JP2009167127A - Pulse wave propagation velocity depressant and application thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicine usable for treatment and prevention of arteriosclerosis, and having excellent safety. <P>SOLUTION: The pulse wave propagation velocity (PWV) depressant is a composition containing at least one dipeptide selected from the group consisting of Val-Tyr, Val-Trp, Tyr-Trp and Ile-Tyr. The dipeptide can be prepared, for example, by treating lees of Sake (rice wine) with a protease. The increased PWV can be decreased by such the PWV depressant, and therefore the PWV depressant is useful as a treating agent of the arteriosclerosis or a preventive agent of the arteriosclerosis. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a pulse wave velocity reducing agent and use thereof.

  Arteriosclerosis refers to a state in which the artery has thickened and hardened, and generally has a pulse wave velocity (PWV) indicating blood vessel hardening as an index. Arteriosclerosis is known to cause various diseases such as ischemic heart disease such as angina pectoris and myocardial infarction, stroke, cerebral infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc. The relationship with cerebrovascular disorder etc. has been reported. In particular, when arteriosclerosis occurs in the coronary artery that plays a role in supplying oxygen to the myocardium, stenosis and occlusion occur, which may cause cardiovascular diseases such as angina pectoris and myocardial infarction. In order to prevent these diseases at an early stage, treatment or prevention of arteriosclerosis is very important in medicine.

On the other hand, pharmaceuticals generally have many artificially designed organic compounds that do not exist in nature (see Patent Document 1). However, due to problems such as side effects, provision of pharmaceuticals having excellent safety to the human body is required (see Patent Document 2 and Patent Document 3).
JP 2004-51639 A JP 2007-31364 A JP 2007-320956 A

  Then, this invention aims at provision of the new chemical | medical agent excellent in safety | security which can be used for treatment and prevention of arteriosclerosis.

  To achieve the above object, the pulse wave velocity lowering agent of the present invention (hereinafter referred to as “PWV lowering agent”) is selected from the group consisting of Val-Tyr, Val-Trp, Tyr-Trp and Ile-Tyr. And at least one dipeptide.

  The therapeutic agent for arteriosclerosis of the present invention or the preventive agent for arteriosclerosis of the present invention comprises the PWV lowering agent of the present invention.

  As a result of intensive studies, the present inventors are able to lower PWV, which is an index of arteriosclerosis, according to at least one dipeptide of four types of Val-Tyr, Val-Trp, Tyr-Trp, and Ile-Tyr. The present invention was found. According to the PWV lowering agent of the present invention containing such a dipeptide, for example, it is possible to lower an elevated PWV or suppress an increase in PWV, and thus is extremely useful as a prophylactic or therapeutic agent for arteriosclerosis. In addition, the dipeptide in the present invention also exists in nature, and for example, it has been confirmed by the present inventors that it is contained in sake lees and the like. And since sake lees have a long history of food in Japan, it can be said that the safety of the various dipeptides described above has been sufficiently proven. Therefore, the PWV lowering agent, arteriosclerosis therapeutic agent and prophylactic agent of the present invention are useful as safe drugs. In addition, the dipeptide in this invention is not restrict | limited at all to the thing derived from sake lees, so that it may mention later.

  As described above, the PWV lowering agent of the present invention comprises at least one dipeptide selected from the group consisting of Val-Tyr, Val-Trp, Tyr-Trp, and Ile-Tyr.

  The PWV lowering agent of this invention should just contain the above-mentioned dipeptide, and another structure is not restrict | limited at all. The dipeptide contained in the PWV lowering agent of the present invention may be, for example, any one of four types, or two or more types. Also, the combination is not limited at all.

  The dipeptide in the present invention may be prepared from a raw material such as a natural product, a processed product thereof, or a brewed product thereof as described later, or may be synthesized by a conventionally known synthesis method. Examples of the synthesis method include a peptide synthesis method using a reverse reaction of a hydrolase, a peptide synthesis method using an organic chemical method, a synthesis method using a genetic engineering method, and the like.

  Examples of the preparation method from the raw material include purification from the raw material itself or a decomposition product of the raw material. The method for decomposing the raw material is not particularly limited, and can be performed, for example, with an acid, an alkali, an enzyme, or the like. In addition, as the purification method, for example, a conventionally known separation and purification method utilizing a difference in adsorption affinity for the adsorbent, a difference in solubility in various solvents, a difference in elution rate due to the molecular sieve effect, and the like can be employed. Moreover, extraction and concentration can also be combined suitably.

  The raw material is not particularly limited, and examples thereof include processed products and brewed products such as cereals such as rice and beans, meats, seaweeds, and milk. Processed rice products include, for example, rice cake flour, shin flour, white ball flour, vine flour, Domyoji flour, rice noodles, rice bran, α rice, rice crackers obtained by further processing these, and pon confectionery. Examples of brewed rice ingredients include sake, miso, miso, soy sauce, and by-products such as koji and koji that are produced during the production process. Of these, sake sake, which is a by-product of sake, is preferred, and examples thereof include general sake lees and liquefied liquors. The liquefied liquor is, for example, sake lees produced in sake production produced by subjecting raw rice liquefied with liquefaction enzymes to alcohol fermentation (Satoshi Imayasu et al .: Journal of Japanese Agricultural Chemical Society, No. 63, page 971, 1989, JP 59-66875). As a specific example, a processed product containing the above-mentioned dipeptide can be obtained by treating a rice protein derived from such rice or a processed rice product or a protein derived from a rice raw material brewed product with a proteolytic enzyme. The obtained treated product may be used as a pulse wave velocity lowering agent as it is, for example, as a composition containing the dipeptide, or by further purifying the dipeptide from the composition and reducing the pulse wave velocity. It can also be used as an agent.

  Below, an example of the method of preparing the above-mentioned dipeptide from sake lees is shown. The present invention is not limited to this.

  First, the sake lees are boiled. The boiling time can be appropriately determined according to the amount of sake lees to be processed, but is usually 5 to 15 minutes.

  Next, the sake cake after the boiling treatment is centrifuged to remove the liquid fraction, and the obtained solid fraction is treated with a proteolytic enzyme. Enzyme treatment is usually performed in a solvent, and examples of the solvent include water and a buffer solution.

  The type of proteolytic enzyme is not particularly limited. For example, protease N Amano (Amano Enzyme Co., Ltd .; titer 150,000 U / g or more), Sumiteam AP (Shin Nippon Chemical Industry Co., Ltd .; titer 50,000 U / g) Etc.) can be used. These enzymes may be used alone or in combination of two or more. Moreover, when using a some enzyme, the process order in particular is not restrict | limited, You may process by several enzymes simultaneously, and you may process using each enzyme separately, respectively. The treatment temperature can be appropriately determined according to, for example, the type of proteolytic enzyme to be used, but is usually 40 to 60 ° C, and preferably 50 to 60 ° C. Moreover, although processing time can be suitably determined according to the quantity etc. of the said solid fraction to process, it is 5 to 20 hours normally, for example. The amount of proteolytic enzyme added can be appropriately determined according to, for example, the amount of sake lees used, but is preferably 0.1 to 1% by weight, more preferably 0.3 to 0. 6% by weight.

  Then, the solid fraction is subjected to an enzyme treatment, and then centrifuged to collect a liquid fraction. This liquid fraction contains the aforementioned Val-Tyr, Val-Trp, Tyr-Trp, and Ile-Tyr. In the present invention, as described above, the liquid fraction containing these dipeptides may be used as the PWV lowering agent of the present invention as it is or after being concentrated or powdered. The fraction may be purified and used. Purification can be performed, for example, by subjecting the liquid fraction to various chromatography. Examples of the chromatography include ion exchange chromatography such as SP-Sephadex C-25 (Pharmacia) and SP-Toyopearl 650S (Tosoh); Sephadex G-15 (Pharmacia), Toyopearl HW Gel filtration such as -40 (manufactured by Tosoh Corporation); high-performance liquid chromatography such as CAPCELL PACK18 (manufactured by Shiseido), Cosmosil C18 (manufactured by Nacalai Tesque), and the like can be employed.

  The PWV lowering agent of the present invention may contain the aforementioned dipeptide as an active ingredient, but may further contain, for example, a pharmaceutically acceptable additive. As the additive, for example, an excipient, a pH adjuster, a preservative, an isotonic agent, and the like can be selected according to the dosage form described later.

  In addition, the PWV lowering agent of the present invention may include a purified product of the dipeptide as the dipeptide, or may include, for example, a processed protein proteolytic enzyme of rice protein or rice raw material brewed protein containing the dipeptide. Good. The content of the dipeptide (total amount of dipeptide) in the PWV lowering agent of the present invention is not particularly limited, but for example, the daily intake may be set to be 0.1 to 1.0 mg. Preferably, it is 0.3-0.7 mg.

  The form of the PWV lowering agent of the present invention is not particularly limited, and may be, for example, a solution, a solid, a powder, or a state dispersed in a solvent. Specific dosage forms include, for example, tablets, fine granules (including powders), capsules, liquids (including syrups), injections and the like depending on the administration method. According to each of these dosage forms, for example, it can be produced according to a usual method described in the Japanese Pharmacopoeia using suitable additives and base materials.

  As described above, the therapeutic agent for arteriosclerosis of the present invention or the preventive agent for arteriosclerosis of the present invention includes the PWV lowering agent of the present invention. The arteriosclerosis therapeutic agent and arteriosclerosis preventive agent of the present invention may contain the PWV lowering agent of the present invention, and other configurations are not limited at all. The form and the like are the same as described above.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

(Preparation of peptide fraction)
A peptide fraction containing the aforementioned dipeptide was prepared from sake lees by the following production method. First, 60 g of sake lees (solid content 58.4%) was boiled for 10 minutes, and then centrifuged under conditions of 12000 rpm and 15 minutes. 42 g of the obtained precipitate was dispersed in 180 ml of water, and 210 mg of protease N Amano (trade name, manufactured by Amano Enzyme Co., Ltd.) was added to this dispersion, followed by treatment at 50 ° C. for 6 hours. Subsequently, Sumiteam AP (trade name, manufactured by Shin Nippon Chemical Industry Co., Ltd.) (250 mg) was further added to the dispersion and treated at 60 ° C. for 16 hours. The obtained enzyme-treated solution was centrifuged at 12000 rpm for 15 minutes, and the resulting supernatant was filtered using membrane filters (manufactured by Toyo Roshi Kaisha, Ltd.) having pore sizes of 0.45 μm and 0.22 μm. . The filtrate was freeze-dried, and the obtained powder was used as a peptide fraction.

  Four types of dipeptides contained in the peptide fraction were quantified by LC / MS analysis. The analysis conditions are shown below, and the results are shown in Table 1 below. In Table 1 below, the total peptide amount is the total amount of peptides having a molecular weight of 3,000 or less, including the four types of dipeptides (Val-Tyr, Val-Trp, Tyr-Trp, Ile-Tyr).

Measurement of standard peak area (1) Preparation of peptide standard solution Weigh 10 mg each of various peptides (Val-Tyr; manufactured by Sigma, Val-Trp, Tyr-Trp, Ile-Tyr; manufactured by Kokusan Chemical Co., Ltd.), water or hydrochloric acid The aqueous solution was added and completely dissolved, and the volume was adjusted to 1 mL. Four types of solutions containing various dipeptides were used as peptide standard solutions.
(2) Preparation of internal standard solution (IS) 10 mg of N-carbamyl-L-Arg (manufactured by Sigma) was weighed and completely dissolved in 1 mL of 0.06N hydrochloric acid aqueous solution to obtain an internal standard solution.
(3) Preparation of standard solution The various peptide standard solutions and the internal standard solution were mixed so that various dipeptides were 0.01 mg / ml and the internal standard substance was 0.1 mg / ml, respectively. These four types of mixed solutions were used as standard solutions.
(4) Measurement of peak area LC / MS analysis was performed for each of the four types of standard solutions under the following conditions, and the MS peak area of each dipeptide was measured.
Equipment: Waters Alliance 2695-2996-ZQ4000
(LC-photodiode array detector-MS detector, manufactured by Waters)
Column: Imtakt UK-C18 75 x 4.6mm (Imtakt)
Mobile phase: Liquid A: 0.05% HCOOH / water Liquid B: Acetonitrile (0-25% / 0-7.5 minutes, 100% / 7.5-8.5 minutes)
Injection volume: 10 μL
Flow rate: 1 mL / min Column temperature: 35 ° C
Detection condition: UV190-300nm
MS electrospray ion (ESI) method positive SIR mode

Measurement of peptide fraction 10 mg of the obtained peptide fraction was dissolved in 1 mL of pure water to prepare a peptide fraction solution. The peptide fraction solution and the internal standard solution were mixed so that the peptide fraction powder was 0.002 g / ml and the internal standard substance was 0.1 mg / mL to prepare a sample solution. This sample solution was subjected to LC / MS analysis in the same manner as the standard solution, and the MS peak area of each dipeptide was measured. Then, the content of each dipeptide in the peptide fraction was calculated from the MS peak area of each obtained dipeptide according to the following formula.
Dipeptide content of peptide fraction (mg / g) = A × {(B / C) / (D / E)} / F
A: Dipeptide concentration of standard solution (mg / ml)
B: Dipeptide peak area of sample solution
C: IS peak area of sample solution
D: Dipeptide peak area of the standard solution
E: IS peak area of standard solution
F: Peptide fraction concentration in the sample solution (g / ml)

(PWV measurement)
A capsule-like PWV lowering agent containing the peptide fraction was prepared and orally administered to the subject, and the change in PWV over time was confirmed. The test was a single blind test comparison test using placebo.

  The PWV lowering agent was set so that the daily dose was 600 mg of the total peptide. Specifically, 1000 mg of the peptide fraction (total amount of peptide 600 mg) was dispensed into 6 tea-transparent hard capsules to make a PWV lowering agent for one day. On the other hand, in place of the peptide fraction, a blend of dextrin and pigment is dispensed into similar brown transparent hard capsules so that the weight and appearance are indistinguishable from the PWV lowering agent, and a placebo is prepared. did. Similarly, 6 capsules were used as a placebo for one day.

  The subjects were 38 adult males and females of paid volunteers recruited at FANCL Corporation and private volunteer recruitment organizations. These subjects include secondary hypertension, patients taking antihypertensive drugs, those who have been diagnosed with diabetes by a doctor, those who are pregnant or breastfeeding, and those who have other severe illnesses. Not included.

  The subjects were randomly divided into two groups using a random assignment table. One group of 19 people (11 men, 8 women) was administered the PWV lowering agent (hereinafter referred to as “PWV lowering agent group”), and the other 19 groups (10 men, 9 women) Administered the placebo (hereinafter referred to as the “placebo group”). Each of the PWV lowering agent group and the placebo group was administered 6 capsules daily for 12 weeks after dinner once a day.

  The PWV measurement was performed 4 times in total: 0 week, 4 weeks, 8 weeks, and 12 weeks from the start of administration. The measurement time period is from 9:00 am to 11:00 am. After a hungry subject who has not eaten breakfast is allowed to rest for 10 minutes or more while sitting, a blood pressure pulse wave test device (trade name form PWV / ABI, model number BP-203RPE II, OMRON Colin Co.).

  The measurement results are shown in Table 2 below. The measured value in each administration period was shown as an average value ± standard deviation (Ave ± SD).

  Further, for each of the PWV lowering agent group and the placebo group, the PWV value before administration (week 0) and the PWV value for each administration period (4 weeks, 8 weeks, and 12 weeks of administration) have corresponding t- The test was performed and the period of each administration was compared. The significance level of the test was 5% on both sides.

  As shown in the table, in the PWV lowering agent group, the PWV value decreased with the lapse of the administration period. In particular, a significant decrease in the PWV value was confirmed at 12 weeks after the start of administration, compared to before the start of administration (week 0).

  As described above, according to the PWV lowering agent of the present invention, by containing at least one dipeptide of four types of Val-Tyr, Val-Trp, Tyr-Trp and Ile-Tyr, it is an index of arteriosclerosis. You can descend a PWV. For this reason, the PWV lowering agent of the present invention is extremely useful as, for example, a prophylactic or therapeutic agent for arteriosclerosis.

Claims (5)

A depressant of pulse wave velocity,
A pulse wave velocity lowering agent comprising at least one dipeptide selected from the group consisting of Val-Tyr, Val-Trp, Tyr-Trp and Ile-Tyr.   The pulse wave velocity lowering agent according to claim 1, comprising a processed product obtained by treating rice protein or rice-derived brewed protein with a proteolytic enzyme, wherein the processed product contains the dipeptide.   The pulse wave velocity lowering agent according to claim 2, wherein the rice raw brew is sake lees. An arteriosclerosis therapeutic agent for treating arteriosclerosis,
A therapeutic agent for arteriosclerosis, comprising the pulse wave velocity lowering agent according to any one of claims 1 to 3. An arteriosclerosis preventive agent for preventing arteriosclerosis,
An arteriosclerosis preventive agent comprising the pulse wave velocity lowering agent according to any one of claims 1 to 3.