KR100715887B1 - Functional food for preventing arrhythmia - Google Patents

Abstract

The present invention relates to a functional food having an effect of preventing arrhythmia.

According to the present invention, there is provided a functional food comprising a composition for preventing arrhythmia, which has decursin as an active ingredient.

Decursin, arrhythmia, hKv1.5 pathway, donkey

Description

Functional food for the prevention of arrhythmia {FUNCTIONAL FOOD FOR PREVENTING ARRHYTHMIA}

FIG. 1A shows control K ⁺ currents recorded at stimulation at 10 mV intervals from +60 mV to -80 mV after fixation of the membrane voltage at −80 mV in Ltk-cells expressing the hKv1.5 pathway gene.

Figure 1b shows the effect of decursine on the recorded K ⁺ currents upon stimulation at 10 mV intervals from +60 mV to -80 mV after fixing the membrane voltage at -80 mV in Ltk-cells expressing the hKv1.5 pathway gene. Drawing.

Figure 1c shows the effect of decursine on the recorded K ⁺ currents upon stimulation at 10 mV intervals from +60 mV to -80 mV after fixing the membrane voltage at -80 mV in Ltk-cells expressing the hKv1.5 pathway gene. It is a figure which shows the relative electric current with respect to a control current in each film voltage.

Figure 1d shows the dose response curves of decursin against the last recorded stable current when stimulating the membrane voltage from -80 mV to +50 mV in Ltk-cells expressing the hKv1.5 pathway gene.

2A shows the effect of decursin (3 μM) on the activation of hKv1.5 pathway in Ltk-cells expressing the hKv1.5 pathway gene.

2B is a diagram showing the effect of decursin (3 μM) on the inactivation of hKv1.5 pathway in Ltk-cells expressing the hKv1.5 pathway gene.

3 is a diagram showing the membrane voltage dependence of hKv1.5 pathway inhibitory effect by decursin in Ltk-cells expressing hKv1.5 pathway gene.

Figure 4 shows the frequency-dependent stimulation of hKv1.5 pathway inhibitory effect by decursin in Ltk-cells expressing hKv1.5 pathway gene.

Fig. 5 shows the path state dependence of hKv1.5 pathway inhibitory effect by decursin (1 μM) in Ltk-cells expressing hKv1.5 pathway gene.

Figures 6a to 6c is a view showing the effect of decursin hKv1.5 channel inhibitory effect on the K ⁺ current of human atrial cells.

The present invention relates to a functional food for preventing arrhythmia, and more particularly, to a functional food that can be usefully used for the prevention of arrhythmia, which shows an excellent effect on selectively blocking the hKv1.5 pathway mainly expressed in the human atria. .

Arrhythmia is a disease in which the heart's pump function is abnormal due to abnormal rhythm of the heart due to abnormal electro pulsation formation or impairment of electric impulse of the heart due to various changes in the electrochemical properties of the myocardium. If the heart rate is severely slowed by these abnormalities, bradycardia arrhythmias, abnormally faster tachyarrhythmias, heart beats come out faster than scheduled one beats are called early beats. In general, arrhythmia causes various types of complications, and therefore accurate diagnosis, prevention and treatment thereof are essential.

Various drugs for the prevention and treatment of such arrhythmias are currently being developed, but are only used in a limited range due to side effects. An ideal antiarrhythmic agent with no side effects should be a drug that acts only on abnormally exciting cardiomyocytes (or cells with an abnormal heart rate) or only on tissues that cause arrhythmia (i.e., atria, ventricles, perkin fibers, etc.) within the heart. However, drugs that meet these conditions have not been developed at present. In order to develop new antiarrhythmic drugs with little or no side effects, a new understanding of the site of action of the antiarrhythmic drugs, that is, the ionic pathways, must be followed. Therefore, new ideal antiarrhythmic drugs can be used in combination with molecular biological cloning and electrophysiological techniques. Efforts are being made to develop.

On the other hand, the action potential period is regulated by several K ⁺ channels, and various genes for the K ⁺ channels that are expressed for each region of the heart are known. The K ⁺ channel current, the most diverse ion channel in the heart, is largely inwardly rectifying (IK ₁ , inward rectifying current; IK _Ach , acetylcholine-activated current; IK _ATP , ATP-sensitive K ⁺ channel, etc.) and voltage-gated. These are classified as K ⁺ (Kv) currents, usually introverted K ⁺ currents are involved in the regulation of the stabilizer voltage, and Kv currents regulate the action potential period.

Kv pathways can contribute to cell repolarization and modulate action potential duration. Clinically, arrhythmias occurring in ventricular muscles are known to result in repolarization disorders of damaged tissues, making them an important target for arrhythmia treatment. Indeed, antiarrhythmic agents such as quinidine, verapamil, nifedipine, sotarol, amiodarone, precanide, and cropyrium are known to act on these Kv pathways (Katz AM.Selectivity and toxicity of antiarrhythmic drugs: molecular interactions with ion channels Am J Med 1998, 104: 179-195). However, since these drugs are known to cause many side effects due to the lack of ion channel selectivity, new drug development with ion channel selectivity is needed.

More than eight Kv pathways (Kv1.1-Kv1.8) are known (Grissner S. Potassium channels still hot.TiPS 1997, 18: 347-350 ). .1, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3 , such as seven kinds of a (Deal such as Molecular physiology of cardiac potassium channels Physiol Rev 1996, 76:.. 49-67 ). Among these genes, the important Kv pathway genes expressed in the human heart are the hKv1.4, hKv1.5, Kv4.3 and HERG genes. These genes are mainly expressed in both the atria and ventricles, but the hKv1.5 gene is dominantly expressed in the human atria, and is known to have the same electrophysiological and pharmacological properties as I _Kur , which is characteristically recorded in the human atria (Fedid et al. ^{The 1997 Stevenson Award Lecture Cardiac K +} channel gating:. cloned delayed rectifier mechanisms and drug modulation Can J Physiol Pharmacol 1998, 76:. 77-89). Thus, the development of selective blockers for the hKv1.5 pathway could be an effective atrial arrhythmia treatment.

The present inventors have conducted extensive research to develop selective blockers for the hKv1.5 pathway that is dominantly expressed in the human atria. As a result, decursin contained in large amounts in the donor hKv1.5 pathway current and I _Kur of human atrial cells. While effectively suppressing the current, the present inventors have found that the effect is increased in proportion to the heart rate, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a functional food for preventing arrhythmia that can be effectively used for the prevention of arrhythmias by acting as a selective blocker for the hKv1.5 pathway.

The present invention in one embodiment for achieving the above object,

Provided is a functional food comprising a composition for preventing arrhythmia having decursin as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for preventing arrhythmia having decursin as an active ingredient in one embodiment, and decursin, which is an active ingredient of the composition according to the present invention, is a compound having Formula 1 below.

In general, decursin having the above formula is Angelica As a material present in a large amount in gigas), anticancer action from the example, neurotoxicity inhibitory action, platelet aggregation inhibitory action, antibacterial action, pain suppression, but is known to have an effect such action, Rezin the K ⁺ channel blocking Deco operation and wherein The fact that it has an arrhythmic effect has not been described or implied in any literature.

The present inventors have found that decursin exhibits an excellent effect on the prevention and treatment of K ⁺ channel mediated diseases, especially arrhythmia, because decursin suppresses hKv1.5 channel current and increases its effect in proportion to heart rate. A composition for preventing arrhythmia according to the present invention was completed.

The composition according to the present invention may further comprise a pharmaceutically acceptable carrier, in addition to decurcin as an active ingredient, such carriers include excipients, binders, lubricants, disintegrants, coatings, emulsifiers, suspensions, solvents, And at least one carrier selected from the group consisting of stabilizers, absorption aids, water for injection, and isotonic agents.

The compositions according to the invention may be formulated in oral or injectable preparations, preferably in oral preparations. Examples of such oral preparations include granules, tablets, capsules, liquids, and the like.

The dosage of the composition according to the present invention may vary depending on the age, sex and weight of the patient, but based on the weight of decursin, it is preferably 2 to 5000 mg / day, more preferably 10 to 1000 mg / day. Do.

The composition according to the present invention can be used not only as a K ⁺ channel blocker and an antiarrhythmic agent, but also applied to the treatment of warm-blooded animals such as rats, dogs, rabbits, cats, and chickens.

In another embodiment, the present invention provides a method for preparing the composition.

In general, decursin is known by known methods (Ryu, KS, Hong, DN, Kim, NJ and Kong, YY, Studies on the coumarin constituents of the root of Angelica gigas Nakai. Kor . J. Pharmacogn . 1990, 21 (1); 64-68) can be prepared and used, but in particular, since it is contained in a large amount in Angelica can also be prepared by extracting from Angelica.

Therefore, the present invention comprises the steps of extracting magnesium chloride, dried by Mgong-Gui, and filtering the filtrate under reduced pressure; Performing the silica gel column chromatography using the concentrated solution using hexane-methylene chloride-ethylacetate as an effluent solvent; And it provides a method for producing a composition for preventing arrhythmia using decursin as an active ingredient, comprising the step of purifying the column using methanol as an effluent solvent.

Preferably, the methylene chloride extraction step is performed 3 to 5 times, the volume ratio of the hexane-methylene chloride-ethyl acetate effluent solvent is 3: 2: 1. In addition, the final column purification step may be performed using purification columns such as, but not limited to, Sephadex LH-20 and JAI-ODS.

In addition, the present invention provides a functional food comprising the composition according to the present invention, and non-limiting examples of such functional foods include tea, health supplements, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example

Example 1.Evaluation of decursin's decursin's effect on hKv1.5 channel current

1) Preparation of selective ion channel expression cell line

A cell line expressing only one K ⁺ pathway gene expressing the heart, each one (Yang, et. al ., Inhibition of cardiac potassium currents by the vesnarinone analog OPC-18790: comparison with quinidine and dofetilide. J Pharmacol Exp Ther 1997, 280: 1170-1175; Kwak, et al ., Phosphorylation is required for alteration of Kv1.5 K ⁺ channel function by the Kvbeta 1.3 subunit. J Biol Chem 1999, 274: 25355-25361). That is, hKv1.5 and HERG were inserted into pMSVneo having a mouse mammary tumor virus promoter induced by dexamethasone, and lipofectamine was added to mouse Ltk-cells. ), And cells were incubated for 2 weeks after 24 hours until a single colony was formed in DMEM medium containing 0.5 μg / ml of G418. Individual colonies were isolated and incubated in a medium containing 0.25 g / ml of G418, and the ion channel expression level was examined by Northern blot and whole-cell voltage clamp method. It was. The cell lines thus selected were cultured in a medium containing 0.25 μg / ml of G418 until used in the experiment. Cells for transient transfection that were not stable cell lines were cultured in DMEM medium.

2) Current record

Currents in human atrial cells and cell lines are recorded by the whole cell method of the gigaohm-seal patch clamp method (Kwak, et. al ., Phosphorylation is required for alteration of Kv1.5 K ⁺ channel function by the Kvbeta 1.3 subunit. J Biol Chem 1999, 274: 25355-25361), amplifying the electrical signal with a patch clamp amplifier (Axon Instruments, Axopatch-1D, Foster, USA), and then converting the current signal into a signal converter (Digidata 1200, Axon Instruments). The digital signal was converted into a digital signal and stored in a computer. In addition, the microelectrode (Kimax-51, 1.5-1.8 x 100 mm) used for current recording was pulled with a 2-stage microelectrode maker (2-stage pipette puller: Narishige, PP-83) and used to heat-treat the microelectrode ends. At this time, a pipette of resistance 1-2 MΩ was used. Meanwhile, the solution in the microelectrode in the whole cell mode contains 100 mM KCl, 10 mM HEPES, 5 mM K ₄ BAPTA, 5 mM K ₂ ATP and 1 mM MgCl ₂ (pH 7.2), and extracellular The solution contained 130 mM NaCl, 4 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM HEPES and 10 mM glucose, pH 7.35.

To the cell line (Ltk-cell) prepared above, the cell membrane voltage was fixed at −80 Hz and depolarized stimulation was performed at +50 Hz for 250 ms, followed by recording the current while repolarizing at −50 Hz. The dose-dependent hKv1.5 channel current inhibitory effects of each compound were compared in terms of the relative magnitude of the stable current measured at the end of the depolarization stimulus at +50 mV when no compound was present.

1A shows the control currents recorded in Ltk-cells expressing the hKv1.5 pathway, and FIG. 1B shows the effect of decursin on hKv1.5 pathway currents expressed in Ltk-cells. FIG. 1C is a graph showing an average path inhibitory effect of decursin at each membrane voltage, and FIG. 1D is a diagram showing a dose response curve of the decurcin effect on the stable current recorded at the end of the depolarization stimulus.

1A-1D, it can be seen that decursin inhibits concentration-dependently the hKv1.5 channel current expressed in Ltk-cells, and preferentially blocks open ion channels.

The IC ₅₀ for hKv1.5 channel inhibitory activity of decursin upon depolarization at 50 mV was 2.7 ± 1.2 μM (n = 8). In addition, the Hill constant value of decursin was 1.31 when fitting the dose response curve to the Hill equation.

Example 2. Activation and Deactivation of hKv1.5 Pathway by Decurin

The activation intermediate voltage from FIG. 2A was -12.1 ± 3.6 mV (n = 8) in the control group and -17.4 ± 3.9 mV (n = 8) in the presence of 3 μM decursin, and the inactivation intermediate voltage from FIG. 2B was -26.3 ± in the control group. It can be seen that -37.1 ± 3.5 mV (n = 8) in the presence of 3.3 mV (n = 8) and 3 μM of decursin.

Example 3 Membrane Voltage Dependence of hKv1.5 Pathway Inhibition by Decurin

Membrane voltage dependence of many drugs acting on the ion channel is important for evaluating the drug's application value. The inhibition effect observed in FIGS. 1 and 2 was compared from the voltage-current relationship curve to the relative magnitude of the current in the presence of decursin relative to the control current when no drug was present at each membrane voltage. As shown in FIG. 3, decursin showed a strong film voltage dependence between −30 kHz and 0 ㎷, and the film voltage dependence was lost between 0 ㎷ and +60 ㎷. That is, when the drug started to appear at the membrane voltage at which the hKv1.5 channel began to open, and the maximum was opened, the inhibitory effect reached the highest.

Example 4 Stimulus Frequency Dependence of hKv1.5 Pathway Blocking Effect by Decurin

4 is a diagram showing the relationship between hKv1.5 pathway blocking effect and stimulation frequency of decursin. FIG. 4 compares the stable current recorded when stimulating 20 times of 1Hz, 2Hz, and 5Hz at +50 mV each time after fixing the membrane voltage at -80 mV by the magnitude of the current at the first stimulation. Compared to the control group, the degree of blocking was increased in the presence of decursin (3 μM), and as the stimulus was repeated, the blocking effect of decursin was increased. That is, when the stimulus frequency increased, the blocking effect of the drug also increased. This suggests that decursin binds to the hKv1.5 pathway better as the heart rate increases, increasing the inhibitory effect.

Example 5 Pathway Dependency of hKv1.5 Pathway Inhibition by Decoursin

5 was obtained by analyzing whether the hKv1.5 channel current inhibitory effect of decursin works better in the ion channel.

FIG. 5 shows the tail current generated when the control and decursin 1 μM are present, when depolarized to −50 mA at a membrane voltage of −80 Hz and then repolarized to −50 Hz.

While the trace current decreases very rapidly in the control group, in the presence of decursin, the initial peak current decreases, but a continuous decrease in current occurs very slowly compared to the control group, and a "crossover" of the trace currents was observed. This strongly suggests that decursin acts as an open pathway blocker for the hKv1.5 pathway. Since this is one of the conditions that an ideal antiarrhythmic drug, it can be seen that decursin has a condition as an ideal antiarrhythmic drug.

Example 6 Effect of Decursin on K ⁺ Current in Human Atrial Cells

We examined whether the depressin hKv1.5 pathway suppressed the same effect on the K ⁺ currents of human atrial cells, which are known to express the hKv1.5 pathway most. Figures 6a to 6c is a view showing the effect of decursin hKv1.5 channel inhibitory effect on the K ⁺ current of human atrial cells.

The extroverted K ⁺ currents measured in human atrial cells consisted of a very fast open and rapidly deactivated current and a continuously open passage current upon depolarization stimulation. FIGS. 6A-6C show the typical currents. 10 μM of decursin reduced the stable current by reducing the peak of K ⁺ currents in human atrial cells and slightly speeding the deactivation process. This confirms the effect of decursin in human atrial cells.

Example 7. Decourin Extract from Angelica

The Angelica gigas was extracted with methylene chloride three times, and the filtrate was concentrated under reduced pressure. The concentrated solution was subjected to silica gel column chromatography with hexane-methylene chloride-ethylacetate (3: 2: 1) as the distillation solvent, and then methanol and Sephadex LH-20 (sephadex LH-20) as the distillation solvent. Purification by a JAI-ODS column yielded decursin.

Example 8. Preparation of Pharmaceutical Composition

Tablets, capsules, powders and injections were prepared according to the following compositions, respectively.

1) tablets

Decursin 500.0 mg

Lactose 500.0 mg

Talc 5.0 mg

Magnesium Stearate 1.0mg

2) Capsule

Capsules were prepared by sieving decursin and mixing with excipients and then filling the gelatine capsules according to the following composition.

Decursin 500.0 mg

Starch 1500 10.0 mg

Magnesium stearate 100.0 mg

3) powder

The following components were mixed in a conventional powder preparation method, put into a bag, and sealed, and powder was prepared.

Decursin 500.0 mg

Lactose 100.0 mg

Talc 5.0 mg

4) Injection

The following ingredients were filled into 2.0 ml ampoules by conventional injection method and sterilized to prepare injections.

Decursin 50.0 mg

Antioxidant 1.0 mg

Tween 80 1.0mg

Distilled water for injection ≤2.0 ml

According to the present invention, it is safe and less toxic to the human body, and the effect is increased when the heart rate is increased by selectively blocking the K ⁺ pathway expressed in the human atrium, and the effect is almost low at low heart rate, thus preventing arrhythmia and A functional food comprising a composition for preventing arrhythmia having an excellent effect as a therapeutic agent can be provided.

Claims (1)

A functional food comprising a composition for preventing arrhythmia, which has decursin as an active ingredient.

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