WO2022097657A1 - Prophylactic or therapeutic agent for atrial fibrillation, and use thereof - Google Patents

Abstract

A prophylactic or therapeutic agent for atrial fibrillation, which contains a compound represented by formula (1) (in formula (1), R¹ to R⁵ independently represent a hydrogen atom or a methyl group, in which at least one of R¹ to R⁵ represents a methyl group) as an active ingredient; a pharmaceutical composition for preventing or treating atrial fibrillation, which comprises a compound represented by formula (1) and a pharmaceutically acceptable carrier; and a food composition for preventing or ameliorating atrial fibrillation, which contains a compound represented by formula (1) as an active ingredient.

Description

Preventive or therapeutic agents for atrial fibrillation and their use

The present invention relates to a prophylactic or therapeutic agent for atrial fibrillation and its use. More specifically, the present invention relates to an agent for preventing or treating atrial fibrillation, a pharmaceutical composition for preventing or treating atrial fibrillation, a food composition for preventing or improving atrial fibrillation, and a method for preventing or improving atrial fibrillation. Regarding. This application claims priority based on Japanese Patent Application No. 2020-184117 filed in Japan on November 4, 2020, the contents of which are incorporated herein by reference.

Atrial fibrillation is a type of arrhythmia caused by the disturbance of the electrical signal flowing in the atrium, and it is a disease in which the atrium trembles like a spasm and cannot pump blood well to the whole body. As a therapeutic agent for atrial fibrillation, amiodarone, sotalol, atrovastatin and the like are known (see, for example, Non-Patent Documents 1 and 2).

However, the antiarrhythmic drug currently on the market has a limited effect on preventing atrial fibrillation, and more than half of the cases repeat recurrence or shift to permanent atrial fibrillation. In addition, although there are reports of suppression of the recurrence rate of atrial fibrillation by steroid hormones, etc., there is a high possibility that various side effects will occur, and long-term use is not expected. As described above, a preventive treatment method (upstream treatment) for atrial fibrillation has not been clearly established, and there is an urgent need to develop a drug having few side effects. Therefore, an object of the present invention is to provide a technique for preventing, improving or treating atrial fibrillation.

The present invention includes the following aspects.
[1] A prophylactic or therapeutic agent for atrial fibrillation, which comprises a compound represented by the following formula (1) as an active ingredient.

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]
[2] A pharmaceutical composition for preventing or treating atrial fibrillation, which comprises the prophylactic or therapeutic agent according to [1] and a pharmaceutically acceptable carrier.
[3] A food composition for preventing or improving atrial fibrillation, which comprises a compound represented by the following formula (1) as an active ingredient.

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]
[4] The food composition for preventing or improving atrial fibrillation according to [3], which is a food with functional claims.
[5] The food composition for preventing or improving atrial fibrillation according to [3], which is a food for specified health use.
[6] A method for preventing or ameliorating atrial fibrillation (excluding medical practice for humans), which comprises a step of ingesting a compound represented by the following formula (1) in a subject.

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a technique for preventing, improving or treating atrial fibrillation.

1 (a) to 1 (c) are diagrams showing the administration schedule of a drug to mice in each group in Experimental Example 1. FIG. 2 is an X-ray photograph of the state of the experiment in Experimental Example 2. FIG. 3A is a graph showing the atrial fibrillation induction rate of the mice of each group measured in Experimental Example 2. FIG. 3B is a graph showing the duration of atrial fibrillation of mice in each group measured in Experimental Example 2. FIG. 3 (c) is a graph showing the effective refractory period of the mice of each group measured in Experimental Example 2. 4 (a) to 4 (c) are representative photographs showing echocardiograms of mice in each group taken in Experimental Example 3. FIG. 4D is a graph showing the results of measuring atrial sutras based on echocardiograms of mice in each group. FIG. 4 (e) is a graph showing a value (mg / g) obtained by dividing the weight of the atrium by the body weight after removing the heart from the mice of each group. 5 (a) to 5 (c) are representative micrographs showing the results of histochemical staining of mouse specimens of each group in Experimental Example 4. FIG. 5D is a graph showing the results of calculating the ratio (%) of the area occupied by the fibrotic region to the total area of the atria based on the results of histochemical staining of the mouse specimens of each group. FIG. 6A is a photograph showing the results of Western blotting in Experimental Example 5. FIG. 6B is a graph in which the expression level of the NFκB protein is quantified based on FIG. 6A. FIG. 6 (c) is a graph in which the expression level of the TPRC protein is quantified based on FIG. 6 (a). 7 (a) to 7 (c) are typical waveforms measured by a multi-electrode array (MEA) system in Experimental Example 6. FIG. 7 (d) is a graph in which the action potential duration is quantified based on the waveform measured by the MEA system in Experimental Example 6. FIG. 8A is a representative image showing the results of Ca ²⁺ line scan imaging of atrial muscle cells in Experimental Example 7. FIG. 8B is a graph showing the results of measuring the frequency of Ca ²⁺ Sparks occurrence in the atrial muscle cells of each group in Experimental Example 7. FIG. 9 is a photograph showing the results of Western blotting in Experimental Examples 8 and 9. 10 (a) to 10 (c) are graphs in which the results of FIG. 9 are quantified. 11 (a) and 11 (b) are graphs showing the results of quantitative RT-PCR in Experimental Example 11. FIG. 12 is a representative image showing the results of Ca ²⁺ line scan imaging of HL-1 cells in Experimental Example 12.

[Preventive or therapeutic agent for atrial fibrillation]
In one embodiment, the present invention provides a preventive agent for atrial fibrillation or a therapeutic agent for atrial fibrillation, which comprises a compound represented by the following formula (1) as an active ingredient.

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]

As used herein, the term "active ingredient" means that the compound contains a sufficient amount to achieve the efficacy or activity of the compound represented by the above formula (1). Alternatively, it means that the compound represented by the above formula (1) is contained as a main active ingredient.

As will be described later in the examples, the inventors have clarified that atrial fibrillation can be prevented or treated by administering the compound represented by the above formula (1).

Further, as will be described later in the examples, the inventors have clarified that the blood pressure of the hypertensive mouse is lowered by administering the compound represented by the above formula (1). Hypertension is one of the risk factors for atrial fibrillation. Therefore, the subject to which the compound represented by the above formula (1) is administered may be a patient having a blood pressure of 140 mmHg or more, which is generally defined as hypertension.

It can also be said that the compound represented by the above formula (1) is a preventive or therapeutic agent for hypertension.

The compound represented by the above formula (1) is a group of compounds known as O-methylated flavonols. Examples of the O-methylated flavonol include isorhamnetin, tamalixetin, 3-O-methylquercetin, azaleatin, rhamnetin and the like.

Among the compounds represented by the above formula (1), the compound in which R ¹ is a methyl group and R ² , R ³ , R ⁴ , and R ⁵ are hydrogen atoms is a kind of polyphenol called isorhamnetin. The chemical formula of isorhamnetin is shown in the following formula (2). Isorhamnetin is known to be contained in arid plants and the like, and is known to be present in ginkgo leaves and Umbelliferae plants in Japan.

In addition, isorhamnetin can be chemically synthesized from quercetin, which is abundant in the onion hull. Quercetin is a compound represented by the formula (1) in which all of R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are hydrogen atoms. The chemical formula of quercetin is shown in the following formula (3).

Among the compounds represented by the above formula (1), the compound in which R ² is a methyl group and R ¹ , R ³ , R ⁴ and R ⁵ are hydrogen atoms is a kind of polyphenol called tamalixetin. The chemical formula of tamalixetin is shown in the following formula (4).

Among the compounds represented by the above formula ( ¹ ), the compound in which R5 is a methyl group and ^R1 , ^R2 , R3 ^, and ^R4 are hydrogen atoms is a kind of polyphenol called rhamnetin. The chemical formula of ramnetin is shown in the following formula (5).

Among the compounds represented by the above formula ( ¹ ), the compound in which ^R4 is a methyl group and ^R1 , ^R2 , R3 ^, and R5 are hydrogen atoms is a kind of polyphenol called azaleatin. The chemical formula of azaleatin is shown in the following formula (6).

Among the compounds represented by the above formula (1), the compound in which R ³ is a methyl group and R ¹ , R ² , R ⁴ , and R ⁵ are hydrogen atoms is a polyphenol called 3-O-methylquercetin. It is a kind. The chemical formula of 3-O-methylquercetin is shown in the following formula (7).

The prophylactic or therapeutic agent of the present embodiment may contain these O-methylated flavonols as an active ingredient. These O-methylated flavonols may be extracted from natural products, synthesized from naturally abundant quercetin, or made from other non-natural products. It may be synthesized.

Quercetin tends to be less effective in preventing or ameliorating atrial fibrillation than O-methylated flavonols such as isorhamnetin. O-methylated flavonols such as isorhamnetin are less likely to be metabolized than quercetin, and by being present in plasma for a long period of time, atrial fibrillation can be effectively and continuously prevented or ameliorated.

O-methylated flavonols such as isorhamnetin have not been reported to have major side effects in studies to date. Furthermore, isorhamnetin and other O-methylated flavonols have various effects such as antioxidant, anti-inflammatory, anti-obesity, and anti-cancer effects even in experiments focusing on diseases other than cardiovascular diseases. Has been reported. Therefore, it is considered that administration of O-methylated flavonols such as isorhamnetin can produce not only effects on the cardiovascular system such as atrial fibrillation but also various beneficial effects on the human body.

[Pharmaceutical composition for prevention or treatment of atrial fibrillation]
In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating atrial fibrillation, which comprises the above-mentioned preventive or therapeutic agent for atrial fibrillation and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present embodiment is orally or injectably prepared in the form of, for example, tablets, capsules, granules, powders, liquids, etc. according to a conventional method (for example, the method described in the Japanese Pharmacopoeia). , Suppositories, external skin preparations, etc. can be administered parenterally.

As the pharmaceutically acceptable carrier, those usually used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragant gum, and rubber arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, and glycerin; Examples thereof include adhesives such as rubber-based adhesives and silicone-based adhesives.

The pharmaceutical composition may contain additives. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and martitol; flavoring agents such as peppermint and akamono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Buffering agents such as salts and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like can be mentioned.

The pharmaceutical composition can be formulated by appropriately combining the above carriers and additives and mixing them in a generally accepted unit dose form required for pharmaceutical practice. The pharmaceutical composition may be formulated, for example, to be orally administered once a day.

The dose of the pharmaceutical composition varies depending on the patient's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but in the case of oral administration, for example, 0.1 to 100 mg / kg body weight per administration unit form. The active ingredient (compound represented by the above formula (1)) may be administered. In the case of parenteral administration, for example, 0.01 to 50 mg of the active ingredient may be administered per administration unit form.

[Food composition for prevention or improvement of atrial fibrillation]
In one embodiment, the present invention provides a food composition for preventing or ameliorating atrial fibrillation, which comprises a compound represented by the following formula (1) as an active ingredient. As will be described later in the examples, atrial fibrillation can be prevented, ameliorated or treated by ingesting the food composition of the present embodiment.

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]

The food composition of the present embodiment may be, for example, in the form of a supplement, in the form of a semi-solid form such as yogurt or gel-like food, or in the form of a beverage. It may be in the form of any cooked food. Examples of the shape of the supplement include shapes of tablets, capsules, and the like.

The food composition of the present embodiment may be a food with functional claims. "Foods with functional claims" means foods notified to the Consumer Affairs Agency as foods with functional claims on product packages based on scientific evidence.

The food composition of this embodiment may be a food for specified health use. Foods for specified health use mean foods that are recognized to be useful for maintaining and improving health based on scientific evidence and are permitted to be labeled with their effects. The indicated effects and safety will be examined by the national government and approved by the Commissioner of the Consumer Affairs Agency for each food. As will be described later in the examples, it has been confirmed that the food composition of the present embodiment has a preventive or ameliorating effect on atrial fibrillation.

[How to prevent or improve atrial fibrillation]
In one embodiment, the present invention provides a method for preventing or ameliorating atrial fibrillation (excluding medical practice for humans), which comprises a step of ingesting a compound represented by the following formula (1).

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]

In the method of this embodiment, the target may be a patient with atrial fibrillation or a patient. The patient is not particularly limited, and examples thereof include companion animals such as dogs and cats, and livestock such as cattle. Here, the medical practice means an act in which a doctor (including a person who has been instructed by a doctor) performs treatment on a human.

As will be described later in the examples, atrial fibrillation can be prevented or ameliorated by ingesting the compound represented by the above formula (1) in the subject.

[Other embodiments]
In one embodiment, the present invention provides a method for preventing or treating atrial fibrillation, which comprises administering an effective amount of a compound represented by the following formula (1) to a patient in need of treatment.

[In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]

In one embodiment, the present invention provides a method for preventing or treating hypertension, which comprises administering an effective amount of a compound represented by the above formula (1) to a patient in need of treatment.

In one embodiment, the present invention provides a compound represented by the above formula (1) for use in the prevention or treatment of atrial fibrillation.

In one embodiment, the present invention provides a compound represented by the above formula (1) for use in the prevention or treatment of hypertension.

In one embodiment, the present invention provides the use of a compound represented by the above formula (1) for producing a prophylactic or therapeutic agent for atrial fibrillation.

In one embodiment, the present invention provides the use of a compound represented by the above formula (1) for producing a prophylactic or therapeutic agent for hypertension.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Animal preparation)
The effect of isorhamnetin administration was investigated using a mouse model of atrial fibrillation with continuous infusion of angiotensin II.

C57BL / 6 mice (male, 8 weeks old, Charles River) are referred to as a control group (hereinafter, may be referred to as "Cont group") and angiotensin II-administered group (hereinafter, may be referred to as "AgII group"). , Angiotensin II and isorhamnetin administration group (hereinafter, may be referred to as "AgII + ISO group") were randomly assigned to three groups.

Mice in the AgII group and the AgII + ISO group were continuously administered with angiotensin II (1,000 ng / kg / hour) for 2 weeks using an osmotic mini-pump (model number "2002", Alzet). Mice in the Cont group were infused with 0.85% saline into an osmotic minipump.

From 1 week before implantation of the osmotic mini-pump, isorhamnetin (5 mg / kg) was intraperitoneally administered to mice in the AgII + ISO group every day for 3 weeks. Isorhamnetin was suspended in physiological saline containing 0.1% dimethyl sulfoxide and 1% polypropylene glycol, and administered in a fixed amount of 0.3 mL daily.

Mice in the Cont group and AgII group were intraperitoneally administered with the same volume of solution (physiological saline containing 0.1% dimethyl sulfoxide and 1% polypropylene glycol) as in the AgII + ISO group every day for 3 weeks. 1 (a) to 1 (c) are diagrams showing a drug administration schedule to mice in each group. FIG. 1 (a) shows the administration schedule of the drug of the Cont group, FIG. 1 (b) shows the administration schedule of the drug of the AgII group, and FIG. 1 (c) shows the administration schedule of the drug of the AgII + ISO group.

Echocardiography of mice in each group was performed before the start of isorhamnetin administration and 3 weeks after the start of administration. In addition, an atrial fibrillation induction experiment was performed 3 weeks after the start of isorhamnetin administration. Immediately after the atrial fibrillation induction experiment, the heart was removed, the left atrium was separated, fixed with 4% paraformaldehyde, and histochemical staining was performed. The left atrium of mice that were not assigned to histochemical staining was stored in liquid nitrogen for further analysis.

[Experimental Example 2]
(Atrial fibrillation induction experiment)
The right jugular vein of each group of mice prepared in Experimental Example 1 was cut, and a 2-Fr quadrupole electrode catheter (Unique Medical) with an electrode distance of 2 mm was inserted into the right atrium for electrophysiological examination, and an electrophysiological examination was performed. It was measured. FIG. 2 is an X-ray photograph of the state of the experiment.

Atrial fibrillation by atrial burst pacing with an amplitude of 6 V, a cycle length of 20 ms, a pulse duration of 6 ms, and a stimulation time of 30 ms using a programmable stimulator (model number "SEN-7203", Nihon Kohden). Induced. Atrial burst pacing was performed 5 times in a row in each group of mice. Atrial fibrillation duration was defined as the interval between the onset and spontaneous termination of atrial fibrillation.

In addition, a programmable stimulator (model number "SEN-7203", Nihon Kohden) is used to stimulate the heart with a constant voltage with a pulse width of 2 ms, which is about twice the diastolic stimulation threshold, and the effective refractory period of the heart. (AERP) was measured.

AERP was measured with a basic cycle length of 150 ms, with eight consecutive basic stimuli (S1 × 8) followed by a single early stimulus (S2) for 5 ms. AERP was defined as the longest S1-S2 interval that failed to capture.

FIG. 3A is a graph showing the atrial fibrillation induction rate of mice in each group. The atrial fibrillation induction rate (%) was calculated by the following formula (F1).
Atrial fibrillation induction rate (%) = number of times atrial fibrillation occurred / number of times atrial burst pacing was performed × 100… (F1)

FIG. 3 (b) is a graph showing the duration of atrial fibrillation in the mice of each group. FIG. 3 (c) is a graph showing the effective refractory period of the mice in each group.

In FIGS. 3A to 3C, "Cont" indicates the result of the control group, "AgII" indicates the result of the angiotensin II administration group, and "AgII + ISO" indicates the result of angiotensin II and isorhamnetin administration. Show that it is the result of the group. In addition, "**" indicates that there is a significant difference at p <0.01 with respect to the control group, and "#" indicates that there is a significant difference at p <0.05 with respect to the angiotensin II-administered group. , "##" indicates that there is a significant difference at p <0.01 with respect to the angiotensin II-administered group.

As a result, it was clarified that the administration of angiotensin II significantly increased the induction rate of atrial fibrillation, and the administration of isorhamnetin significantly suppressed the induction rate of atrial fibrillation. It was also clarified that the administration of angiotensin II significantly prolonged the duration of atrial fibrillation, and the administration of isoramnetin significantly shortened the duration of atrial fibrillation. It was also clarified that the effective refractory period was significantly shortened by the administration of angiotensin II, and the effective refractory period was significantly recovered by the administration of isorhamnetin.

The above results indicate that administration of isorhamnetin can prevent, improve or treat atrial fibrillation.

[Experimental Example 3]
(Examination of atrial hypertrophy)
Echocardiography of mice in each group prepared in Experimental Example 1 was performed to examine the effect of isorhamnetin administration on atrial hypertrophy. Echocardiography was performed using a Doppler echocardiography system (model number "Vevo 2100", Visual Sonics).

First, the mice in each group were anesthetized with 1% isoflurane. Subsequently, 2D echocardiograms of the parasternal short axis and parasternal long axis were obtained at the level of the papillary muscle, and the atrial sutra was measured on the B mode diagram.

FIGS. 4 (a) to 4 (c) are typical photographs showing echocardiograms of mice in each group. Further, FIG. 4 (d) is a graph showing the results of measuring the atrial sutra based on the echocardiograms of the mice of each group. Further, FIG. 4 (e) is a graph showing a value (mg / g) obtained by dividing the weight of the atrium by the body weight after removing the heart from the mice of each group.

In FIGS. 4A to 4E, "Cont" indicates the result of the control group, "AgII" indicates the result of the angiotensin II administration group, and "AgII + ISO" indicates the result of angiotensin II and isorhamnetin administration. Show that it is the result of the group. Further, in FIGS. 4A to 4C, "LV" indicates the left ventricle, "Ao" indicates the aorta, and "LA" indicates the left atrium. Further, in FIGS. 4 (d) and 4 (e), “*” indicates that there is a significant difference at p <0.05 with respect to the control group, and “**” indicates p <0 with respect to the control group. At 0.01, there is a significant difference, and "#" indicates that there is a significant difference at p <0.05 with respect to the angiotensin II-administered group.

As a result, it was clarified that the administration of angiotensin II significantly prolonged the atrial sutra, and the administration of isorhamnetin significantly shortened the atrial sutra. It was also revealed that the administration of angiotensin II significantly increased the weight of the atrium, and the administration of isorhamnetin significantly decreased the weight of the atrium.

The above results indicate that administration of isorhamnetin can suppress hypertrophy of the heart due to administration of angiotensin II.

[Experimental Example 4]
(Histochemical staining)
Sections of 4% paraformaldehyde-fixed specimens of the left atrium of mice in each group prepared in Experimental Example 1 were stained with Masson's trichrome and hematoxylin / eodin and observed under a microscope.

FIGS. 5 (a) to 5 (c) are representative micrographs of mouse specimens of each group. The scale bar is 50 μm. Further, FIG. 5 (d) is a graph showing the result of calculating the ratio (%) of the area occupied by the fibrotic region to the total area of the atria based on the results of histochemical staining of the mouse specimens of each group. ..

In FIGS. 5A to 5D, "Cont" indicates the result of the control group, "AgII" indicates the result of the angiotensin II administration group, and "AgII + ISO" indicates the result of angiotensin II and isorhamnetin administration. Show that it is the result of the group. Further, in FIG. 5 (d), "**" indicates that there is a significant difference at p <0.01 with respect to the control group, and "#" indicates p <0.05 with respect to the angiotensin II-administered group. Indicates that there is a significant difference in.

As a result, it was clarified that the administration of angiotensin II significantly increased the ratio (%) of the area occupied by the fibrotic region to the total area of the atrium, and the administration of isorhamnetin significantly decreased the above ratio.

This result indicates that administration of isorhamnetin can suppress fibrosis of the heart due to administration of angiotensin II.

[Experimental Example 5]
(Western blotting 1)
Proteins were extracted from the left atrium of the mice of each group prepared in Experimental Example 1, and the expression levels of proteins related to inflammation, myocardial hypertrophy and fibrosis were examined by Western blotting.

NFκB protein was detected as a protein related to inflammation. In addition, TPRC6 protein was detected as a protein associated with myocardial hypertrophy and fibrosis. In addition, β-actin protein was detected as a loading control.

FIG. 6A is a photograph showing the result of Western blotting. Further, FIG. 6 (b) is a graph in which the expression level of the NFκB protein is quantified based on FIG. 6 (a). Further, FIG. 6 (c) is a graph in which the expression level of the TPRC6 protein is quantified based on FIG. 6 (a).

In FIGS. 6 (a) to 6 (c), “Cont” indicates the result of the control group, “AgII” indicates the result of the angiotensin II administration group, and “AgII + ISO” indicates the result of angiotensin II and isorhamnetin administration. Show that it is the result of the group. Further, in FIGS. 6 (b) and 6 (c), "*" indicates that there is a significant difference at p <0.05 with respect to the control group, and "**" indicates p <0 with respect to the control group. At 0.01, there is a significant difference, and "#" indicates that there is a significant difference at p <0.05 with respect to the angiotensin II-administered group.

As a result, it was clarified that the administration of angiotensin II significantly increased the expression level of proteins related to inflammation, hypertrophy and fibrosis in the heart, and the administration of isorhamnetin significantly decreased the expression level.

This result indicates that administration of isorhamnetin can suppress inflammation, hypertrophy and fibrosis of the heart due to administration of angiotensin II.

[Experimental Example 6]
(Examination using cultured cells)
In vitro studies were performed using HL-1 cells, which are mouse atrial muscle-derived cell lines.

The surface of a 6-well multi-electrode array (MEA) dish (model number "60-6wellMEA", Multichannel Systems) was precoated with a solution of 0.02% (w / v) gelatin containing 5 μg / mL fibronectin.

Subsequently, HL-1 cells were seeded and 5% CO ₂ in Claycomb medium supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, 100 μM norepinephrine, 30 mM L-ascorbic acid, 2 mM L-glutamine, and 10% FBS. The cells were cultured at 37 ° C. under humidification for 2 days.

Subsequently, the HL-1 cells are referred to as a control group (hereinafter, may be referred to as “Cont group”), angiotensin II administration group (hereinafter, may be referred to as “AgII group”), angiotensin II and isorhamnetin administration group (hereinafter, may be referred to as “AgII group”). Hereinafter, it may be referred to as "AgII + ISO group").

Angiotensin II 1 μM was added to the medium for the cells of the AgII group. To the cells of the AgII group + ISO group, 1 μM of angiotensin II and 10 μM of isorhamnetin were added to the medium. Subsequently, after 24 hours, the experiment was performed when the cell density became about 80% confluent.

Place the MEA dish of each group in a chamber set at 37 ° C. and use the MEA2100 system (Multichannel Systems) and MC-Rack software (Multichannel Systems) to ± the HL-1 cells of each group. A bipolar stimulus of 7500 mV was applied 30 times at 3 Hz, and the action potential duration (APD) was measured from the waveform.

7 (a) to 7 (c) are typical waveforms measured by the MEA system. FIG. 7 (d) is a graph in which the action potential duration is quantified based on the waveform measured by the MEA system.

In FIGS. 7 (a) to 7 (d), “Cont” indicates the result of the control group, “AgII” indicates the result of the angiotensin II administration group, and “AgII + ISO” indicates the result of angiotensin II and isorhamnetin administration. Show that it is the result of the group. Further, in FIG. 7 (b), the region surrounded by a circle shows the waveform of post-delayed depolarization (DAD). Further, in FIG. 7 (d), "**" indicates that there is a significant difference at p <0.01 with respect to the control group, and "##" indicates p <0. With respect to the angiotensin II-administered group. 01 indicates that there is a significant difference.

As a result, it was clarified that the administration of angiotensin II significantly prolonged the action potential duration of HL-1 cells, and the administration of isoramnetin significantly restored the prolongation of the action potential duration.

In addition, delayed post-depolarization (DAD), which is known as a factor that induces atrial fibrillation, was observed by administration of angiotensin II, but DAD disappeared by administration of isorhamnetin.

The above results further support that administration of isorhamnetin can prevent, improve or treat atrial fibrillation.

[Experimental Example 7]
(Ca ²⁺ imaging using isolated atrial muscle cells)
Atrial muscle cells were isolated from the hearts of mice in the Cont group, AgII group, and AgII + ISO group prepared in Experimental Example 1 using the Langendorff perfusion heart experiment method.

Subsequently, Fluo 4-AM, which is an intracellular calcium ion measuring reagent, was dissolved in Tyrode buffer, and the isolated atrial muscle cells were loaded for 10 minutes. Subsequently, the cells were washed with a Tyrode buffer containing 1.8 mM Ca ²⁺ . Subsequently, confocal Ca ²⁺ imaging was performed with the cells maintained in a Tyrode buffer containing 1.8 mM Ca ²⁺ .

Ca ²⁺ line scan imaging (1.82 ms / line) was performed using a Zeiss LSM 800 confocal microscope (Zeiss) focusing on a single cell.

After baseline was stable, isoproterenol (1 μM) was added to the cells. Subsequently, the frequency of Ca ²⁺ Sparks occurrence was evaluated using Spark master, which is a plug-in software of ImageJ software.

FIG. 8 (a) is a representative image of atrial muscle cells of each group when Ca ²⁺ line scan imaging was performed. Further, FIG. 8B is a graph showing the results of measuring the Ca ²⁺ Sparks occurrence frequency (Sparks / 100 μm / s) of each group.

In FIGS. 8A and 8B, "Cont" indicates the result of the control group, "AgII" indicates the result of the angiotensin II administration group, and "AgII + ISO" indicates the result of angiotensin II and isorhamnetin administration. Show that it is the result of the group. Further, in FIG. 8 (b), the vertical axis of the graph indicates the frequency of Ca ²⁺ Sparks occurrence, “*” indicates that there is a significant difference at p <0.05 with respect to the control group, and “#” indicates that there is a significant difference. It is shown that there is a significant difference at p <0.05 with respect to the angiotensin II-administered group.

As a result, it was clarified that the frequency of Ca ²⁺ Sparks occurrence was significantly increased by the administration of angiotensin II, and the frequency of Ca ²⁺ Sparks occurrence was significantly decreased by the administration of isorhamnetin.

This result indicates that isorhamnetin administration suppressed Ca ²⁺ leakage from the angiotensin II-induced type 2 ryanodine receptor (RyR2).

[Experimental Example 8]
(Western blotting 2)
The type 2 ryanodine receptor (RyR2) is an important molecule for calcium handling. A protein was extracted from the left atrium of the mice of each group prepared in Experimental Example 1, and the protein expression of CAMKII, which is the molecule most contributing to the activity of RyR2, was detected by Western blotting. Β-actin protein was detected as a loading control. It is known that CAMKII is converted into an active type of oxidized-CAMKII by active oxygen or the like.

FIG. 9 is a photograph showing the result of Western blotting. Further, FIGS. 10A to 10C are graphs in which the results of FIG. 9 are quantified. In FIGS. 9 and 10 (a) to 10 (c), "Cont" indicates the result of the control group, "AgII" indicates the result of the angiotensin II administration group, and "AgII + ISO" indicates the result of the angiotensin II. And the result of the isorhamnetin administration group.

As a result, it was clarified that the administration of angiotensin II significantly increased the expression level of oxygenized-CAMKII (ox-CAMKII), which is a protein related to calcium handling, and the administration of isorhamnetin significantly decreased the expression level. became.

This result indicates that administration of isorhamnetin can regulate abnormalities in calcium handling due to administration of angiotensin II and regulate electrical remodeling.

[Experimental Example 9]
(Western blotting 3)
The activity of the MAPK pathway is known to cause inflammation, fibrosis and hypertrophy of tissues and cells. Proteins were extracted from the left atrium of the mice of each group prepared in Experimental Example 1, and protein expression of JNK and ERK, which are molecules contributing to the activity of the MAPK pathway, was detected by Western blotting. In addition, β-actin protein was detected as a loading control. It is known that JNK and ERK become active forms by being phosphorylated.

FIG. 9 is a photograph showing the result of Western blotting. Further, FIGS. 10A to 10C are graphs in which the results of FIG. 9 are quantified. In FIGS. 9 (a) to 10 (c), “Cont”, “AgII”, and “AgII + ISO” are as described above. Further, p-JNK and p-ERK indicate phosphorylated JNK and phosphorylated ERK.

As a result, it was clarified that the administration of angiotensin II significantly increased the expression level of the protein related to the structural abnormality of the tissue, and the administration of isorhamnetin significantly decreased the expression level.

This result indicates that administration of isorhamnetin can suppress structural abnormalities due to administration of angiotensin II and suppress structural remodeling.

[Experimental Example 10]
(Examination of the effect on blood pressure)
Three weeks after the start of the experiment in Experimental Example 1, the blood pressure in the tail of the mouse was measured using a Non-Invasive Blood Pressure System (Kent Scientific).

As a result, the blood pressure in the tail of the mice in the control group was about 150 mmHg. The blood pressure in the tail of the mice in the angiotensin II-administered group was about 200 mmHg. On the other hand, the blood pressure in the tail of the mice treated with angiotensin II and isorhamnetin was about 180 mmHg.

The above results indicate that the administration of isorhamnetin suppressed the increase in blood pressure due to the administration of angiotensin II.

[Experimental Example 11]
(Examination of isorhamnetin derivatives)
Quantitative RT-PCR using HL-1 cells, which are cell lines derived from mouse atrial muscle, is used to express the Tgfb1 gene involved in inflammation, fibrosis, and hypertrophy of cells and tissues, and the col1a1 gene, which is a marker for fibrosis. , The effect of administration of isoramnetin derivative was investigated.

First, HL-1 cells are seeded on a 6-well plate, and a control group (hereinafter, may be referred to as “Cont group”), angiotensin II administration group (hereinafter, may be referred to as “AgII group”), and angiotensin II. And isorhamnetin derivative administration group (hereinafter, may be referred to as "AgII + ISO derivative group").

Subsequently, 23 hours after seeding of the cells, isorhamnetin derivatives (isoramnetin, 3-O-methylquercetin, tamalixetin, ramnetin, azaleatin, 10 μM each) were added to the medium of the AgII + ISO derivative group.

Subsequently, 24 hours after seeding of the cells, angiotensin II (1 μM) was added to the medium of the AgII group and the AgII + ISO derivative group, and the cells were further cultured for 24 hours.

Subsequently, cells of each group were collected, cDNA was extracted from each group, and quantitative RT-PCR was performed. 18S ribosomal RNA was used as an endogenous control for quantitative RT-PCR.

11 (a) and 11 (b) are graphs showing the results of quantitative RT-PCR. FIG. 11 (a) shows the result of the Tgfb1 gene, and FIG. 11 (b) shows the result of the col1a1 gene. In FIGS. 11 (a) and 11 (b), "Cont" indicates the result of the control group, "AgII" indicates angiotensin II, "ISO" indicates isorhamnetin, and "O-Methyl" indicates 3-. "Tama" indicates tamalixetin, "Rham" indicates rhamnetin, and "Aza" indicates azaleatin. In addition, "*" indicates that there is a significant difference at p <0.05 with respect to the angiotensin II-administered group.

As a result, regarding the Tgfb1 gene, it was confirmed that 3-O-methylquercetin, tamalixetin, and azaleatin had a significant decrease in the expression level similar to that of isorhamnetin. It was confirmed that the expression level of the Col1a1 gene also decreased, similar to that of isorhamnetin. This result suggests that O-methylated quercetin other than isorhamnetin also has an inhibitory function on structural remodeling of atrial tissue, similar to isorhamnetin.

[Experimental Example 12]
(Ca ²⁺ imaging using HL-1 cells)
HL-1 cells, which are mouse atrial muscle-derived cell lines, were seeded in glass bottom dish. Subsequently, the HL-1 cells are referred to as a control group (hereinafter, may be referred to as “Cont group”), angiotensin II administration group (hereinafter, may be referred to as “AgII group”), angiotensin II and isorhamnetin administration group (hereinafter, may be referred to as “AgII group”). Hereinafter, it may be referred to as "AgII + ISO derivative group").

Subsequently, 24 hours after seeding of the cells, isorhamnetin (10 μM) was added to the medium of the AgII + ISO derivative group. In addition, 24 hours after seeding of the cells, angiotensin II (1 μM) was added to the media of the AgII group and the AgII + ISO derivative group, and the cells were further cultured for 1 hour.

Subsequently, Fluo 4-AM, which is an intracellular calcium ion measuring reagent, was dissolved in Tyrode buffer and loaded onto HL-1 cells for 10 minutes. Subsequently, the cells were washed with a Tyrode buffer containing 1.8 mM Ca ²⁺ . Subsequently, confocal Ca ²⁺ imaging was performed with the cells maintained in a Tyrode buffer containing 1.8 mM Ca ²⁺ .

Ca ²⁺ line scan imaging (1.82 ms / line) was performed using a Zeiss LSM 800 confocal microscope (Zeiss) focusing on a single cell.

FIG. 12 is a representative image of cells in each group when Ca ²⁺ line scan imaging was performed. In FIG. 12, "Cont" indicates that it is the result of the control group, "AgII" indicates that it is the result of the angiotensin II administration group, and "AgII + ISO" indicates that it is the result of the angiotensin II and isorhamnetin administration group. show.

As a result, it was clarified that the irregular waveform by the administration of angiotensin II changed to the regular waveform by the administration of isorhamnetin. This result indicates that isorhamnetin has a therapeutic effect on atrial fibrillation in the acute phase.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a technique for preventing, improving or treating atrial fibrillation.

Claims (6)

1. A prophylactic or therapeutic agent for atrial fibrillation containing a compound represented by the following formula (1) as an active ingredient.
   

   

   [In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]
2. A pharmaceutical composition for preventing or treating atrial fibrillation, which comprises the prophylactic or therapeutic agent according to claim 1 and a pharmaceutically acceptable carrier.
3. A food composition for preventing or ameliorating atrial fibrillation, which comprises a compound represented by the following formula (1) as an active ingredient.
   

   

   [In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]
4. The food composition for preventing or improving atrial fibrillation according to claim 3, which is a food with functional claims.
5. The food composition for preventing or improving atrial fibrillation according to claim 3, which is a food for specified health use.
6. A method for preventing or ameliorating atrial fibrillation (excluding medical practice for humans), which comprises a step of ingesting a compound represented by the following formula (1) to a subject.
   

   

   [In the formula (1), R ¹ to R ⁵ each independently represent a hydrogen atom or a methyl group, and at least one of R ¹ to R ⁵ is a methyl group. ]

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