JP4915833B2 - Dipeptidyl peptidase IV inhibitor - Google Patents

Description

The present invention relates to a novel dipeptidyl peptidase used as a therapeutic compound in the treatment of type 2 diabetes called non-insulin dependent diabetes mellitus (NIDDM) and diseases associated with this disease (eg obesity and lipid disorders). It relates to IV (DPPIV) inhibitors. Moreover, this invention relates to the peptide which has DPPIV inhibitory activity which exists in the water-soluble fraction of cheese. The DPPIV inhibitor of the present invention is characterized in that the peptide present in the water-soluble fraction of cheese or the water-soluble fraction of cheese is an active ingredient, and the blood glucose level is lowered by acting antagonistically on DPPIV.

Diabetes is a disease that is induced by multiple causes and is characterized by high plasma glucose levels following glucose administration during fasting or during an oral glucose tolerance test, ie, hyperglycemia. If hyperglycemia persists and blood glucose levels are not normally controlled, the morbidity and mortality of other metabolic and hemodynamic diseases increases. Abnormal glucose homeostasis alters lipid, lipoprotein and apolipoprotein metabolism and develops other metabolic and hemodynamic diseases. Thus, diabetic patients are particularly at risk of developing macrovascular and microvascular complications including coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy and retinopathy. Therefore, it is important to control glucose homeostasis, lipid metabolism and hypertension when clinically managing and treating diabetes.

It is recognized that there are two types of diabetes. In type 1 diabetes, or insulin-dependent diabetes (IDDM), patients produce little to no insulin, a hormone that regulates glucose utilization. In type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM), the patient's plasma insulin levels are often the same as or higher than those who are not diabetic. However, these patients are resistant to insulin-stimulating effects on glucose and lipid metabolism in the main insulin-sensitive tissues muscle, liver and adipose tissue, with high plasma insulin levels but significant insulin resistance Is not enough to solve.

Insulin resistance is said to be mainly due to a post-insulin receptor binding defect, not a decrease in the number of insulin receptors. When resistance to this insulin responsiveness occurs, insulin activation of glucose uptake, oxidation and storage in muscle is insufficient, and insulin suppression of lipolysis in adipose tissue and glucose production and secretion in the liver is insufficient. .
Currently, treatment for type 2 diabetes has not changed substantially over the years, but there are limitations. Although exercise and dietary caloric restriction improves the diabetic state, compliance with this treatment is very low. This is because an inactive lifestyle has been established, and foods, particularly foods rich in saturated fat, are consumed excessively. By administering sulfonylureas (eg, tolbutamide and glipizide) or meglitinides that stimulate pancreatic beta cells to secrete more insulin, the insulin concentration is high enough to stimulate highly insulin resistant tissues. It becomes. When sulfonylurea or meglitinide is not effective, insulin is increased by injecting insulin. However, administration of insulin or insulin secretagogues (sulfonylurea and meglitinide) can result in very low plasma glucose levels, and high insulin levels can increase insulin resistance. Biguanides increase insulin sensitivity and slightly improve hyperlipidemia. However, the two biguanides phenformin and metformin can induce lactic acidosis, nausea and diarrhea, respectively.

Insulin secreted from pancreatic β cells plays an important role in glucose homeostasis, as it is the only hormone that lowers blood sugar and is involved in the regulation of glucose metabolism in the body. Incretin is thought to be involved in insulin secretion. Incretin is a collective term for gastrointestinal hormones, and information that food has been ingested is quickly transmitted to the pancreatic β-cells via incretin secretion, stimulating insulin secretion and suppressing postprandial blood glucose levels And has a role of keeping blood sugar constant. So far, two hormones of gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are known as incretins. Therefore, by the action of incretin, insulin secretion can be efficiently increased early after meals when blood sugar begins to rise, and postprandial hyperglycemia can be suppressed. In patients with type 2 diabetes, postprandial hyperglycemia is one of the characteristics, and incretin action disorder has been reported as one factor.

DPPIV present in the blood is an enzyme that specifically acts on a physiologically active peptide having the second proline or alanine from the N-terminus and releases the dipeptide from the N-terminus, and is used for the degradation of various hormones and chemokines. It is known to be involved. GIP and GLP-1 are also rapidly degraded by DPPIV in the blood and lose their activity. This is a test using DPPIV-deficient mice, in which glucose tolerance was deteriorated in the oral glucose tolerance test (OGTT), and DPPIV affected in vivo glucose metabolism through degradation of GLP-1 and GIP. (For example, refer nonpatent literature 1).
Therefore, the blood glucose level can be lowered by inhibiting the incretin degradation activity by DPPIV in the blood.

As DPPIV inhibitors, many chemical compounds have been reported. For example, there are fluoropyridines, pyrazines, sulfostins, and the like, and some of them have already been put into practical use as oral agents (for example, see Patent Documents 1, 2, 3, and 4). .) However, these chemically synthesized products must always pay attention to safety issues.
Only natural natto extract has been reported to have DPPIV inhibitory activity (see, for example, Non-Patent Document 2).

Physiological actions such as calcium absorption promoting action, cell proliferation action, and antibacterial action have been clarified for the water-soluble fraction of cheese. However, there is no report that the water-soluble fraction of cheese has DPPIV inhibitory activity. Moreover, regarding the physiological function of cheese, although antitumor action, antimutagenic action, etc. are reported, it is not known about having DPPIV inhibitory activity. Thus, there is no report that the water-soluble fraction of cheese has DPPIV inhibitory activity.
Special table 2005-500321 gazette Special table 2004-535455 gazette Special Table 2004-536115 Publication JP 2000-327689 Scrocchi, LA et al., Nat.med., 2: 1254-1258, 1996 Mitsuru Ichinose Laboratory, 2002 graduation thesis, Prefectural Siebold University, search for enzyme inhibitors related to type 2 diabetes, DPPIV inhibitory activity found in natto extract

It is an object of the present invention to provide a DPPIV inhibitor obtained from a natural product, having low toxicity and high safety, having a remarkable DPPIV inhibitory activity, and a method for producing the same. Another object of the present invention is to provide a DPPIV inhibitor that can be used for pharmaceuticals, foods for specified health use, health foods, and the like.

As a result of intensive studies to solve the above problems, the present inventors have found that the water-soluble fraction obtained by suspending cheese in an aqueous solvent and then removing insoluble substances has significant DPPIV inhibitory activity. It was found that a substance having Further, the OASIS elution fraction obtained by treating the water-soluble fraction with an OASIS column has DPPIV inhibitory activity, and the inhibitory activity tends to increase as the ripening period of cheese increases. Furthermore, OASIS elution fraction of Gouda type 12-month-aged cheese with high activity was fractionated by HPLC and the activity of the fraction was measured. As a result, there were several fractions that were particularly active. The active ingredient was fractionated. This highly active fraction was analyzed by MS, and the presence of casein (α, β) -derived peptide was confirmed. As a result of synthesizing several confirmed peptides and determining their IC50s, they found that they were 1/8 to 1/500 times more active than Diprotin A, a commercially available inhibitor.
That is, this invention relates to the DPPIV inhibitor which uses the water-soluble fraction of cheese as an active ingredient.
Moreover, this invention relates to the DPPIV inhibitor which uses the water-soluble fraction of ripening natural cheese as an active ingredient.
The present invention also relates to a peptide having any of the following sequences having DPPIV inhibitory activity.
(1) Ile-Pro-Asn
(2) Gly-Pro-Ile-Pro-Asn
(3) Val-Pro-Ile-Thr-Pro-Thr
(4) Val-Pro-Gly-Glu-Ile-Val-Glu
(5) Leu-Pro-Gln-Asn-Ile-Pro-Pro
(6) Thr-Pro-Val-Val-Val-Pro-Pro
(7) Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro
(8) Val-Ala-Pro-Phe-Pro-Glu
(9) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(10) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser
(11) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn
(12) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val
(13) Met-Pro-Phe-Pro-Lys-Tyr
(14) Phe-Pro-Gly-Pro-Ile-Pro-Asn
(15) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(16) Val-Pro-Ile-Thr-Pro-Thr-Leu
(17) Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn
(18) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(19) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu
(20) Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
(21) Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
(22) Thr-Pro-Val-Val-Val-Pro-Pro-Phe
(23) Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp
(24) Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
Moreover, this invention relates to the peptide which consists of one of the said sequences which has DPPIV inhibitory activity which exists in the water-soluble fraction of cheese.
The present invention also relates to a DPPIV inhibitor comprising as an active ingredient any one or more of the aforementioned peptides having DPPIV inhibitory activity.

The water-soluble fraction of cheese of the present invention or the peptide having DPPIV inhibitory activity present in the water-soluble fraction of cheese can be used as a hyperglycemia inhibitor having DPPIV inhibitory activity, etc., which is highly safe and has no side effects. It is a fraction and can be expected to be used for various pharmaceuticals, special health foods, functional foods, etc. The DPPIV inhibitor of the present invention can be obtained by a simple production method in which the water-soluble fraction of cheese obtained by suspending cheese in an aqueous solvent and then removing insoluble substances is used as it is, Furthermore, cheese as a raw material has an advantage that it can be easily obtained at low cost, and is extremely useful in practical use.

The DPPIV inhibitor of the present invention contains, as an active ingredient, a water-soluble fraction of cheese or a peptide having DPPIV-inhibiting activity present in the water-soluble fraction of cheese. More preferably, the water-soluble fraction of cheese is a water-soluble fraction of ripened natural cheese.
The water-soluble fraction of cheese having DPPIV inhibitory activity of the present invention can be obtained, for example, by suspending cheese in an aqueous solvent and then removing insoluble substances. Suspending cheese in an aqueous solvent in the water-soluble fraction of the cheese of the present invention means adding an aqueous solvent to the cheese and homogenizing it with a homogenizer or crushing it in an aqueous solvent, To make it easy to get the minutes. The supernatant obtained by suspending cheese in an aqueous solvent and then centrifuging, or the supernatant further filtered to remove insoluble substances is the water-soluble fraction of the cheese of the present invention. A permeate obtained by passing this water-soluble fraction through ultrafiltration may be used. Further, the water-soluble fraction may be desalted with a dialysis membrane, an ion exchange resin, or the like, or may be a fraction obtained by eluting the fraction adsorbed through an OASIS column or the like. In addition, the water-soluble fraction of cheese having DPPIV inhibitory activity is separated into a fraction containing a large amount of peptide consisting of any one of the amino acid sequences represented by formulas (1) to (24) using a C18 column. It is also possible to draw.
A peptide having DPPIV inhibitory activity consisting of any one of the amino acid sequences represented by the sequence formulas (1) to (24) that can be used in the present invention uses, for example, the above water-soluble fraction using a C18 column. Can be obtained by fractionation. Furthermore, the peptide obtained by synthesis can also be used. As an aqueous solvent, solvents such as water and phosphate buffer can be used. The water-soluble fraction may be pulverized by drying by freeze drying or spray drying.

The DPPIV inhibitory activity present in the water-soluble fraction of cheese having DPPIV inhibitory activity of the present invention or the water-soluble fraction of cheese comprising any one of the amino acid sequences represented by the sequence formulas (1) to (24) Natural cheese and processed cheese made from natural cheese can be used as the cheese raw material for obtaining the peptide having, but Parmesan cheese, Gruyère cheese, Maribo cheese, Gouda cheese, cheddar cheese, Emmental cheese, Edam cheese, Camembert It is desirable to use aged cheeses such as cheese, brie cheese, manstail cheese, pon levec cheese, stilton cheese, dana blue cheese, blue cheese, and processed cheese made from these aged natural cheeses. Ripening period It is more desirable to use a long ripened natural cheese.
Thus, since the DPPIV inhibitor of this invention uses cheese as a raw material, it has the advantage that acquisition of a raw material is very easy and it is cheap. In addition, cheese is used as a raw material and only an aqueous solvent is used, which is extremely advantageous in terms of safety and toxicity.

The DPPIV inhibitor of the present invention can utilize the water-soluble fraction of cheese obtained by suspending cheese in an aqueous solvent and then removing insoluble substances by degreasing, centrifugation, filtration, etc. Those obtained by desalting with a dialysis membrane or an ion exchange resin or the like, and further powdered by drying by freeze drying or spray drying can be used.
Moreover, the fraction which has DPPIV inhibitory activity of this invention can be mix | blended with food-drinks, and can be used as food-drinks for DPPIV inhibition. When blended in food or drink, the water-soluble fraction of cheese can be blended as it is, desalted with a dialysis membrane, ion exchange resin, etc., and further dried by freeze drying, spray drying, etc. Powdered products can also be blended.
Thus, the DPPIV inhibitor of the present invention uses the water-soluble fraction of cheese obtained by suspending cheese in an aqueous solvent and then removing insoluble substances by degreasing, centrifugation, filtration, etc. Since it can be obtained by a simple production method, it is extremely useful in practical use. Furthermore, there is an advantage that cheese as a raw material can be easily obtained.

The DPPIV inhibitor of the present invention can be orally administered to inhibit blood glucose level by inhibiting DPPIV in a living body. When administered orally, examples of the dosage form of the DPPIV inhibitor of the present invention include tablets, capsules, fine granules, powders, pills, troches, sublingual or liquid preparations. Can do.

The oral dose of the DPPIV inhibitor of the present invention may be appropriately determined in consideration of the purpose of treatment and prevention, symptoms, body weight, age, sex, etc., but usually the water soluble fraction of cheese per adult day When administered as a solid content of 50 mg to 100 g, a therapeutic or prophylactic effect inhibiting DPPIV can be obtained.

By ingesting the DPPIV inhibitor of the present invention, or a food or drink containing them, the effect of lowering blood glucose level in vivo is exhibited. Examples of the food and drink containing the DPPIV inhibitor of the present invention include cheese, butter, milk drink, juice, yogurt, jelly, bread, ice cream, noodle, sausage, infant formula and baby food.

The activity of DPPIV can be measured using, for example, the -Glo ^™ protease method. DPPIV-Glo ^™ Reagent (Promega) is used as the substrate. In this method, the activity of DPPIV is measured by adding a test sample to DPPIV, adding a substrate solution, mixing, and measuring the amount of luminescence after a certain time. Thus, when DPPIV-Glo ^™ Reagent is added by the operation of addition-mixing-measurement, a luminescence signal is generated by luciferase reaction following the cleavage of the substrate by DPPIV. Since the signal generated in the homogeneous type assay due to the coupling of the enzyme reaction is proportional to the amount of DPPIV activity, it is possible to measure the activity of DPPIV.

EXAMPLES The present invention will be described in more detail below with reference to examples and test examples, but these are merely illustrative and the present invention is not limited by these.

(Preparation of water-soluble fraction of cheese)
Lacticococcus lactis subsp. Gouda type cheese was used as a raw material. This cheese is cut into fine pieces, 10 times the amount of water is added, pulverized (10,000 rpm, 10 minutes) with a homogenizer (AM-3: Nippon Seiki Seisakusho), suspended, and then centrifuged (6,000 rpm, 10 The supernatant from which the precipitate was removed at 4 ° C for 4 minutes was further filtered (No. 2 filter paper: ADVANTEC) to remove insoluble substances, followed by ultrafiltration (Centriplus YM-30: Millipore) to obtain a permeate, This was made into the water-soluble fraction of cheese. The water-soluble fraction of cheese thus obtained can be used as it is as a DPPIV inhibitor.

(Preparation of water-soluble fraction of cheese)
A water-soluble image of cheese in the same manner as in Example 1 except that Italian type cheese produced by a conventional method using Streptococcus thermophilus and Lactobacillus helveticus as a lactic acid bacteria starter was used as a raw material. Minutes were prepared. The water-soluble fraction of cheese thus obtained can be used as it is as a DPPIV inhibitor.

[Test Example 1]
(Measurement of DPPIV activity)
About each water-soluble fraction of the Gouda type cheese obtained in Example 1, and the Italian type cheese obtained in Example 2, the DPPIV inhibitory activity was measured by the DPPIV-Glo ^™ protease method. Gouda-type cheese was prepared as described in Example 1 for each of the water-soluble fractions that had been ripened immediately after production, for 1, 2, 6, and 12 months, and used for the test. DPPIV-Glo ^™ Reagent (G8350; Promega) was used as a substrate, DPPIV (317630, derived from human placenta; Calbiochem) was used as an enzyme, and diprotin A (Diprotin A, 4132-v; Peptide) was used as a control inhibitor. That is, add 1 μl of DPPIV (10 μU / μl) and 6 μl of the Gouda-type cheese water-soluble fraction to 100 μl, 96-well black plate, make up to 50 μl with ultrapure water, and add 50 μl of substrate solution. And mixed. The amount of luminescence was measured after 30 minutes. The relative activity when the sample was added was calculated by setting the activity (luminescence amount) of the sample not added to 100%.
In addition, Italian type cheese is prepared as described in Example 2 for each of water-soluble fractions that have been ripened immediately after production, for 1, 2, 6, and 12 months, and is similar to the water-soluble fraction of Gouda-type cheese. The test was conducted.
The results are shown in FIG.

It was confirmed that both the water-soluble fraction of Gouda-type cheese and the water-soluble fraction of Italian-type cheese have an effect of inhibiting DPPIV activity.
Moreover, the inhibitory effect became stronger as the ripening of each cheese progressed, and especially the water-soluble fraction of cheese with a ripening period of 6 months or more showed a remarkable inhibitory effect on DPPIV activity. This is considered to be an active ingredient is a peptide produced by aging. Thus, the water-soluble fraction of cheese can be used as a DPPIV inhibitor as it is.

(Preparation of water-soluble fraction of cheese)
The same cheese as the gouda type cheese of Example 1 and the Italian type cheese of Example 2 was used as a raw material. After cutting these cheeses finely, adding 10 times the amount of water, pulverizing (10,000 rpm, 10 minutes) with a homogenizer (AM-3: Nippon Seiki Seisakusho Co., Ltd.) and suspending, then stirring with a stirrer 40 Heating treatment was performed for 1 hour in a constant temperature bath at ℃. The supernatant after removing the precipitate by centrifugation (6,000 rpm, 10 minutes, 4 ° C.) is further filtered (No. 2 filter paper: ADVANTEC) to remove insoluble substances, and then ultrafiltered (Centriplus YM-30: Millipore ) To obtain a permeate.
This permeate was applied to an OASIS column (HLB 6cc: Waters) equilibrated with distilled water, washed with distilled water, and the adsorbed fraction was eluted with 100% methanol. The recovered eluate was dried with a centrifugal evaporator (CVE-200D: EYELA), and then dissolved in 1/10 amount of distilled water supplied to the OASIS column.
The OASIS-eluted fraction thus obtained can be used as the water-soluble fraction of cheese as it is as the DPPIV inhibitor of the present invention.

[Test Example 2]
(Measurement of DPPIV activity)
With respect to the water-soluble fraction of Gouda type cheese and Italian type cheese obtained in Example 3, DPPIV inhibitory activity was measured using a DPPIV-Glo ^™ protease kit (G8350, Promega). Gouda-type cheese and Italian-type cheese were prepared as described in Example 3 for each water-soluble fraction after ripening for 1, 2, 4, 6 and 12 months immediately after production and subjected to the test. DPPIV-Glo ^™ Reagent (Promega) was used as a substrate, and DPPIV (317630, derived from human placenta; Calbiochem) was used as an enzyme. That is, 10 μl of the Gouda type cheese or Italian type cheese water-soluble fraction was added to a 96-well black plate, and 50 μl of the substrate solution was added and mixed. Furthermore, 39 μl of ultrapure water and 1 μl of DPPIV (10 μU / μl) were added to start the reaction, and the amount of luminescence was measured after 30 minutes. The relative activity when the sample was added was calculated with the amount of luminescence of the sample not added as 100%. The measurement results of DPPIV inhibitory activity are shown in FIG.

(Change in activity due to increased aging period)
It was confirmed that any water-soluble fraction of Gouda-type cheese or Italian-type cheese has an effect of inhibiting DPPIV activity. Moreover, the inhibitory effect became stronger as the ripening of each cheese progressed, and in particular, the gouda-type 12-month-aged cheese showed a very remarkable effect of inhibiting DPPIV activity. This is considered to be an active ingredient is a peptide produced by aging.

(Fractionation by reverse phase HPLC and measurement of DPPIV inhibitory activity)
In order to identify the inhibitory active ingredient, the OASIS elution fraction of Gouda type 12-month-aged cheese having a high inhibitory activity was first fractionated by reverse phase HPLC. A water soluble fraction of Gouda type 12 month ripened cheese was prepared as described in Example 3 and subjected to testing. For the fractionation, an HPLC system (HP 1050; Hewlett Packard) was used, and a PROTEIN & PEPTIDE C18 column (4.6 mm ID × 250 mm: VYDAC) was used at 30 ° C. Elution was performed at a gradient of 5 → 65% acetonitrile (0.1% TFA) / 60 minutes, a flow rate of 1 ml / minute, and detected at UV 220 nm. The change in absorbance at 220 nm is shown in FIG.
After injecting 100 μl of OASIS elution fraction, the eluate was collected at intervals of 1 minute for 3 to 43 minutes. Each was dried with a centrifugal evaporator (CVE-200D: EYELA), then dissolved in 10 μl of distilled water or 1 μl of DMSO (048-21985: Wako Pure Chemical Industries), and the total amount was added per well to add each DPPIV Inhibitory activity was determined. The change in DPPIV inhibitory activity of the HPLC fraction is shown in FIG.
As a result, a broad inhibitory activity was observed, but the activity was particularly strong in some fractions. It is also possible to fractionate the active ingredient in this way.

[Test Example 3]
(Analysis of active fraction by MS)
Fractions with high activity were analyzed by mass spectrum (MS). Each fraction fractionated by HPLC was appropriately diluted with a buffer, and then subjected to MS analysis by LCQ Advantage MSsystem. MS / MS analysis was performed with Xcalibur 1.3 & Bioworks 3.1. For the database, only protein sequences derived from Bos taurus were extracted from nr.fasta (NCBI) and used.
Table 1 shows the identified casein-derived peptides as a result of MS analysis of the fraction obtained by HPLC fractionation of the water-soluble fraction of Gouda type 12-month ripened cheese.
As a result, it was shown that there were many casein-derived peptides in each fraction, and these identified peptides are considered to be involved in DPPIV inhibitory activity.

[Table 1]
───────────────────────────────────
Flax amino acid sequence casein position (min)
───────────────────────────────────
6 Ile-Pro-Asn β 66-68
───────────────────────────────────
10 Gly-Pro-Ile-Pro-Asn β 64-68
───────────────────────────────────
12 Val-Pro-Ile-Thr-Pro-Thr αS2 117-122
───────────────────────────────────
15 Val-Pro-Gly-Glu-Ile-Val-Glu β 8-14
Leu-Pro-Gln-Asn-Ile-Pro-Pro β 70-86
───────────────────────────────────
17 Thr-Pro-Val-Val-Val-Pro-Pro β 80-86
Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro β 178-186
───────────────────────────────────
18 Val-Ala-Pro-Phe-Pro-Glu αS1 25-30
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro β 74-81
───────────────────────────────────
21 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser β 60-69
Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn β 60-68
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val β 74-82
Met-Pro-Phe-Pro-Lys-Tyr β 109-114
───────────────────────────────────
22 Phe-Pro-Gly-Pro-Ile-Pro-Asn β 62-68
Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-P β 70-84
ro
Val-Pro-Ile-Thr-Pro-Thr-Leu αS2 117-123
───────────────────────────────────
23 Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn αS1 106-114
───────────────────────────────────
4 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn β 60-68
Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu β 70-78
Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu β 84-91
───────────────────────────────────
25 Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys αS1 26-34
───────────────────────────────────
27 Thr-Pro-Val-Val-Val-Pro-Pro-Phe β 80-87
───────────────────────────────────
29 Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp αS2 102-109
───────────────────────────────────
30 Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-G β 80-91
lu
───────────────────────────────────

(Peptide synthesis and activity measurement)
Some of the peptides estimated by MS were synthesized and dissolved in distilled water or DMSO (048-21985: Wako Pure Chemical Industries) to determine their DPPIV inhibitory activity. First, serially dilute each peptide solution to determine the inhibitory activity of each peptide, and calculate the 50% inhibitory concentration (IC50) of each sample by calculating back from the relational expression of inhibitory activity (%) and logarithm of sample concentration (Log10). It was. The inhibitory activity of Diprotin A (4132-v; Peptide) was also measured as a control inhibitor.
DPPIV inhibitory activities of the peptides synthesized as a result of MS analysis were compared. The results are shown in Table 2.
As a result, there was a difference in the inhibitory activity depending on the peptide. In particular, the peptide with the sequence of Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu (# 9) has a high inhibitory activity of IC50 = 45.6μM. Indicated. It is thought that each fraction shows inhibitory activity by containing these components.

[Test Example 4]
(Accumulation of active peptide by aging period)
For some of the peptides that showed DPPIV inhibitory activity, in order to examine the amount change during cheese ripening, the cheese water-soluble fraction during each ripening period was appropriately diluted, and then subjected to MS analysis by LCQ Advantage MSsystem. MS / MS analysis was performed with Xcalibur 1.3 & Bioworks 3.1. For the database, only protein sequences derived from Bos taurus were extracted from nr.fasta (NCBI) and used. A signal corresponding to each peptide was extracted from the obtained data, and the intensity of the signal was compared for each aging period.
As a result, it was shown that these peptides increased with aging. From the above, some peptides with inhibitory activity were generated and increased with aging, and the total activity in the cheese supernatant It was shown that the inhibitory activity of was increased.

It is the figure which showed the effect | action which inhibits DPPIV activity of the water-soluble fraction of Gouda type cheese and Italian type cheese from which a ripening period differs. (Test Example 1) It is the figure which showed the effect | action which inhibits DPPIV activity of the water-soluble fraction of Gouda type cheese and Italian type cheese from which a ripening period differs. (Test Example 2) It is the figure which showed the light absorbency at the time of analyzing the water-soluble fraction of Gouda type 12-month ripening cheese by reverse phase HPLC. (Example 4) It is the figure which showed the DPPIV inhibitory activity of the fraction fractionated by the 1-minute space | interval at the time of analyzing the water-soluble fraction of Gouda type 12-month ripening cheese by reverse phase HPLC. (Example 4)

Claims (3)

A dipeptidyl peptidase IV inhibitor comprising as an active ingredient a water-soluble fraction of cheese obtained by a purification method comprising at least the following steps;
Step (1) A gouda-type cheese produced using a starter selected from Lactococcus lactis subspices lactis, Lactococcus lactis subspices cremolith and Leuconostok mesenteroides and aged for 6 months or more is an aqueous solvent The step of suspending in,
Step (2) centrifuging the suspension obtained in step 1 to obtain a supernatant;
Step (3) A step of removing insoluble substances from the supernatant obtained in Step 2 to obtain a water-soluble fraction. A dipeptidyl peptidase IV inhibitor comprising as an active ingredient a water-soluble fraction of cheese obtained by a purification method comprising at least the following steps;
Step (1) suspending Gouda type cheese produced using a starter selected from Lactococcus lactis subspices lactis, Lactococcus lactis subspices cremolith, Leuconostoc mesenteroides in an aqueous solvent,
Step (2) heating the suspension obtained in step 1 at 40 ° C .;
Step (3) centrifuging the suspension obtained in step 2 to obtain a supernatant;
Step (4) removing insoluble substances from the supernatant obtained in step 3 to obtain a water-soluble fraction;
Step (5) A step of subjecting the water-soluble fraction obtained in Step 4 to an OASIS column. A dipeptidyl peptidase IV inhibitor comprising any one or more of the following peptides as an active ingredient.
(1) Ile-Pro-Asn
(2) Gly-Pro-Ile-Pro-Asn
(3) Val-Pro-Ile-Thr-Pro-Thr
(4) Val-Pro-Gly-Glu-Ile-Val-Glu
(5) Leu-Pro-Gln-Asn-Ile-Pro-Pro
(6) Thr-Pro-Val-Val-Val-Pro-Pro
(7) Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro
(8) Val-Ala-Pro-Phe-Pro-Glu
(9) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(10) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser
(11) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn
(12) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val
(13) Met-Pro-Phe-Pro-Lys-Tyr
(14) Phe-Pro-Gly-Pro-Ile-Pro-Asn
(15) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(16) Val-Pro-Ile-Thr-Pro-Thr-Leu
(17) Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn
(18) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(19) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu
(20) Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
(21) Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
(22) Thr-Pro-Val-Val-Val-Pro-Pro-Phe
(23) Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp
(24) Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu

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