KR20110130397A - Dipeptide having antiatherosclerotic action - Google Patents

Abstract

It is an object of the present invention to provide an atherosclerotic agent which can be used without worrying about the effect on the heart of atherosclerosis patients having a heart disease, or to provide a food having the above function. To this end, there is provided an anti- atherosclerotic agent having a dipeptide Trp-His and / or a salt thereof as an active ingredient or a food having the above function.

Description

Dipeptide HAVING ANTIATHEROSCLEROTIC ACTION}

The present invention uses dipeptide represented by Trp-His or a salt thereof as an active ingredient, and can be used even when a heart disease in which calcium antagonists are avoided is used. Atherosclerosis It relates to compositions such as health foods, functional foods, pharmaceuticals, etc., having the action of preventing or inhibiting its progress.

Calcium channel blockers are drugs that have vasorelaxing or vasodilating action, and there is a document that describes the effect on atherosclerosis. For example, Non-Patent Document 1 relates to azelnidipine, which is a representative calcium channel blocker, and has a description of its anti- atherosclerotic action. The minimum effective dose for which the effect is shown here is 3 mg / kg / day, which is significantly larger than 8-16 mg / day (60 kg body weight patient), which is required for vasodilation. Data on azelnidipine ("Kalblock (Azelnidipine) and Calblock Tablet 8mg / 16mg") Ubekosan Co., Ltd., Sankyo Corporation (http://www.info.pmda.go.jp/shinyaku/g030102) (Obtained on February 23, 2009 by the Internet)), in an experiment administered to rats, side effects including an increase in heart weight were confirmed by administration of 10 mg / kg / day.

Calcium channel blockers are agents that are concerned about their effects on the heart due to their mechanism of action. When a calcium channel blocker is used as a vasodilator, there is a small difference between the amount of side effects such as an increase in heart weight and its effective amount, and it seems to be a problem. However, a calcium channel blocker is used as an atherosclerotic agent. In this case, as described above, there is only a 3.3-fold difference between the amount of adverse effects such as an increase in heart weight and the amount of effective action as an atherosclerotic agent, which is a concern when a calcium channel blocker is used as an atherosclerotic agent. have.

Non-Patent Document 1: Atherosclerosis 196 (2008) 172-179

An object of the present invention is to provide a substance having an anti- atherosclerotic effect, which is relatively easily available and has little effect on the heart.

The present inventors earnestly researched about the said subject. Therefore, to find out that the dipeptide Trp-His, which is known to have vasodilation as a calcium blocker, exhibits anti-atherogenic atherosclerosis without causing a significant effect that was naturally expected to occur in the heart as a calcium blocker, to complete the present invention. Reached.

That is, the present invention

(1) A prophylactic or ameliorating agent for atherosclerosis, wherein the dipeptides or salts thereof represented by Trp-His are used as an active ingredient and no adverse effects are caused to the heart even when an excessive amount of the effective amount is administered.

(2) A prophylactic or ameliorating agent for atherosclerosis, wherein the dipeptide represented by Trp-His or a salt thereof is used as an active ingredient and is for patients with heart disease avoided by calcium antagonists.

  (3) The agent for preventing or improving atherosclerosis according to any one of (1) to (2), wherein the dosage per day is 5 to 100 mg per kg of body weight.

It is about.

According to the present invention, by the dipeptides represented by Trp-His and its salts which can be prepared relatively easily by synthesis or the like, a composition such as a medicament having an anti- atherosclerotic effect that does not significantly affect the heart Can be provided.

Fig. 1 shows the arrangement of "(beta) conglycinin α 'subunit".
Figure 2, in Comparative Example 1 (Control), Example 1 (Trp-His dosage 10mg / kg / day), Example 2 (Trp-His dosage 100mg / kg / day), after breeding for 9 weeks, Atherosclerotic lesions in the aortic tree (white part). It can be seen that the lesions of Examples 1 and 2 are small compared to Comparative Example 1 (Control).
3 is numerical analysis data of the lesion area of FIG. 2. Example 1 (Trp-His dose 10mg / kg / day), Example 2 (Trp-His dose 100mg / kg / day) has a smaller lesion area compared to Comparative Example 1 (Control).
FIG. 4 shows that in Comparative Example 1 (Control), Example 1 (Trp-His dose 10 mg / kg / day) and Example 2 (Trp-His dose 100 mg / kg / day), after 9 weeks of breeding, It is an atherosclerotic lesion in the aortic sinus. No big difference is seen between Comparative Example 1, Example 1 and Example 2.
5 is numerical analysis data of the lesion area of FIG. 4. No significant difference was seen between Comparative Example 1, Example 1 (Trp-His dose 10 mg / kg / day) and Example 2 (Trp-His dose 100 mg / kg / day).

The term "anti-atherosclerotic action" as used in the present invention refers to suppressing the development of atherosclerosis or suppressing the development of atherosclerosis once developed.

  The "effective amount" as used in the present invention means a dose that exhibits an anti-atherosclerosis action. "Excessive amount administration with respect to the effective amount of action" means administration of about 10 times with respect to an amount as small as possible among the amount | actions considered to be effective.

In addition, `` it does not cause side effects to the heart '' is a cardiac disease in which a calcium blocker is avoided in view of the phenomenon that a drug called a calcium blocker is avoided to be used in patients with a heart disease due to its mechanism of action. It indicates that the heart is small enough to be used in the same patient. More specifically, as in Example 2 of the present invention, the administration of 100 mg / kg / day corresponding to the 10-fold amount of 10 mg / kg / day, which is the dose for which the desired effect is recognized, is especially effective for the heart weight. When it does not affect, it determines with "does not cause a side effect to a heart" said by this invention. This invention is one of the characteristics that "it does not cause a side effect to a heart," and it is especially preferable as a use for patients with a heart disease, and a patient avoiding a calcium blocker. Moreover, as a cardiac disease to which a calcium blocker is avoided, an atrial block (ariculo-ventricular block), a cardiogenic shock, etc. are mentioned.

Arteries emerge from the heart, and in humans, they begin at the entrance to the aorta in the left ventricle, ascend, and then migrate to the aortic arch at the height of the sternum or second cartilage. In the beginning of the ascending aorta, the diameter of blood vessels is larger than the peripheral side, so it is called abula (bulb of aorta), and its lumen is called the sinus of aorta or foot salva. It is called the sinus of Valsalva. In the present invention, the effect has not been confirmed in the aortic sinus.

The artery to which the effect of this invention exerts is called an aortic tree. The aortic tree here does not include the aortic sinus.

Dipeptide Trp-His, an active ingredient of the present invention, is an arrangement also present in soy protein, which can be obtained by digesting soy protein with an appropriate enzyme, fractionating it and purifying the portion containing the desired peptide, and furthermore, the peptide chain length (chain length). Since it is short as 2, it can also be obtained by the normal method of peptide synthesis. The Trp-His array is present in the 125-126th soybean protein β-conglycinin α ′ subunit primary structure. The amino acid sequence of the corresponding soy protein is shown in FIG. Trp-His prepared by decomposition by enzymes or the like from soy protein is useful as a material for supplements, health supplements, specific health foods, etc., because it is derived from a material having a past food experience.

When obtaining the dipeptide of the present invention by the synthesis method, a biological synthesis method using a chemical synthesis method (solid phase method, liquid phase method), enzyme synthesis method and DNA recombination method used for ordinary peptide synthesis This is known, but any method may be used. Regarding the enzyme method, a method using a reverse reaction of a protease (protease) (J. Biol. Chem., 707-720 (1937)) A method using a heat-resistant aminoacyl t-RNA synthetase [JP-A] Japanese Patent Application Laid-Open No. 59-106298], and the like. Recently, a manufacturing method using microorganisms belonging to the genus Escherichia or Bacillus (International Publication No. WO2004 / 058960) is disclosed. .

The peptide of the present invention obtained by the synthesis is prepared by reverse phase high performance liquid chromatography, chromatography using an ion exchange resin or a high porous polymer resin, affinity chromatography, or the like. It can be purified. Moreover, it is preferable that amino acid is L type.

The route of administration and the dosage form of the pharmaceutical composition of the present invention can be appropriately designed by those skilled in the art. Examples of routes of administration include oral, oral, injection (intradermal, subcutaneous, muscle, vein), airway respirators, and body pores (mucosa) I)) and examples of the formulation include powdered medicines, granules, granules, tablets, capsules, pills, potions, suspensions, emulsions, inhalants, sprays, aerosols and suppositories. Etc. When formulated, various carriers that are acceptable as pharmaceuticals, such as excipients, binders, shells (coatings), disintegrants, lubricants, suspending agents, Formulation components, such as surfactant, a flavoring substance, a coloring agent, a preservative, and a stabilizer, can be used. This pharmaceutical composition can be orally administered and is effective.

In addition, administration in the form of food is also possible, and a contemplated form includes beverages, tablet confectionery, baked confectionery, ice cream, chocolate and the like.

If a predetermined amount of the dipeptide or salt thereof represented by Trp-His is contained, even if other peptides and other components are mixed, there is no problem as long as the effects of the present invention are not adversely affected. In addition, the expression "dipeptide or its salt" also points out the state in which a dipeptide and its salt were mixed.

The amount of Trp-His required for anti-atherosclerosis is preferably 5 to 100 mg / day, more preferably 7 to 100 mg / day, and more preferably 10 to 100 mg / day. In the case where the amount of Trp-His is too small, it is often undesirable because of insufficient anti-atherosclerosis. In addition, for doses over 100 mg / kg / day, the effect on the heart is not validated at the stage until February 23, 2009.

An example is described below. Unless otherwise indicated,% refers to% by weight.

Example

Experiment 1 (Examples 1 and 2, Comparative Example 1)

`` Anti-atherosclerosis of Dipeptide Trp-His ''

Obtaining Peptides, Reagents

Trp-His was synthesize | combined by the Fmoc solid-phase synthesis method (made by the domestic chemical company). Reagents other than that were used as reagent grades.

Animal specimens were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and used ApoE-deficient mice (8-18 weeks old male) bred in the Animal Experiment Laboratory at the Kyushu University School of Medicine. This was divided into three groups and fed for nine weeks with the food shown in Table 1. There was no significant difference in the age of mice between the groups (Comparative Example 1 15.0 ± 0.8, Example 1 14.3 ± 1.2, Example 2 14.6 ± 1.3).

[Table 1] Formulation of food

Prey was made by AIN-76 formula.

In the administration of Trp-His, 10-100 mg / kg of Trp-His was dissolved in 1 ml of deionized water and orally intubated with respect to the body weight of each mouse, and once a day. Administered. Control mice received deionized water in the same manner. In addition, in Comparative Example 1, Trp-His was administered at 10 mg / kg / day for Example 1 and 100 mg / kg / day for Example 2, and only deionized water was administered. Mice were bred repeatedly at a room temperature of 22 degrees for 12 hours. In addition, the food and deionized water in Table 1 were freely consumed.

After breeding for 9 weeks, the blood was collected from the heart after 6 hours of fasting. The liver, the white adipose tissue of the peritoneum, the mesentery, the subcutaneous part, and the testicular epithelial part were extracted and cooled with liquid nitrogen. Similarly, the aorta and the heart were extracted and fixed with formalin.

Triacylglycerol, total cholesterol, and HDL-cholesterol in plasma were measured by an enzyme assay kit (Triglyceride E test from Wako Pure Chemicals, T-CHO kainos from Kainos). Plasma MCP-1 concentration was measured by ELISA kit (Mouse MCP-1 ELISA kit from Invitrogen, California, USA). Soy lipids were extracted with CHCl 3 -CH 3 OH mixed solution, and were prepared using "Br J Nutr. 94,896-901 (2005) ".

Quantitative analysis of atherosclerotic lesions was performed by the method described in J Nurt 128, 1884-1889 (1998). The results are expressed as mean and standard error of the mean (SEM). It was also evaluated using one-way batch analysis according to Tukey-Kramer's multiple comparison. P (probability) <0.05 was judged to be meaningful. Analysis was by software (Stat View J5.0 (SAS Institute, Cary, NC, USA)).

result

Weight and feeding efficiency

After 9 weeks of breeding, body weight, organ weight, food intake and feeding efficiency were not different in the three groups (Table 2). Body fat amount (Comparative Example 1 Control: Average 8.9 (SEM1.2)%, n8; Example 1 10 mg / kg Trp-His: Average 7.1 (SEM0.7)%, n7; Example 2 100 mg / kg Trp-His: Average 7.5 (SEM0.6)%, n8) showed no difference between the groups as in the mesentery, testis and subcutaneous fat mass.

[Table 2] State after breeding for 9 weeks

Blood lipids

In the analysis of blood, there were no differences in the three groups in the amounts of total cholesterol, HDL-cholesterol and MCP-1 (Table 3). Plasma cholesterol is relatively high (> 1000 mg / day), but this is thought to be due to high cholesterol.

[Table 3] Cholesterol content after breeding for 9 weeks

In the aortic tree after administration of the dipeptide Trp-His for 9 weeks, the Trp-His administration group (dose 10 mg / kg (Example 1), 100 mg / kg (Example) was used in the aortic tree than the control group (Comparative Example 1). 2) In both cases, atherosclerotic lesions were small (Fig. 2). Numerically, as shown in FIG. 3, the ratio of lesion area in the Trp-His administration group (10 mg / kg: average 9.5 (SEM 1.3)%, n7, 100 mg / kg: average 8.1 (SEM 1.6)%, n8, P < 0.05) was clearly smaller than the control group (average 13.0 (SEM 1.0)%, n8). This indicates that the dipeptide Trp-His inhibits the progression of atherosclerotic lesions. There was no significant difference depending on the dose between the Trp-His administration groups. This indicates that, when the dose of Trp-His is 10 mg / kg / day, the effect is sufficient for showing effect.

In the atherosclerotic lesions of the aortic sinus (Fig. 4) and the numerical data of the lesion area (Fig. 5), there was no difference in the three groups (P> 0.05).

Review

As described above, in apoE deficient mice administered with the dipeptide Trp-His for 9 weeks, the lesion area of atherosclerosis of the aortic tree was smaller than that of the dipeptide. This indicates that the dipeptide Trp-His has a function of inhibiting the development of atherosclerosis in the aortic tree. The same effect is thought to be found for other blood vessels, especially arteries, which are similar in basic structure to the aortic tree. In addition, the effect was not shown by the difference in the dose per body weight of Trp-His, and it is thought that the effect appears in the dose of 5-100 mg / kg / day.

In this experiment, there was no particular change in heart weight even at the dose of 100 mg / kg / day corresponding to the 10-fold dose with respect to the dose of 10 mg / kg / day for which sufficient effect was recognized. This is not known at all, and it is assumed that the dipeptide Trp-His of the present invention exhibits anti-atherogenic atherosclerosis by a mechanism of action other than the conventionally known calcium block action.

In the present invention, anti- atherosclerotic action by dipeptide Trp-His was not confirmed in the aortic sinus. This is also extremely unusual when considered from the mechanism of action previously considered. In other words, if the arterial antiarterial atherosclerosis is exhibited only by the known calcium antagonistic action, the aortic sinus should naturally exhibit the atherosclerosis as well as other arteries. Proof of existence. In addition, the presence of the aortic sinus in the vicinity of the heart suggests that the dipeptide Tpr-His may have a specific function not previously known to the heart.

Moreover, since the above effects are shown by Trp-His, it can be estimated by a person skilled in the art that the same effect also appears in the salt. In addition, although this experiment is about a mouse, it can be estimated by a person skilled in the art that the same effect can be expected with respect to mammals in general.

INDUSTRIAL APPLICABILITY According to the present invention, in atherosclerosis patients having a heart disease, an anti-atherosclerosis that can be taken even by a patient who could not take a calcium antagonist in the past because of concern about the effect on the heart can be provided.

<110> FUJI OIL COMPANY, LIMITED          KYUSHU UNIVERSITY, NATIONAL UNIVERSITY CORPORATION <120> DIPEPTIDE HAVING ANTIATHEROSCLEROTIC ACTION <130> P110-57JP <150> JP2009 / 060978 <151> 2009-03-13 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 639 <212> PRT <213> Soybean <400> 1 Met Met Arg Ala Arg Phe Pro Leu Leu Leu Leu Gly Val Val Phe Leu   1 5 10 15 Ala Ser Val Ser Val Ser Phe Gly Ile Ala Tyr Trp Glu Lys Gln Asn              20 25 30 Pro Ser His Asn Lys Cys Leu Arg Ser Cys Asn Ser Glu Lys Asp Ser          35 40 45 Tyr Arg Asn Gln Ala Cys His Ala Arg Cys Asn Leu Leu Lys Val Glu      50 55 60 Glu Glu Glu Glu Cys Glu Glu Gly Gln Ile Pro Arg Pro Arg Pro Gln  65 70 75 80 His Pro Glu Arg Glu Arg Gln Gln His Gly Glu Lys Glu Glu Asp Glu                  85 90 95 Gly Glu Gln Pro Arg Pro Phe Pro Phe Pro Arg Pro Arg Gln Pro His             100 105 110 Gln Glu Glu Glu His Glu Gln Lys Glu Glu His Glu Trp His Arg Lys         115 120 125 Glu Glu Lys His Gly Gly Lys Gly Ser Glu Glu Glu Gln Asp Glu Arg     130 135 140 Glu His Pro Arg Pro His Gln Pro His Gln Lys Glu Glu Glu Lys His 145 150 155 160 Glu Trp Gln His Lys Gln Glu Lys His Gln Gly Lys Glu Ser Glu Glu                 165 170 175 Glu Glu Glu Asp Gln Asp Glu Asp Glu Glu Gln Asp Lys Glu Ser Gln             180 185 190 Glu Ser Glu Gly Ser Glu Ser Gln Arg Glu Pro Arg Arg His Lys Asn         195 200 205 Lys Asn Pro Phe His Phe Asn Ser Lys Arg Phe Gln Thr Leu Phe Lys     210 215 220 Asn Gln Tyr Gly His Val Arg Val Leu Gln Arg Phe Asn Lys Arg Ser 225 230 235 240 Gln Gln Leu Gln Asn Leu Arg Asp Tyr Arg Ile Leu Glu Phe Asn Ser                 245 250 255 Lys Pro Asn Thr Leu Leu Leu Pro His His Ala Asp Ala Asp Tyr Leu             260 265 270 Ile Val Ile Leu Asn Gly Thr Ala Ile Leu Thr Leu Val Asn Asn Asp         275 280 285 Asp Arg Asp Ser Tyr Asn Leu Gln Ser Gly Asp Ala Leu Arg Val Pro     290 295 300 Ala Gly Thr Thr Phe Tyr Val Val Asn Pro Asp Asn Asp Glu Asn Leu 305 310 315 320 Arg Met Ile Ala Gly Thr Thr Phe Tyr Val Val Asn Pro Asp Asn Asp                 325 330 335 Glu Asn Leu Arg Met Ile Thr Leu Ala Ile Pro Val Asn Lys Pro Gly             340 345 350 Arg Phe Glu Ser Phe Phe Leu Ser Ser Thr Gln Ala Gln Gln Ser Tyr         355 360 365 Leu Gln Gly Phe Ser Lys Asn Ile Leu Glu Ala Ser Tyr Asp Thr Lys     370 375 380 Phe Glu Glu Ile Asn Lys Val Leu Phe Gly Arg Glu Glu Gly Gln Gln 385 390 395 400 Gln Gly Glu Glu Arg Leu Gln Glu Ser Val Ile Val Glu Ile Ser Lys                 405 410 415 Lys Gln Ile Arg Glu Leu Ser Lys His Ala Lys Ser Ser Ser Arg Lys             420 425 430 Thr Ile Ser Ser Glu Asp Lys Pro Phe Asn Leu Gly Ser Arg Asp Pro         435 440 445 Ile Tyr Ser Asn Lys Leu Gly Lys Leu Phe Glu Ile Thr Gln Arg Asn     450 455 460 Pro Gln Leu Arg Asp Leu Asp Val Phe Leu Ser Val Val Asp Met Asn 465 470 475 480 Glu Gly Ala Leu Phe Leu Pro His Phe Asn Ser Lys Ala Ile Val Val                 485 490 495 Leu Val Ile Asn Glu Gly Glu Ala Asn Ile Glu Leu Val Gly Ile Lys             500 505 510 Glu Gln Gln Gln Arg Gln Gln Gln Glu Glu Gln Pro Leu Glu Val Arg         515 520 525 Lys Tyr Arg Ala Glu Leu Ser Glu Gln Asp Ile Phe Val Ile Pro Ala     530 535 540 Gly Tyr Pro Val Met Val Asn Ala Thr Ser Asp Leu Asn Phe Phe Ala 545 550 555 560 Phe Gly Ile Asn Ala Glu Asn Asn Gln Arg Asn Phe Leu Ala Gly Ser                 565 570 575 Lys Asp Asn Val Ile Ser Gln Ile Pro Ser Gln Val Gln Glu Leu Ala             580 585 590 Phe Pro Arg Ser Ala Lys Asp Ile Glu Asn Leu Ile Lys Ser Gln Ser         595 600 605 Glu Ser Tyr Phe Val Asp Ala Gln Pro Gln Gln Lys Glu Glu Gly Asn     610 615 620 Lys Gly Arg Lys Gly Pro Leu Ser Ser Ile Leu Arg Ala Phe Tyr 625 630 635

Claims (3)

Dipeptide or salt thereof represented by Trp-His is used as an active ingredient and does not cause adverse effects on the heart even when an excessive amount of the effective amount is administered.
An agent for preventing or improving atherosclerosis.
It is for patients with heart disease avoided by calcium antagonists using dipeptide or salt thereof represented by Trp-His as an active ingredient.
An agent for preventing or improving atherosclerosis.
The method according to claim 1 or 2,
A prophylactic or ameliorating agent for atherosclerosis, characterized in that the dosage per day is 5-100 mg / kg body weight.

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