MX2008010328A - Vasodilator - Google Patents

Abstract

It is intended to provide a vasodilator having a vascular endothelium-dependent vasodilating action, a preventive agent for arteriosclerosis, and a functional food having a vascular endothelium-dependent vasodilating action which can be continuously used on a daily basis and is excellent in safety. The vasodilator and the functional food of the invention are characterized by comprising, as an activeingredient, at least one of tripeptides of Val Pro Pro and Ile Pro Pro or a protein degradation product containing Val Pro Pro and/or Ile Pro Pro.

Description

VASODILATOR FIELD OF THE INVENTION The present invention relates to a vasodilator having a vasodilator effect dependent on the endothelium, and the functional food having such an effect.

BACKGROUND OF THE INVENTION Ischemic diseases, such as myocardial or cerebral infarction, which are the final development of arteriesclerotic diseases, account for the majority of causes of death in Japan, along with cancers. Risk factors for arteriosclerosis include hyperlipidemia, hyperlipidemia, hypertension, diabetes, smoking, obesity, hyperuricemia, aging, stress and the like, which are interrelated to cause angiopathy. In this way, even if each risk factor is low, the accumulation of the factors additively and synergistically increases the risk, which also increases the risk of ischemic diseases. On the other hand, it is considered that the mere mitigation of one of the aforementioned risk factors will not present the onset of arteriosclerosis. For example, Publications 1 and 2 non-patent report the absence of interrelation between blood cholesterol level and Ref .: 195387 beginning of arteriosclerosis, non-patent publication 3 teaches that the suppression of hypertension does not change the degree of arteriosclerosis, and the publication does not patent 4 discloses that administration of an angiotensin-converting enzyme inhibitor containing enalapril as an active component does not result in the inhibitory effect of arteriosclerosis. Furthermore, when the infarct occurs, ischemic diseases, for example, acute heart failure develops, according to Non-patent Publication 5, the medicament, such as a vasodilator that includes nitroprusside or nitroglycerin, a diuretic, or a cardiotonic, it is necessary to stabilize the hemodynamics. Thus, although Patent Publications 1 and 2, for example, describe that the tripeptides Val-Pro-Pro and Ile-Pro-Pro have an inhibitory activity on the angiotensin I conversion enzyme, which leads to an effect hypotensive, and also to an anti-stress effect, this does not mean that these tripeptides have an anti-arteriosclerotic effect or a vasodilator effect. Arteriosclerosis is a pathology in which the arterial wall is thickened, losing its elasticity. One of the factors for such a symptom is recently considered as the damaging or diminished functions of vascular endothelial cells.

Thus, it is expected that a vasodilator dependent on the endothelium has an inhibitory effect on arteriosclerosis. Patent Publication 1: JP-6-197786-A Patent Publication 2: JP-11-100328-A Non-Patent Publication 1: Shoku no Kagaku 257 (1999), p20-25 Non-Patent Publication 2: Atherosclerosis 151 (2000), p501-508 Non-patent publication 3: Circulation 104 (2001), p2391-2394 Non-patent publication 4: International Journal of Cardiology 81 (2001), pl07-115 Non-patent publication 5: Bessatsu Igaku no Ayumi , Junkanki Sftikkann, state of arts see. 2 (2001), p332-334 BRIEF DESCRIPTION OF THE INVENTION An object of the present invention is to provide a vasodilator that has a vasodilator effect dependent on the endothelium. Yet another objective of the present invention is to provide functional food that has an endothelial vasodilator effect, which can be subjected to daily and regular ingestion, and is excellent in safety.

According to the present invention, a vasodilator comprising at least one of the Val-Pro-Pro and Ile-Pro-Pro peptides is provided as an active component. According to the present invention, there is also provided a vasodilator comprising a proteolytic product containing Val-Pro-Pro and / or Ile-Pro-Pro as an active component. According to the present invention, functional food comprising at least one of the Val-Pro-Pro and Ile-Pro-Pro peptides is also provided as an active component, and having a vasodilatory effect. According to the present invention, there is further provided functional food comprising a proteolytic product containing Val-Pro-Pro and Ile-Pro-Pro as an active component, and having a vasodilatory effect. According to the present invention, the use of at least one of the peptides Val-Pro-Pro and Ile-Pro-Pro, or the use of a proteolytic product containing Val-Pro-Pro and Ile-Pro-Pro is provided. , in the manufacture of a vasodilator or functional food that has a vasodilatory effect. According to the present invention, there is also provided a method for dilating blood vessels, comprising the step of administering to an animal, an effective amount of at least one of the peptides Val-Pro-Pro and Ile-Pro-Pro, or a proteolytic product that contains Val-Pro-Pro and / or Ile-Pro-Pro. This contains, as an active component, Val-Pro-Pro and / or Ile-Pro-Pro derived from animal milk casein or the like, or a proteolytic product containing at least one of these peptides, the vasodilator and the functional food of according to the present invention, they are excellent in safety, and have a vasodilator effect dependent on the endothelium. In particular, the functional food can be taken regularly. The vasodilator according to the present invention is useful for ensuring blood circulation when a patient suffers from ischemic disease, such as cardiac or cerebral infarction, relaxes blood vessels that are more prone to constriction due to aging, diseases related to lifestyle or the like, or can be expected to prevent arteriosclerosis, or neck stiffness, cold constitution, thrombosis or similar symptoms associated with blood flow dysfunction. The functional food according to the present invention is useful as healthy foods or foods for specific health uses, which claim the vasodilatory effect, as well as effects on the various symptoms or diseases associated with blood flow dysfunction, such as stiffness of the neck, cold constitution, thrombosis or the like.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing the results of the vasodilation tests conducted in Example 1 and Comparative Example 1. Figure 2 is a graph showing the results of a confirmatory test for the dependence of the endothelium of the vasodilator response conducted in Example 1.

DETAILED DESCRIPTION OF THE INVENTION The present invention will now be explained in detail. The vasodilator and the functional food according to the present invention contains, as an active component, Val-Pro-Pro and / or Ile-Pro-Pro (these tripeptides are abbreviated as VPP and IPP, respectively, in the following) or a A proteolytic product that contains at least one of these tripeptides. Tripeptides may be those to which a pharmacologically acceptable salt has been added, including salts of inorganic acids, such as hydrochlorides, sodium and phosphate salts, or salts of organic acids, such as citrates, maleates, fumarates, tartrates and lactates. The tripeptides can be prepared by digestion and purification of peptides or proteins containing the amino acid sequence of VPP and / or IPP through fermentation with microorganisms, by enzymatic hydrolysis of such peptides or proteins, or by synthesis. For details, see the aforementioned Patent Publications 1 and 2. Thus, the active component of the present invention can be a fermentation product containing at least one of the peptides VPP and IPP obtained by fermenting the peptides or proteins containing the amino acid sequence VPP and / or IPP with the microorganisms , or a purified product thereof, or a hydrolyzate containing at least one of the VPP and IPP peptides obtained by digestion of the peptides to proteins containing the amino acid sequence VPP and / or IPP with enzymes, or a purified product thereof. The effective dose of the vasodilator of the present invention is usually 10 pg to 10 g, preferably about 1 mg to 1 g per day for humans in terms of the tripeptides, to achieve the effect in a single dose. The scheme of administration of the vasodilator can be adjusted to the symptoms of a disease. For acute symptoms, simple or continuous parenteral administration is adequate. For chronic symptoms or for the use prophylactic, regular oral administration is preferred for 30 days or longer. The vasodilator administration route according to the present invention can be either oral or parenteral. Parenteral administration can be topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, or intraspinal. Direct administration to the sick area is also possible. The vasodilator form according to the present invention may be decided depending on the route of administration and may be in the form of a formulation, such as tablets, pills, hard capsules, soft capsules, microcapsules, powders, granules, liquids, suspensions. or emulsions. The formulation may be with, for example, a carrier, adjuvant, excipient, auxiliary excipient, antiseptic, stabilizer, binder, pH regulator, buffer, thickener, gelatinizer, preservative, antioxidant, or the like, which are acceptable for pharmaceutical use, as desired, in a unit dosage form that is required in the generally approved formulation. The functional food according to the present invention can be assortment as health food or food for specific uses for health, which claim or announce the vasodilatory effect as well as the effects on various diseases associated with blood flow dysfunction. The amount ingestion for obtaining such an effect, in particular the vasodilator effect or the associated improving effect on a disease, is usually from 10 pg to 10 g, preferably from approximately 1 mg to 1 g per day in terms of the tripeptides, taking into account the fact that the present functional element can be taken regularly or daily in a prolonged period of time The simple ingestion of the functional food may be less than the previous amount in terms of tripeptides, depending on the number of ingestions per day. The period for ingestion of the functional food of the present invention is not particularly limited, and it is preferred to take it for a prolonged period of time to ameliorate chronic symptoms or for prophylactic use. In order to obtain the effect discussed above, regular ingestion is preferred for 30 days or more, particularly about 3 to 12 months. The functional food according to the present invention can be produced by the addition of the tripeptide active components, or a food material containing the tripeptides, to various foods and beverages, and can thus be elaborated in a variety of food forms. and drink. For example, the present food functional can be in the form of candies in tablets, yogurt, milk drinks, dairy products, alcoholic beverages, soft drinks, powdered or granulated food, encapsulated food, various fortified foods, or supplements. The functional food of the present invention may optionally contain various additives usually used in food.

EXAMPLES The present invention will now be explained in more detail with reference to the Examples, which are illustrative only and are not intended to limit the present invention.

EXAMPLE OF SYNTHESIS IPP and VPP were synthesized through the following organic chemical synthesis by the solid phase method in an automated peptide synthesizer (PSSM-8) manufactured by SHIMADZU CORPORATION. 50 mg of 2-chlorotrityl-polystyrene resin to which the proline having its protected amino group was linked with a fluorenylmethyloxycarbonyl group (hereinafter abbreviated as Fmoc) (trademark SynProPep Resin, manufactured by SHIMADZU CORPORATION), used as a solid support. 100 mol each of Fmoc-Ile, Fmoc-Pro and Fmoc-Val, where the amino groups were protected with the Fmoc group, were reacted sequentially by a routine method according to the amino acid sequence mentioned above to obtain a resin linked to the peptide. The resin bound to the peptide was suspended in 1 ml of reaction liquid A (10% by volume of acetic acid, 10% by volume of trifluoroethanol, and 80% by volume of dichloromethane), reacted at room temperature for 30 to 60 minutes. minutes to cleave the peptides from the resin, and filtered through a glass filter. The solvent in the resulting filtrate was removed under reduced pressure, and immediately 1 ml of the reaction liquid B (82.5% by volume of trifluoroacetic acid, 3% by volume of ethylmethyl sulfide, 5% by volume of purified water, was added). % by volume of thioanisole, 2.5% by volume of ethanedithiol, and 2% by volume of thiophenol). The resulting mixture was reacted at room temperature for 6 hours to remove the side chain protecting groups, to which 10 ml of the anhydrous ether was added to precipitate the peptides. The precipitate was separated by centrifugation at 3000 rpm for 5 minutes, washed several times with anhydrous ether, and dried under nitrogen gas. All the crude synthesized peptides obtained in this way were dissolved in 2 ml of a 0.1 N aqueous solution of hydrochloric acid, and purified by C18 reverse phase HPLC under the following conditions. Pump: intelligent pump model L6200 (manufactured by HITACHI, LTD.); Detector: ultraviolet absorption at 215 nm was detected with the UV detector model L4000 (manufactured by HITACHI, LTD.); Column: μBondasphere 5 μ C18 (manufactured by Nihon aters K.K.); Eluate: Liquid A of 0.1% by weight of aqueous solution of TFA and Liquid B of 0.1% by weight of acetonitrile containing TFA, (B / A + B) x 100 (%): 0 to 40% (in 60 minutes); Flow rate: 1 ml / minute. The eluted fraction having the maximum absorption was collected and lyophilized to obtain the peptides synthesized Ile-Pro-Pro and Val-Pro-Pro in the yields of 5.7 mg and 6.5 mg, respectively. The purified peptides were analyzed from the N-terminus in a protein automation sequencer (model PPSQ-10, manufactured from SHIMADZU CORPORATION), and further analyzed in an amino acid analyzer (model 800 series, manufactured by JASCO CORPORATION). It was confirmed that the peptides were prepared as designed.

Example 1 < Vasodilation test > The thoracic aorta of a Wistar rat was taken, cut in 2 mm in length, and elaborated in an aortic ring.

The ring was placed in a Magnus device (trade name "MTB-1Z microtech organ bath", manufactured by LABO SUPPORT CO., LTD.), And allowed to equilibrate with a constant tension. The response to constriction of the aortic annulus was confirmed with 50 mM KC1. The aortic annulus was then left to constrict with 1 μ? of phenylephrine, and stably constricted samples were observed for the endothelium-dependent vasodilator response using 1 μ? of acetylcholine to confirm that endothelial functions were tained. Next, the aortic annulus was preliminarily constrained with 1 uM phenylephrine, and VPP and IPP prepared in the Synthesis Example were added at concentrations in 10-fold increments of 10"9 M to observe the vasodilator response through the change in stress The dose dependence of the vasodilator response was also studied.The result was that the vasodilator response to PPV and PPI was observed from the concentration of 1 mM, and a transient, clear vasodilatory response was confirmed. 10 mM The results were shown in Figure 1. < Confirmatory Test for the Endothelium Dependence of the Vasodilator Response to PPV and IPP > In order to confirm that the vasodilatory effect of PPV and PPI was associated with the vascular endothelium, He conducted a vasodilation test similar to the previous one, with VPP, used as a control, a blood vessel from which the vascular endothelium was physically removed by a routine method. The result was that the vasodilator response was weakened due to the elimination of the endothelium, indicating that the vasodilatory effect of PPV and IPP was highly dependent on the endothelium. The results are shown in Figure 2.

Comparative Example 1 The vasodilation test was conducted in the same manner as in Example 1, except that the tripeptides VPP and IPP were replaced with amino acids, valine (Val) and proline (Pro), at the same concentration. The results were that no vasodilator response was observed at 10 m either with Val or Pro. The results are shown in Figure 1. From the results discussed above, it is understood that VPP and IPP are active as vasodilators in the peptide forms .

Reference Example JP-2004-244359-A reports a vasodilator medical composition and a vasodilator health food composition that contains, as a active component, the peptides obtained by the hydrolysis of proteins derived from various milk proteins. Based on conventional wisdom that ß-casein and -casein have the amino acid sequences that include VPP and IPP, the peptides obtained in the Production Examples described in this publication were measured for VPP and IPP. The commercially available skim milk, which is a protein material containing ß-casein and -casein was tested among the material used in the Production Examples. 1 kg of the skim milk was suspended in 2 liters of hot water, adjusted to pH 7.5, mixed with 40 g of thermoase (manufactured by DAIWA FINE CHEMICALS CO., LTD.), And reacted at 50 ° C for 16 hours . After the reaction, the reaction liquid was heated at 100 ° C for 10 minutes to inactivate the enzyme, to obtain the hydrolyzed peptides. Through analysis with a high resolution liquid chromatograph-mass spectrometer, it was confirmed that the obtained hydrolyzed peptides did not include VPP or IPP. From the results discussed above, it was shown that the vasodilation reported in JP-2004-244359-A was caused by the different components of VPP and IPP.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (2)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A vasodilator, characterized in that it comprises at least one of the peptides Val-Pro-Pro and Ile-Pro-Pro as an active component.
2. 2. A vasodilator, characterized in that it comprises a proteolytic product containing Val-Pro-Pro and / or Ile-Pro-Pro as an active component.