was measured by mass spectrometry to have a molecular weight (M) of 374.2, and by MS / MS analysis using m / z = 375.2 (MH +) as a precursor ion, H-Met-Lys-Pro It was found to be -OH.

Test Example 1: Dipeptidyl peptidase-IV inhibitory activity confirmation test of each peptide The DPP-4 inhibitory activity confirmation test of the tripeptide Met-Lys-Pro (MKP: SEQ ID NO: 1) obtained in Production Example 1 shown in Table 1 went. Further, tripeptide Val-Pro-Pro (hereinafter referred to as “VPP”: SEQ ID NO: 2), tripeptide Ile-Pro-Pro (hereinafter referred to as “IPP”: SEQ ID NO: 3), dipeptide Leu-Tyr (hereinafter referred to as “LY”). ": DPP-4 inhibitory activity confirmation test of SEQ ID NO: 4) was conducted. VPP, IPP, and LY were prepared according to the above-mentioned Production Example 2: MKP peptide chemical synthesis.

Reference Example 1: Angiotensin converting enzyme inhibitory activity confirmation test of each peptide In addition, an ACE inhibitory activity confirmation test of tripeptide MKP, tripeptide VPP, tripeptide IPP and dipeptide LY shown in Table 1 was conducted.

<Method for Measuring Dipeptidyl Peptidase-IV Inhibitory Activity>
Measurement of dipeptidyl peptidase-IV (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 (DPP-4) was performed using Recombinant Human DPPIV / CD26 (R & D Systems, Inc.) and the substrate was H-Gly-Pro-AMC (Biomol GmbH).
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>
Angiotensin converting enzyme inhibition (ACE inhibition) was measured according to the method of Dash Cushman et al. [DW Cushman, HS Cheung, Biochemical Pharmacology, Vol 20 (7), p1637-1648, 1971].
The sample was dissolved in 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3), 0.08 ml was placed in a test tube, and then 0.1 M borate buffer (containing 0.3 M NaCl). 0.2 ml of enzyme substrate (Hiprilhistidylleucine, Sigma) adjusted to 5 mM with pH 8.3) was added and incubated at 37 ° C. for 3 minutes. Subsequently, 0.02 ml of rabbit lung angiotensin converting enzyme (manufactured by Sigma) adjusted to 0.1 U / ml by adding distilled water was added and reacted at 37 ° C. for 30 minutes.
Thereafter, 0.25 ml of 1N hydrochloric acid was added to complete the reaction, 1.7 ml of ethyl acetate was added, and the mixture was vigorously stirred for 20 seconds, centrifuged at 3000 rpm for 10 minutes, and 1.4 ml of the ethyl acetate layer was collected. did. The obtained ethyl acetate layer was heated to remove the solvent, and then 1.0 ml of distilled water was added, and the absorption of the extracted hyprilic acid (absorbance at 228 nm) was measured to determine the enzyme activity.
Inhibitory activity was calculated from the following formula, and IC ₅₀ [sample concentration (μg / ml, or μM) necessary for inhibiting the activity of angiotensin converting enzyme by 50%] was determined.
Inhibition rate = (A−B) / (A−C) × 100%
A: Enzyme activity when sample (peptide) is not included (absorbance at 228 nm)
B: Enzyme activity when adding sample (absorbance at 228 nm)
C: Enzyme activity when no enzyme and sample are added (absorbance at 228 nm)

  Since the peptide MKP of the present invention can be isolated from a casein hydrolyzate, it is highly safe and can be used in a wide range of fields such as pharmaceuticals, foods, skin external preparations and functional foods. It is.

Further, the present technology can also take the following configurations.
[1] A dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, an antidiabetic agent, a vascular endothelial disorder inhibitor or a vascular ameliorating agent containing a peptide comprising Met-Lys-Pro as an active ingredient.
[2] Use of a peptide comprising Met-Lys-Pro for the production of a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, an antidiabetic agent, a vascular endothelial disorder inhibitor or a vascular ameliorating agent.
[3] Use of a peptide comprising Met-Lys-Pro for the production of a food for inhibiting dipeptidyl peptidase-IV, a food for lowering blood sugar, a food for antidiabetics, a food for suppressing vascular endothelial injury or a food for improving blood vessels.
[4] Use of a peptide comprising Met-Lys-Pro as a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, an antidiabetic agent, a vascular endothelial disorder inhibitor or a vascular ameliorating agent.
[5] Use of a peptide comprising Met-Lys-Pro in a food for inhibiting dipeptidyl peptidase-IV, a food for lowering blood sugar, a food for antidiabetics, a food for suppressing vascular endothelial injury or a food for improving blood vessels.
[6] From Met-Lys-Pro for prevention, amelioration, or treatment of diseases caused by dipeptidyl peptidase-IV, diseases caused by hyperglycemia, diabetes, or diseases caused by vascular endothelial or vascular disorders Peptide.
[7] Prevention of a disease caused by dipeptidyl peptidase-IV, a disease caused by a hyperglycemic state, diabetes or a vascular endothelial disorder or a vascular disorder, using a peptide comprising Met-Lys-Pro as an active ingredient, Improvement or treatment method.
[8] Met-Lys- for use in the prevention, amelioration, or treatment of a disease caused by dipeptidyl peptidase-IV, a disease caused by a hyperglycemic state, diabetes or a disease caused by vascular endothelial disorder or vascular disorder Peptide consisting of Pro.
[9] From Met-Lys-Pro for prevention, amelioration, or treatment of diseases caused by dipeptidyl peptidase-IV, diseases caused by hyperglycemia, diabetes, or diseases caused by vascular endothelial or vascular disorders Use of the peptide
[10] It is preferable that the disease and / or symptom caused by the dipeptidyl peptidase-IV is selected from hyperglycemia, diabetes, diabetic complications, vascular endothelial disorder and vascular disorder.
[11] A peptide comprising Met-Lys-Pro obtained by hydrolyzing casein and a method for producing the same.
[12] A peptide comprising Met-Lys-Pro obtained by separating and purifying casein in the following steps and a method for producing the same.
(A) Performing with a hydrolase (preferably endopeptidase). It is preferably carried out under conditions of 10 to 85 ° C. and 0.1 to 48 hours.
(B) Separating and purifying the hydrolyzate by chromatography. Preferably, one or two kinds selected from ion exchange chromatography, reverse phase chromatography, partition chromatography and the like are used.

Claims (10)

  A dipeptidyl peptidase-IV inhibitor containing a peptide consisting of Met-Lys-Pro as an active ingredient.   A hypoglycemic agent comprising a peptide comprising Met-Lys-Pro as an active ingredient.   A vascular endothelial disorder inhibitor comprising a peptide comprising Met-Lys-Pro as an active ingredient.   Use of a peptide comprising Met-Lys-Pro for the production of a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, an antidiabetic agent, a vascular endothelial disorder inhibitor or a vascular ameliorating agent.   Use of a peptide comprising Met-Lys-Pro for the production of a food for inhibiting dipeptidyl peptidase-IV, a food for lowering blood sugar, a food for anti-diabetes, a food for suppressing vascular endothelial injury or a food for improving blood vessels.   Use of a peptide comprising Met-Lys-Pro as a dipeptidyl peptidase-IV inhibitor, a hypoglycemic agent, an antidiabetic agent, a vascular endothelial disorder inhibitor or a vascular ameliorating agent.   Use of a peptide comprising Met-Lys-Pro as a food for inhibiting dipeptidyl peptidase-IV, a food for lowering blood sugar, a food for anti-diabetics, a food for suppressing vascular endothelial injury or a food for improving blood vessels.   Consists of Met-Lys-Pro for use in prevention, amelioration or treatment of diseases caused by dipeptidyl peptidase-IV, diseases caused by hyperglycemic conditions, diabetes or diseases caused by vascular endothelial or vascular disorders peptide.   A peptide comprising Met-Lys-Pro for the prevention, amelioration or treatment of a disease caused by dipeptidyl peptidase-IV, a disease caused by a hyperglycemic state, diabetes or a disease caused by vascular endothelial disorder or vascular disorder use.   Prevention, amelioration or treatment of diseases caused by dipeptidyl peptidase-IV, diseases caused by hyperglycemia, diabetes, or diseases caused by vascular endothelial or vascular disorders, using a peptide comprising Met-Lys-Pro as an active ingredient Method.