Composition for treating heart disease

Abstract

To provide compositions for use in suppressing cardiomyocyte hypertrophy, and to provide compositions for use in the treatment of heart disease and the like caused by cardiomyocyte hypertrophy.SOLUTION: According to the present invention, provided is a composition for suppressing cardiomyocyte hypertrophy, containing cocoa polyphenol as an active ingredient. According to the present invention, also provided is a composition for use in the treatment, prevention or amelioration of heart disease caused by cardiomyocyte hypertrophy, containing cocoa polyphenol as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for treating heart disease, particularly to a composition for treating heart disease caused by cardiomyocyte hypertrophy. The present invention also relates to a composition for inhibiting cardiomyocyte hypertrophy. [Background technology]

[0002] Heart disease has long been the second leading cause of death in Japan, and is one of the diseases that must be overcome. Among these, heart failure is the most common cause of death, and in Japan, where the aging population is driving an increase in the number of people with heart disease, the development of a new treatment for heart failure has been desired. Heart failure is the final stage of all heart diseases, including ischemic heart disease and hypertensive heart disease, which are on the rise. Of these, ischemic heart disease is primarily caused by coronary arteriosclerosis, and various drug therapies have been developed to prevent risk factors such as hypercholesterolemia and hypertension.

[0003] However, once cardiomyocytes are lost due to ischemic heart disease, they rarely regenerate, so the remaining cardiomyocytes are burdened. The remaining cardiomyocytes respond by hypertrophy, but this is also a limited compensatory mechanism, and the patient eventually progresses to heart failure. As such, the pathology of heart failure caused by hypertrophy of cardiomyocytes is more complex than that of ischemic heart disease, and although cardiac stimulants, ACE inhibitors, angiotensin receptor antagonists, vasodilators, etc. are used, the current situation is that sufficient effects have not been obtained.

[0004] The effects of cacao polyphenols on arteriosclerosis model mice (Non-Patent Document 1) and on hypertension model mice (Non-Patent Document 2) have been reported, but their effect on cardiomyocyte hypertrophy has not been reported. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Natsume M et al., Subcell Biochem, 77:189-198(2014) [Non-Patent Document 2] Quinones M et al., Food Funct. 11:649-653(2011) Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a composition for use in inhibiting cardiomyocyte hypertrophy and a cardiomyocyte hypertrophy inhibitor. The present invention also aims to provide a composition for use in treating heart diseases caused by cardiomyocyte hypertrophy and a therapeutic agent for heart diseases caused by cardiomyocyte hypertrophy. [Means for solving the problem]

[0007] The present inventors have now found that when a cocoa bean extract rich in cocoa polyphenols was added to primary cultured rat cardiomyocytes in which cardiomyocyte hypertrophy had been induced, the cardiomyocyte hypertrophy was significantly suppressed. The present inventors have also found that the transcription of cardiac hypertrophy response genes and the phosphorylation of ERK1 / 2, a kinase that induces cardiomyocyte hypertrophy, were significantly suppressed by the addition of cocoa polyphenols. The present inventors have further found that the administration of cocoa polyphenols suppressed the increase in heart weight and the increase in left ventricular wall thickness, which are characteristic of heart disease caused by cardiomyocyte hypertrophy, in a mouse model of induced cardiac hypertrophy. The present invention is based on these findings.

[0008] That is, according to the present invention, the following inventions are provided. [1] A composition for inhibiting cardiomyocyte hypertrophy and a cardiomyocyte hypertrophy inhibitor comprising cocoa polyphenols as an active ingredient. [2] The composition and agent described in [1] above, which is ingested or administered to a subject who has developed a disease or condition that causes cardiomyocyte hypertrophy or a subject who is at risk of developing said disease or condition. [3] The composition and agent according to [1] or [2] above, which is in the form of an oil or fat processing composition. [4] The composition and agent according to any one of [1] to [3] above for use in the treatment, prevention or amelioration of a disease or symptom that can be treated, prevented or ameliorated by suppressing cardiomyocyte hypertrophy. [5] The composition and agent described in [4] above, wherein the disease or symptom is a cardiac disease caused by cardiomyocyte hypertrophy. [6] A composition for inhibiting the activation of ERK1 protein and / or ERK2 protein, and an inhibitor of the activation of ERK1 protein and / or ERK2 protein, comprising cocoa polyphenols as an active ingredient.

[0009] The composition and the inhibitor described in [1] and [6] above will be referred to hereinafter as the "composition of the present invention" and the "agent of the present invention", respectively, and may be collectively referred to as the "composition and agent of the present invention".

[0010] The compositions and agents of the present invention utilize polyphenols contained in cacao, which has been used as a food ingredient for many years, and are therefore advantageous in that they have few side effects and are highly safe, even when taken over a long period of time in the hope of suppressing cardiomyocyte hypertrophy and treating heart diseases caused by cardiomyocyte hypertrophy. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the cell area of ​​photographic images of each cell after immunostaining (expressed as mean ± standard deviation; One-way ANOVA, Dunnett's multiple comparison test). [Diagram 2] FIG. 2 is a graph showing the induction of atrial natriuretic factor (ANF) gene expression by phenylephrine (PE) and the transcriptional repression by CBP (expressed as mean ± standard deviation; One-way ANOVA, Dunnett's multiple comparison test). [Diagram 3]FIG. 3 is a graph showing the induction of gene expression of brain natriuretic peptide (BNP) by phenylephrine (PE) and the transcriptional repression by CBP (expressed as mean ± standard deviation; One-way ANOVA, Dunnett's multiple comparison test). [Figure 4] Figure 4 is an electrophoretic photograph (Western blotting) showing activation of ERK signaling by phenylephrine (PE) and inhibition of ERK signaling by CBP. pERK1 / 2 shows the band of phosphorylated ERK1 / 2. tERK1 / 2 shows the band of modified and unmodified ERK1 / 2 combined. [Diagram 5] FIG. 5 is a graph showing activation of ERK1 signaling by phenylephrine (PE) and inhibition of ERK1 signaling activation by CBP (expressed as mean ± standard deviation; One-way ANOVA, Dunnett's multiple comparison test). [Figure 6] FIG. 6 is a graph showing activation of ERK2 signaling by phenylephrine (PE) and inhibition of ERK2 signaling activation by CBP (expressed as mean ± standard deviation; One-way ANOVA, Dunnett's multiple comparison test). [Figure 7] 7 is a graph showing the increase in heart weight due to transverse aortic coarctation surgery (hereinafter referred to as "TAC surgery") and the suppression of heart weight by CBP. Sham: control group, TAC: TAC surgery group, TAC+High: TAC surgery group administered with high-dose CBP, TAC+Middle: TAC surgery group administered with medium-dose CBP, TAC+Low: TAC surgery group administered with low-dose CBP. [Figure 8] Figure 8 is a graph showing the increase in heart weight / body weight ratio due to TAC surgery and the suppression of heart weight / body weight ratio by CBP. Sham: control group, TAC: TAC surgery group, TAC+High: TAC surgery group administered with high-dose CBP, TAC+Middle: TAC surgery group administered with medium-dose CBP, TAC+Low: TAC surgery group administered with low-dose CBP. [Figure 9]Figure 9 shows echocardiograms showing the increase in left ventricular wall thickness due to TAC surgery and the inhibition of the increase in left ventricular wall thickness by CBP. Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 10] Figure 10 is a graph showing the increase in left ventricular wall thickness (LVPWd) due to TAC surgery and the inhibition of the increase in left ventricular wall thickness by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 11] Figure 11 is a graph showing the decrease in cardiac fractional shortening (FS) due to TAC surgery and the inhibition of the decrease in cardiac fractional shortening due to CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 12] Figure 12 is a graph showing the decline in left ventricular ejection fraction (EF) due to TAC surgery and the inhibition of the decline in left ventricular ejection fraction by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 13] Figure 13 is a graph showing the increase in heart weight-tibial length ratio (HW / TL) by TAC surgery and the inhibition of the increase in heart weight-tibial length ratio by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 14]Figure 14 is a graph showing the hypertrophy of cardiomyocytes caused by TAC surgery and the inhibition of hypertrophy of cardiomyocytes by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 15] Figure 15 is a graph showing the induction of atrial natriuretic factor (ANF) gene expression by phenylephrine (PE) and transcriptional repression by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 16] Figure 16 is a graph showing the induction of gene expression of brain natriuretic peptide (BNP) by phenylephrine (PE) and the transcriptional repression by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 17] Figure 17 is an electrophoretic photograph (Western blotting) showing activation of ERK signaling by phenylephrine (PE) and inhibition of ERK signaling by CBP. pERK1 / 2 shows the band of phosphorylated ERK1 / 2. tERK1 / 2 shows the band of modified and unmodified ERK1 / 2 combined. [Figure 18] Figure 18 is a graph showing activation of ERK1 signaling by phenylephrine (PE) and inhibition of ERK1 signaling activation by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. [Figure 19]Figure 19 is a graph showing activation of ERK2 signaling by phenylephrine (PE) and inhibition of ERK2 signaling activation by CBP (expressed as mean ± standard deviation. Dunnett's multiple comparison test). Sham: control group, TAC: TAC surgery group, TAC+Low: TAC surgery group administered with low-dose CBP, TAC+High: TAC surgery group administered with high-dose CBP. Description of the Invention

[0012] In the present invention, "cocoa polyphenol" refers to polyphenols contained in cocoa. Thus, typically, cocoa polyphenols extracted (including crude extraction) or purified (including crude purification) from the cocoa plant or its processed products can be used as the active ingredient of the present invention, but polyphenols prepared by chemical synthesis may also be used as part or all of the cocoa polyphenols. Examples of cocoa polyphenols include monomers such as catechin, procyanidins formed by polymerization of catechin, and oligomers (dimers or higher) such as tannin.

[0013] In the present invention, examples of the cacao plant or its processed products that can be used as the raw material for cacao polyphenols include various parts of the plant or processed products of cacao beans, such as cacao bark, cacao leaves, cacao beans, cacao shells, cacao mass, defatted cacao mass, and cocoa powder. Cacao mass is obtained by grinding cacao beans, and defatted cacao mass can be obtained by removing fats and oils from cacao mass. The method for removing fats and oils is not particularly limited, and can be performed according to a known method such as squeezing. Cocoa powder can be obtained by crushing the defatted cacao mass. In addition, when extracting using the cacao plant or its processed products as the raw material, it is preferable to use cacao mass or cocoa powder that has been subjected to a micronization treatment such as grinding or pulverization from the viewpoint of extraction efficiency. In addition, the cacao plant can also include things other than the cacao plant, whether intentionally or unintentionally. In addition, when extracting cacao plants or their processed products as raw materials, substances other than cacao plants may be included, either intentionally or unintentionally. Furthermore, cacao mass or cocoa powder may also contain substances other than cacao plants, either intentionally or unintentionally.

[0014] The extraction method using the cacao plant or its processed product as the raw material is known, and for example, the cacao polyphenol-containing composition can be prepared according to the description in JP 2009-183229 A or JP 2011-93807 A. The extraction solvent is not particularly limited, but it is preferable to use water or alcohol such as ethanol. In addition, the purification method using the cacao plant or its processed product as the raw material is not particularly limited, and may use known methods such as synthetic adsorbents, ion exchange resins, ultrafiltration, and activated clay treatment.

[0015] In the present invention, the total mass of polyphenols can be measured by the Prussian Blue method. For example, the total mass can be calculated using commercially available epicatechin as a standard substance according to the method described in Martin L. Price and Larry G. Butler, J. Agric Food Chem., Vol. 25, No. 6, 1268-1273, 1977. The content of each component of cocoa polyphenols can be measured by high performance liquid chromatography (HPLC) using commercially available epicatechin as a standard substance.

[0016] In order to efficiently administer or ingest cocoa polyphenols, it is preferable to use a composition enriched in cocoa polyphenols in the present invention. In this case, a cocoa polyphenol enriched composition obtained according to a known method (for example, the method described in JP 2009-183229 A) can be used in the present invention.

[0017] Since cacao polyphenols can be prepared using cacao plants as raw materials, the compositions and preparations of the present invention may contain components derived from cacao beans other than cacao polyphenols. Such components include theobromine, caffeine, proteins, peptides, amino acids, sugars, carbohydrates, lipids, dietary fiber, minerals, etc. However, it is known that theobromine has physiological effects such as headache, and caffeine has physiological effects such as insomnia, nausea, and headache (when used regularly). In addition, as shown in the examples below, it has been shown that cacao polyphenols that are substantially free of theobromine and caffeine exhibit cardiomyocyte hypertrophy suppression effects, etc. Therefore, the compositions and preparations of the present invention can be substantially free of either or both of theobromine and caffeine. In such compositions and preparations, the content of theobromine and caffeine can be below the lower limit of quantification, preferably below the detection limit. The compositions and preparations of the present invention, which are substantially free of either or both of theobromine and caffeine, are advantageous in that they can suppress myocardial cell hypertrophy while avoiding side effects such as headache and nausea.

[0018] As shown in the examples below, cacao polyphenols have an inhibitory effect on myocardial cell hypertrophy. Therefore, cacao polyphenols can be used to inhibit myocardial cell hypertrophy. In the present invention, the degree of "inhibition of myocardial cell hypertrophy" can be evaluated using the area of ​​myocardial cells induced by phenylephrine (PE) as an index (see Example 1). Specifically, myocardial cell hypertrophy is induced by phenylephrine in the presence or absence of a test sample, and when the area of ​​myocardial cells in the presence of the test sample is significantly reduced (significance level 5%) compared to the area of ​​myocardial cells in the absence of the test sample, it can be determined that the test sample inhibits myocardial cell hypertrophy. In the present invention, the degree of "inhibition of myocardial cell hypertrophy" can also be evaluated using the gene expression level of cardiac hypertrophy response genes (cardiac hypertrophy markers) such as atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) as an index (see Example 2). Specifically, phenylephrine is used to induce hypertrophy of cardiomyocytes in the presence or absence of a test sample, and when the expression level of the cardiac hypertrophy response gene in the presence of the test sample is significantly (significance level 5%) lower than the expression level of the cardiac hypertrophy response gene in the absence of the test sample, it can be determined that the test sample has suppressed hypertrophy of cardiomyocytes. In the present invention, the degree of "suppression of cardiomyocyte hypertrophy" can be evaluated using the diameter of cardiomyocytes of the heart excised from a pressure overload-induced cardiac hypertrophy model mouse as an index (see Example 5). Specifically, the hearts are excised from a group of pressure overload-induced cardiac hypertrophy model mice administered with a test sample and a group of mice not administered with the test sample, and the diameter of cardiomyocytes is measured for each group. When the diameter of cardiomyocytes of the test sample-administered group is significantly (significance level 5%) lower than the diameter of cardiomyocytes of the test sample-unadministered group, it can be determined that the test sample has suppressed hypertrophy of cardiomyocytes.

[0019] As mentioned above, cocoa polyphenols have an inhibitory effect on myocardial cell hypertrophy, but it is known that myocardial cell hypertrophy plays an important role in the onset and progression of heart failure and myocarditis (Heineke J & Molkentin JD, Nat Rev Mol Cell Biol. 7:589-600(2006) and Marian AJ & Braunwald E, Circ. Res. 121:749-770(2017)). In addition, as shown in the examples below, cocoa polyphenols actually suppressed the increase in heart weight in a pressure overload-induced model mouse in which cardiac hypertrophy and resulting heart failure were induced, suppressed the expression of cardiac hypertrophy response genes, and suppressed the increase in left ventricular wall thickness, decrease in cardiac shortening fraction, and decrease in left ventricular ejection fraction, which are characteristic of heart disease caused by myocardial cell hypertrophy. Therefore, cocoa polyphenols can be used to treat, prevent, or improve diseases or symptoms that can be treated, prevented, or improved by suppressing myocardial cell hypertrophy. Here, examples of diseases or symptoms that can be treated, prevented, or improved by suppressing cardiomyocyte hypertrophy include heart diseases caused by cardiomyocyte hypertrophy, such as heart failure caused by cardiomyocyte hypertrophy and myocarditis caused by cardiomyocyte hypertrophy. Note that, in the present invention, "heart diseases caused by cardiomyocyte hypertrophy" are to be distinguished from cardiovascular diseases such as arteriosclerosis.

[0020] In addition, it is known that ischemic heart disease, hypertension, valvular disease, etc., can cause hypertrophy of cardiomyocytes (Hill JA and Olson EN, N Engl J Med. 358:1370-80(2008)). Therefore, the composition and agent of the present invention can be ingested or administered to a subject who has developed a disease or condition that causes hypertrophy of cardiomyocytes (e.g., ischemic heart disease, hypertension, valvular disease) or a subject who is at risk of developing the disease or condition, thereby more effectively suppressing hypertrophy of cardiomyocytes.

[0021] Furthermore, as shown in the Examples below, cocoa polyphenols can inhibit the phosphorylation of ERK1 protein and ERK2 protein, which are extracellular signal-regulated kinases, and inhibit their activation. Therefore, according to the present invention, there are provided a composition for inhibiting the activation of ERK1 protein and / or ERK2 protein, and an inhibitor of the activation of ERK1 protein and / or ERK2 protein, which contain cocoa polyphenols as an active ingredient. In the present invention, the degree of "inhibition of the activation of ERK1 protein and / or ERK2 protein" can be evaluated using the degree of phosphorylation of ERK1 protein and / or ERK2 protein as an index (see Examples 3 and 5). Specifically, cardiomyocytes are stimulated with phenylephrine in the presence or absence of a test sample, and if the amount of phosphorylation of ERK1 protein and / or ERK2 protein or the ratio of phosphorylated protein to total ERK1 protein and / or ERK2 protein in the presence of the test sample is significantly reduced (significance level 5%) compared to the amount of phosphorylation or the ratio of phosphorylated protein to total ERK1 protein and / or ERK2 protein in the absence of the test sample, it can be determined that the test sample has inhibited the phosphorylation of ERK1 protein and / or ERK2 protein. Alternatively, hearts are removed from a group of pressure overload-induced cardiac hypertrophy model mice administered a test sample and a group of mice not administered the test sample, and the amount of phosphorylation of ERK1 protein and / or ERK2 protein is measured for each.If the amount of phosphorylation of ERK1 protein and / or ERK2 protein or the ratio of phosphorylated protein to total ERK1 protein and / or ERK2 protein in the test sample-administered group is significantly lower (significance level 5%) than the amount of phosphorylation or the ratio of phosphorylated protein to total ERK1 protein and / or ERK2 protein in the test sample-administered group, it can be determined that the test sample has inhibited the phosphorylation of ERK1 protein and / or ERK2 protein.It is known that ERK1 protein and ERK2 protein are activated by phosphorylation and induce cardiomyocyte hypertrophy (Liang Q et al., Mol. Cell. Biol. 21:7460-7469(2001)), and the above-mentioned composition and agent are advantageous in that they can suppress the activation of ERK1 protein and ERK2 protein, which are factors in cardiomyocyte hypertrophy.

[0022] The composition and preparation of the present invention may contain cocoa polyphenols alone or in combination with other ingredients. The amount of cocoa polyphenols in the composition and preparation of the present invention may be determined as desired depending on the purpose, use, form, dosage form, symptoms, body weight, etc., and the present invention is not limited thereto. The content may be 0.1-90% (w / w) based on the total amount, and more preferably 0.1-50% (w / w). In the present invention, the preparation of the present invention may be made of cocoa polyphenols, and the composition of the present invention may contain cocoa polyphenols and other ingredients.

[0023] The compositions and agents of the present invention can be provided in the form of pharmaceuticals (eg, pharmaceutical compositions), quasi-drugs, foods and beverages, feeds, etc., and can be implemented according to the description below.

[0024] The cacao polyphenols, which are the active ingredients of the present invention, can be orally administered to humans and non-human animals. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavors, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

[0025] The active ingredient of the present invention, cacao polyphenol, can be administered to humans and non-human animals by a route other than oral administration (e.g., feeding tube administration, nasogastric tube administration, drip infusion, suppository, etc.) depending on the form of the composition and preparation of the present invention. For example, the composition and preparation of the present invention can be administered to humans and non-human animals with reduced chewing and swallowing functions and unable to take orally orally by making them take orally by making them take or administer them by a route other than oral administration, even if the chewing and swallowing functions are reduced due to aging, etc.

[0026] The cacao polyphenols, which are the active ingredients of the present invention, can be orally ingested by humans and non-human animals. When cacao polyphenols are orally ingested, they may be in an isolated, purified or crude form, or in the form of a food or food ingredient containing them. When cacao polyphenols are orally ingested by humans and non-human animals, they may be in any state selected from room temperature, warm, cold, etc. For example, by making them into a viscous liquid composition containing cacao polyphenols, such as a cocoa drink, or a semi-solid composition containing cacao polyphenols, such as chocolate, they can be ingested by humans and non-human animals who have reduced mastication and swallowing functions and cannot take orally orally administer them.

[0027] When the active ingredient of the present invention, cacao polyphenol, is provided as a food, the cacao polyphenol can be directly contained in the food, and the food contains an effective amount of cacao polyphenol. Here, "containing an effective amount" of cacao polyphenol refers to a content in which the cacao polyphenol is ingested in a range described below when the amount of each food is normally consumed. The term "food" is used to mean health food, functional food, health functional food (e.g., food for specified health uses, food with nutritional function claims, food with functional claims), and special purpose food (e.g., food for infants, food for pregnant women, food for sick people). The form of the "food" is not particularly limited, and may be, for example, a beverage, a semi-liquid or gel-like form, or a solid form. For example, by making it into a viscous liquid composition containing cacao polyphenol, such as a cocoa drink, or a semi-solid composition containing cacao polyphenol, such as chocolate, it can be ingested by humans and non-human animals who have reduced mastication and swallowing functions and cannot take orally orally.

[0028] The active ingredient of the present invention, cacao polyphenol, has the effect of suppressing myocardial cell hypertrophy and cardiac hypertrophy, and therefore can be provided by being contained in foods taken daily or foods taken as supplements. The food provided by the present invention is not particularly limited in its form or shape, but preferably includes foods made mainly from cacao beans, more preferably oil-processed compositions, and even more preferably oil-processed foods such as chocolate and cocoa. For example, by making it into a viscous liquid composition containing cacao polyphenol, such as a cocoa drink, or a semi-solid composition containing cacao polyphenol, such as chocolate, it can be ingested by humans and non-human animals who have reduced mastication and swallowing functions and cannot take orally orally.

[0029] As described above, in order to efficiently ingest cocoa polyphenols, a composition enriched with cocoa polyphenols can be used in the present invention. Therefore, the food and supplements made mainly from cocoa beans are preferably those containing high concentrations of cocoa polyphenols, more preferably oil-processed compositions containing high concentrations of cocoa polyphenols, and even more preferably chocolate and cocoa containing high concentrations of cocoa polyphenols.

[0030] Here, the content of cocoa polyphenols in foods and supplements is not particularly limited as long as intake of cocoa polyphenols is possible, but from the viewpoint of efficient intake of cocoa polyphenols, the content in the oil or fat processed composition may be, for example, 1 to 10 mass%, preferably 1.2 to 8 mass%, more preferably 1.4 to 7 mass%, even more preferably 1.6 to 6 mass%, even more preferably 1.8 to 5 mass%, and particularly preferably 2 to 4.5 mass%, based on the solid content of the composition.

[0031] The food provided by the present invention is not limited to any food that contains cacao beans as the main ingredient, such as chocolate and cocoa, but is also not limited to any food that can contain cacao polyphenols as an active ingredient. Examples of the food include starch-based foods such as bread, biscuits, noodles, crackers, and nutritional bars; various confectioneries such as candies, gums, gummies, and snacks; milk and dairy products such as milk, processed milk, ice cream, fermented milk (yogurt, etc.), milk drinks, cheeses, butters, and creams; desserts such as puddings, jellies, bavarois, and mousses; beverages such as non-alcoholic and alcoholic beverages; processed meat products such as ham and sausages; processed fish products such as kamaboko, chikuwa, and fish sausages; processed fruit products such as jams and purees; and seasonings such as roux and sauces. Cacao polyphenols can be used in a variety of ways depending on the characteristics and purpose of each food. Depending on the purpose, they can be appropriately mixed at a suitable stage of the manufacturing process.

[0032] The pharmaceuticals and foods of the present invention utilize polyphenols contained in cacao beans, which have long been used as a food, and therefore can be safely used by mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.) that require them. The dosage or intake of cacao polyphenols can be determined depending on the recipient's sex, age, and weight, symptoms, administration time, dosage form, administration route, and drugs to be combined, etc.

[0033] For example, when cacao polyphenols are orally administered as a medicine, the amount administered to an adult per day is preferably in the range of 10 to 2000 mg, more preferably 50 to 1800 mg, even more preferably 100 to 1500 mg, even more preferably 125 to 1400 mg, particularly preferably 200 to 1200 mg, even more particularly preferably 220 to 1100 mg, and most preferably 500 to 1000 mg.

[0034] When cacao polyphenols are ingested as a food product, they can be ingested so that the amount per day for an adult is preferably in the range of 10 to 2000 mg, more preferably 50 to 1800 mg, even more preferably 100 to 1500 mg, even more preferably 125 to 1400 mg, particularly preferably 200 to 1200 mg, even more particularly preferably 220 to 1100 mg, and most preferably 500 to 1000 mg.

[0035] The composition and agent of the present invention may be used in combination with other compositions and agents that can be orally ingested, without any restrictions. For example, by using the composition and agent in combination with materials or compositions that are expected to suppress myocardial cell hypertrophy, or to prevent, treat, and improve heart diseases caused by myocardial cell hypertrophy, the effect of suppressing myocardial cell hypertrophy, or the effect of preventing, treating, and improving heart diseases caused by myocardial cell hypertrophy can be further enhanced.

[0036] The composition and agent of the present invention can be provided as a composition comprising a one-day or two-day intake of cocoa polyphenols effective for suppressing myocardial cell hypertrophy. In this case, the composition and agent of the present invention may be packaged so that one-day or two-day effective intake can be taken, and the package form may be a single package or multiple packages as long as one-day or two-day effective intake can be taken. When provided in a packaged form, it is desirable that the package has a description regarding the intake amount so that one-day or two-day effective intake can be taken, or that a document with such description is provided together. In addition, when providing one-day or two-day effective intake in multiple packages, multiple packages of one-day or two-day effective intake can be provided as a set for convenience of intake.

[0037] In this case, a composition containing a one-day amount of cocoa polyphenols is preferably taken once per day, and a composition containing a two-day amount of cocoa polyphenols is preferably taken once every two days.

[0038] The packaging form for providing the compositions and agents of the present invention is not particularly limited as long as it specifies a certain amount, and examples include wrapping paper, bags, soft bags, paper containers, cans, bottles, capsules, and other containers that can accommodate the composition and agents of the present invention.

[0039] In order for the composition and agent of the present invention to exert its effect more effectively, the administration and ingestion period is preferably continuous administration or ingestion for 6 weeks or more, more preferably continuous administration or ingestion for 6 to 18 weeks, and particularly preferably continuous administration or ingestion for 9 to 15 weeks. Here, "continuously" means continuous administration every day or administration once every two days. When the composition and agent of the present invention are provided in a packaged form, an effective intake amount for a certain period (e.g., 1 week) may be provided as a set for continuous ingestion.

[0040] According to another aspect of the present invention, there is provided a method for inhibiting cardiomyocyte hypertrophy, comprising administering or administering an effective amount of cocoa polyphenols to a human or non-human animal. According to another aspect of the present invention, there is provided a method for inhibiting the activation of ERK1 protein and / or ERK2 protein, comprising administering or administering an effective amount of cocoa polyphenols to a human or non-human animal. The method of the present invention can be carried out according to the description of the composition and preparation of the present invention.

[0041] In accordance with yet another aspect of the present invention, there is provided a method for treating, preventing or ameliorating a disease or condition that can be treated, prevented or ameliorated by inhibiting cardiomyocyte hypertrophy, comprising ingesting or administering an effective amount of cocoa polyphenols to a human or non-human animal. The method for treating, preventing or ameliorating a disease or condition can be carried out according to the description of the compositions and preparations of the present invention.

[0042] According to yet another aspect of the present invention, there is provided the use of cocoa polyphenols for the manufacture of an inhibitor of cardiomyocyte hypertrophy, and the use of cocoa polyphenols as an inhibitor of cardiomyocyte hypertrophy. According to the present invention, there is also provided the use of cocoa polyphenols for the manufacture of an inhibitor of ERK1 protein and / or ERK2 protein activation, and the use of cocoa polyphenols as an inhibitor of ERK1 protein and / or ERK2 protein activation. According to the present invention, there is also provided the use of cocoa polyphenols for the manufacture of a therapeutic, preventive or ameliorating agent for a disease or symptom that can be treated, prevented or ameliorated by the inhibition of cardiomyocyte hypertrophy, and the use of cocoa polyphenols as a therapeutic, preventive or ameliorating agent for a disease or symptom that can be treated, prevented or ameliorated by the inhibition of cardiomyocyte hypertrophy. The use of the present invention can be carried out according to the description of the composition and the agent of the present invention.

[0043] According to still further aspects of the present invention there are provided cocoa polyphenols for use in inhibiting cardiomyocyte hypertrophy, cocoa polyphenols for use in inhibiting activation of ERK1 protein and / or ERK2 protein, and cocoa polyphenols for use in the treatment, prevention or amelioration of diseases or symptoms that can be treated, prevented or ameliorated by inhibiting cardiomyocyte hypertrophy. The above cocoa polyphenols can be used in accordance with the description of the compositions and preparations of the present invention.

[0044] The method and use of the present invention may be used in mammals, including humans, and both therapeutic and non-therapeutic uses are intended. As used herein, "non-therapeutic" means not including surgical, therapeutic or diagnostic procedures for humans (i.e., medical procedures for humans), and specifically means not including methods of surgical, therapeutic or diagnostic procedures for humans performed by a physician or a person under the supervision of a physician. EXAMPLES

[0045] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0046] Example 1: Inhibitory effect of cocoa bean extract on myocardial cell hypertrophy (1) (1) Preparation of cocoa bean extract First, 150 kg of cacao beans were heated at 80°C for 1 hour. Next, the heated cacao beans were defatted using an expeller to obtain 80 kg of cacao powder. 800 kg of ethanol (50%) was added to the defatted cacao powder and then heated. Then, after the temperature reached 50°C, stirring and extraction was performed for 1 hour, and the extract was left at 4°C overnight. The extract was filtered using a screw decanter and a filter press, and the filtrate was concentrated under reduced pressure to 27 kg, and then 13 kg of ethanol (95%) was added. Then, the concentrated liquid was stirred at 40°C for 1 hour, and then refrigerated and stored overnight. The concentrated liquid was centrifuged (25°C, 12000 rpm) using a Sharpless centrifuge. Then, 35 kg of acid clay (Mizuka Ace #20, manufactured by Mizusawa Chemical Industries Co., Ltd.) was added to the supernatant of this centrifugation and then stirred for 1 hour. The mixture was filtered using a Nutsche, the filtrate was concentrated under reduced pressure, and then freeze-dried to obtain 6.3 kg of powder. 25 L of anion exchange resin and 50 L of water were added to 0.5 kg of the powder, and the mixture was stirred for 30 minutes or more. After collecting the resin, 75 L of ethanol (80%) was added and the mixture was stirred for 20 to 30 minutes. The mixture was filtered using a Nutsche, the filtrate was concentrated under reduced pressure, and then freeze-dried to obtain a cacao bean extract rich in cacao polyphenols (hereinafter sometimes referred to as "CBP" (Cacao Bean Polyphenol Extract)).

[0047] The dried CBP was redissolved in 100 μL of methanol aqueous solution (50 mass%) to prepare an analytical sample. The polyphenol content and procyanidin polymer content of the analytical sample were analyzed. Specifically, total polyphenols were analyzed by the Prussian blue colorimetric method, procyanidins were analyzed by the reversed-phase HPLC method, procyanidin polymers were analyzed by the normal-phase HPLC method based on the method of Kelm et al. (see Kelm, MA et al. J. Agric Food Chem 2006, 54(5); p.1571-1576), and theobromine was analyzed by the HPLC method. Each measurement value was expressed as epicatechin equivalent.

[0048] The results are shown in Tables 1 and 2. The levels of caffeine and theobromine were below the detection limit.

[0049] [Table 1]

[0050] [Table 2]

[0051] (2) Evaluation of the inhibitory effect of cocoa bean extract on cardiomyocyte hypertrophy 2-1: Test method Cardiac cardiomyocytes derived from neonatal rats (SD rats, 1 day old, obtained from Japan SLC) were cultured as primary cultures. The CBP prepared in (1) above was added to the primary cultured rat cardiomyocytes to a total polyphenol concentration (epicatechin equivalent equivalent) of 3 μM, 10 μM, or 30 μM, and the cells were treated for 2 hours. Next, phenylephrine (PE, Wako Pure Chemical Industries, Ltd.), an adrenergic α1 receptor stimulant that induces cardiomyocyte hypertrophy, was added to a final concentration of 30 μM, and the cells were treated for 48 hours to induce cardiomyocyte hypertrophy (experimental group). The control group was treated in the same manner as the experimental group, without adding CBP and / or PE. After completion of the cardiomyocyte hypertrophy induction stimulation, the cells were fixed, permeabilized, and immunostained using anti-β myosin heavy chain (β-MHC) antibody. After staining, the cells were photographed under a microscope, and the cell area was measured using image analysis software ImageJ (obtained from the National Institutes of Health, USA; reference URL: https: / / imagej.nih.gov / ij / ).

[0052] 2-2: Test results The test results are shown in Figure 1.

[0053] From the results in Figure 1, it was confirmed that phenylephrine (PE)-induced cardiomyocyte hypertrophy was significantly suppressed by 30 μM CBP. The cell area of ​​cardiomyocytes treated with 30 μM CBP in the absence of phenylephrine (PE) was not changed from that in the absence of phenylephrine (PE) and CBP, suggesting that CBP does not have cardiomyocyte injury or cardiomyocyte hypertrophy induction effects.

[0054] Example 2: Inhibitory effect of cocoa bean extract on myocardial cell hypertrophy (2) In Example 2, the effect of cocoa bean extracts on transcriptional suppression of cardiac hypertrophy response genes was evaluated.

[0055] 1-1: Test method Induction of cardiomyocyte hypertrophy was performed in the same manner as described in 2-1: Test Method in Example 1(2). After completion of cardiomyocyte hypertrophy induction stimulation, RNA was extracted from rat primary cultured cardiomyocytes using a commercially available reagent containing guanidine thiocyanate (GTC), reverse transcribed, and the gene expression levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), which are cardiac hypertrophy response genes, were quantified by quantitative reverse transcription PCR. Specifically, the relative expression levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) genes to the expression level of 18S ribosomal RNA were determined using 18S ribosomal RNA as an internal standard gene according to the procedure described in Suzuki H, et al., Biochim. Biophys. Acta. 1862:1544-1557(2016).

[0056] 1-2: Test results The test results are shown in Figures 2 and 3.

[0057] The results of Figures 2 and 3 show that gene expression of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), cardiac hypertrophy response genes induced by phenylephrine (PE), was significantly suppressed by 30 μM CBP.

[0058] Example 3: Inhibitory effect of cocoa bean extract on myocardial cell hypertrophy (3) In Example 3, the inhibitory effect of cocoa bean extracts on extracellular signal-regulated kinase (ERK) activation was evaluated.

[0059] 1-1: Test method Cardiac cardiomyocytes derived from neonatal rats (SD rats, 1 day old, obtained from Japan SLC) were cultured as primary cultures. The CBP prepared in Example 1 (1) was added to the primary cultured rat cardiomyocytes to a total polyphenol concentration (epicatechin equivalent equivalent) of 10 μM and 30 μM, and the mixture was left to stand for 2 hours. Next, phenylephrine (PE) was added to a final concentration of 30 μM, and the mixture was left to stand for 10 minutes to stimulate the cardiomyocytes (experimental group). The control group was treated in the same manner as the experimental group, without adding CBP and / or PE. Next, the cardiomyocytes were lysed with a cell lysis buffer, and the total proteins of the cardiomyocytes were collected, denatured, and separated by SDS-PAGE. Next, Western blotting was performed using anti-phosphorylated ERK1 / 2 antibody and anti-ERK1 / 2 antibody (Cell Signaling Technology), and the activation of ERK1 / 2 was detected by chemiluminescence. The detected bands were quantified by taking images using a C-Digit Blot scanner (Li-COR) and acquiring and quantifying the images using Image Studio Digits Ver.5.2 (Li-COR). The ratio of phosphorylated ERK1 protein (pERK1) to ERK1 protein (tERK1) and the ratio of phosphorylated ERK2 protein (pERK2) to ERK2 protein (tERK2) were calculated.

[0060] 1-2: Test results The test results are shown in Figures 4 to 6.

[0061] From the results in Figures 4 to 6, it was confirmed that the activation of ERK1 / 2 activated by phenylephrine (PE) was significantly inhibited by 30 µM CBP.

[0062] Example 4: Inhibitory effect of cocoa bean extract on myocardial cell hypertrophy (4) In Example 4, the cardiac hypertrophy suppressing effect of a cacao bean extract was evaluated using a pressure overload-induced cardiac hypertrophy model mouse.

[0063] 1-1: Test method Transverse aortic coarctation (TAC) was performed on 17 7-week-old male C57BL / 6J mice (obtained from Japan SLC Co., Ltd.) and pressure overload-induced cardiac hypertrophy model mice were created by applying afterload. As a control group, 3 7-week-old male C57BL / 6J mice were subjected to thoracotomy without aortic ligation (sham surgery). The mice that underwent TAC surgery were randomly divided into 4 groups (non-CBP group (5 mice), high-dose CBP group (4 mice), medium-dose CBP group (4 mice), and low-dose CBP group (4 mice)) on the day after surgery. The CBP prepared in Example 1(1) was dissolved in sterilized water, and the high-dose CBP group was administered 1200 mg / kg body weight / day (solid content equivalent), the medium-dose CBP group was administered 300 mg / kg body weight / day (solid content equivalent), and the low-dose CBP group was administered 50 mg / kg body weight / day (solid content equivalent), twice a day (morning and evening) for two consecutive weeks, by forced oral administration (3.3 mL / kg body weight) using a sonde and a syringe. Body weight was measured before surgery, one week and two weeks after surgery. Two weeks after surgery, the heart was removed under anesthesia, the organ weight was measured, and the heart weight / body weight ratio was calculated.

[0064] 1-2: Test results The test results are shown in Figures 7 and 8.

[0065] From the results of FIG. 7 and FIG. 8, it was confirmed that the heart weight and the heart weight / body weight ratio, which were significantly increased by pressure overload (TAC surgery), tended to be suppressed by administration of high-dose CBP.

[0066] Example 5: Inhibitory effect of cocoa bean extract on myocardial cell hypertrophy (5) In Example 5, the cardiac hypertrophy suppressing effect of a cacao bean extract was evaluated using a pressure overload-induced cardiac hypertrophy model mouse.

[0067] 1-1: Test method Transverse aortic coarctation (TAC) was performed on 17 male C57BL / 6J mice (obtained from Japan CLEA) aged 8 to 10 weeks, and pressure overload-induced cardiac hypertrophy model mice were created by applying afterload. As a control group, 15 male C57BL / 6J mice were subjected to thoracotomy without tying the aorta (Sham surgery). The mice that underwent TAC surgery were randomly divided into three groups (CBP non-administration group (10 mice), low-dose CBP administration group (12 mice), and high-dose CBP administration group (10 mice)) on the day after surgery. CBP prepared in Example 1 (1) was dissolved in sterile water, and 1200 mg / kg body weight / day (solid content equivalent) was administered to the high-dose CBP administration group, and 600 mg / kg body weight / day (solid content equivalent) was administered to the low-dose CBP administration group, once a day (morning) for 8 consecutive weeks, by forced oral administration (6.6 mL / kg body weight) using a sonde and a syringe. Administration began the day after surgery. Body weight was measured before surgery and once a week. There was no significant difference in the mean body weight of each group 8 weeks after surgery, confirming that high-dose CBP administration did not affect normal development of mice. Eight weeks after surgery, echocardiography was performed under anesthesia to measure the left ventricular wall thickness. Specifically, measurements were performed using the HP SONOS 5500 imaging system (Philips) at 10-12 MHz M-mode. Fractional shortening (FS) and left ventricular ejection fraction (EF), which are indicators of cardiac function, were also measured using the HP SONOS 5500 imaging system (Philips) at 10-12 MHz M-mode. The heart was then removed under anesthesia, the organ weight was measured, and the heart weight / tibia length ratio was calculated.

[0068] Next, the excised heart was embedded in paraffin, cut into sections, and stained with HE. After staining, photographs were taken under a microscope, and the diameter of myocardial cells was measured using the image analysis software ImageJ (see above). In addition, RNA was extracted from the excised heart, reverse transcribed, and the gene expression levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), which are cardiac hypertrophy response genes, were measured by quantitative reverse transcription PCR. Specifically, the relative expression levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) genes to the expression level of 18S ribosomal RNA were calculated using 18S ribosomal RNA as an internal standard gene according to the procedure described in Suzuki H, et al., Biochim. Biophys. Acta. 1862:1544-1557 (2016). The excised hearts were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate), denatured, and separated by SDS-PAGE. Phosphorylated ERK1 / 2 and total ERK1 / 2 were detected by chemiluminescence using Western blotting. Quantification of the detected bands was performed by taking images using a C-Digit Blot scanner (Li-COR), and image acquisition and quantification were performed using Image Studio Digits Ver.5.2 (Li-COR). Quantification of the obtained signals was performed by calculating the ratio of phosphorylated ERK1 protein to ERK1 protein and the ratio of phosphorylated ERK2 protein to ERK2 protein.

[0069] 1-2: Test results The test results are shown in Figures 9 to 19.

[0070] From the results of Figures 9 to 12, it was confirmed that the wall thickness significantly increased by pressure overload (TAC surgery) was significantly improved by high-dose CBP administration. It was also confirmed that the cardiac function significantly decreased by pressure overload (TAC surgery) was significantly improved by high-dose CBP administration. From the results of Figure 13, it was confirmed that the heart weight to tibia length ratio increased by pressure overload (TAC surgery) was significantly improved by high-dose CBP administration. From the results of Figure 14, it was confirmed that the cardiomyocyte hypertrophy induced by pressure overload (TAC surgery) was significantly suppressed by CBP administration. From the results of Figures 15 and 16, it was confirmed that the expression level of cardiac hypertrophy-related genes increased by pressure overload (TAC surgery) was significantly suppressed by CBP administration. From the results of Figures 17 to 19, it was confirmed that ERK activation in the heart was suppressed by CBP administration.

Claims

1. A composition for inhibiting myocardial cell hypertrophy (excluding myocardial cell hypertrophy caused by hypertension), comprising as an active ingredient an alcohol extract of defatted cocoa powder containing cocoa polyphenols in a total polyphenol content (Prussian blue colorimetric method) of at least 792 mg / g (converted to solids).

2. The composition of claim 1, which is ingested or administered to a subject who has or is at risk of developing a disease or condition (excluding hypertension) that causes cardiomyocyte hypertrophy.

3. 3. The composition according to claim 1 or 2, in the form of an oil or fat processing composition.

4. The composition according to any one of claims 1 to 3 for use in treating, preventing or ameliorating a disease or symptom which can be treated, prevented or ameliorated by suppressing cardiomyocyte hypertrophy.

5. The composition of claim 4, wherein the disease or condition is a cardiac disease caused by cardiomyocyte hypertrophy.

6. A composition for inhibiting the activation of ERK1 protein and / or ERK2 protein (excluding cardiomyocyte hypertrophy caused by hypertension), comprising as an active ingredient an alcohol extract of defatted cocoa powder containing cocoa polyphenols in a total polyphenol content (Prussian blue colorimetric method) of at least 792 mg / g (converted to solids).

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