KR101298787B1 - A pharmaceutical composition for regulating the potassium channel in cardiac muscle cell and its preparation method and its uses - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions for regulating potassium channels in cardiomyocytes, methods for their preparation and their use. The pharmaceutical composition includes 45-180 parts by weight of ginseng, 50-200 parts by weight of ophiopononis, 125-450 parts by weight of corni, 125-450 parts by weight of mild ginseng (miltiorrhizae), fried semen ziziphi spinosae 95- 400 parts by weight, 95-400 parts by weight of taxilli, 45-200 parts by weight of rubra, and 35-150 parts by weight of steleophaga.

Description

Pharmaceutical compositions for controlling potassium channels in cardiomyocytes and methods for preparing the same and methods for their use {A PHARMACEUTICAL COMPOSITION FOR REGULATING THE POTASSIUM CHANNEL IN CARDIAC MUSCLE CELL AND ITS PREPARATION METHOD AND ITS USES}

The present invention relates to pharmaceutical compositions, methods for their preparation and their use for modulating potassium channels in cardiomyocytes.

Cardiovascular diseases, such as hypertension, coronary heart disease, and arrhythmia, are major threats to human health, and morbidity and mortality among all diseases worldwide. mortality is highest. Severe arrhythmias such as fast ventricular tachycardia (VT), ventricular fibrillation (VF), and sudden cardiac death (SCD) are the leading causes of cardiovascular disease-related deaths.

The normal blood pump function of the heart depends on the periodic movement of the heart muscle, which alternately contracts and relaxes. Formation and conduction of excitability on cardiomyocyte membranes is an early cause for periodic contraction and relaxation of the heart. Excitability of the excitability and delivery on the myocardial cell membrane is based on the change in electrical potential on the myocardial cell membrane. Abnormal electrical potential changes in the membrane cause changes in the heart's cycle, or arrhythmia. Arrhythmia seriously affects the blood pump function of the heart, which needs to be treated as early as possible.

Depending on the lesion, arrhythmia may be classified into sinus arrhythmia, atrial arrhythmia, atrioventricular junctional arrhythmia, ventricular arrhythmia and heart blocks. Atrial arrhythmias include premature atrial contractions, atrial tachycardia, atrial flutter, and atrial fibrillation. Ventricular arrhythmias include premature ventricular contractions, ventricular tachycardia, ventricular flutter, ventricular fibrillation (Ren-gao Ye, Internal medicine, People's medical publishing house, Oct 1984, 2nd edition, p172-212).

Currently, depending on the effects and mechanisms on cardiac muscle electrophysiology, the anti-arrhythmic agents in clinical use and improvement are divided into four groups: I-sodium channel blockers. , Heart and agents that selectively extend II-β receptor blockers, action potential duration (APD) and effective refractory period (ERP) in III-cardiomyocytes Agents that selectively block potassium channels in muscle, IV-calcium channel blockers. Agents from groups I, II and IV may reduce the rate of delivery and may even increase the likelihood of reentry activation and thereby induce conduction blocks that may cause arrhythmia. . Agents from group III have no substantial effect on automaticity (Rui Li, Pharmacology (4th edition), People's medical publishing house, Nov. 1999, 14th print, p159-171).

Pharmaceutical compositions and methods for their preparation are disclosed in Chinese patent ZL02146572.X, which relates only to the study of premature ventricular beat of the coronary heart.

Surprisingly, Applicant has discovered a pharmaceutical composition that modulates potassium channels in cardiomyocytes, which can inhibit potassium channels and inward rectifying potassium currents and transient outward potassium currents in a voltage dependent manner. transient outward potassium current) can be suppressed and the commutated potassium current can be delayed. Antiarrhythmic effects are achieved by reducing the excitability of cardiomyocytes, inhibiting repolarization of action potentials (APs) in cardiomyocytes, and extending action potential duration (APD). The pharmaceutical composition of the present invention is useful for various atrial arrhythmias, ventricular arrhythmias and supraventricular arrhythmia.

It is an object of the present invention to provide a method for preparing the pharmaceutical composition.

Another object of the present invention is to provide a use of the pharmaceutical composition.

The present invention is a pharmaceutical composition for regulating potassium channels in cardiomyocytes, comprising the following components:

Ginseng 45-180 parts by weight, 50-200 parts by weight of ophiopogonis, 125-450 parts by weight of corni, 125-450 parts by weight of Miltiorrhizae, 95-400 parts by weight of fried semen ziziphi spinosae 95-400 parts by weight of the taxilli, 45-200 parts by weight of rubra, 35-150 parts by weight of steleophaga.

The pharmaceutical composition also comprises the following components: 45-200 parts by weight of Rhizoma Nardostachyos, 25-90 parts by weight of Rhizoma Coptidis, 35-150 parts by weight of Omija (Fructus Schisandrae Sphenanthenae), Os draconis ) 75-300 parts by weight further.

The pharmaceutical composition preferably comprises the following components:

45-90 parts by weight of ginseng, 112-135 parts by weight of Macmundong, 224-270 parts by weight of cornus oil, 200-225 parts by weight of sweet ginseng, 150-186 parts by weight of acetic acid, 150-186 parts by weight, 89-89 parts by weight, Insects 35-100 parts by weight, 45-95 parts by weight of saengsonghyang, 45-60 parts by weight of yellow lotus, 67-75 parts by weight of Schizandra chinensis, 145-150 parts by weight of keel.

The pharmaceutical composition preferably comprises the following components: ginseng 89 parts by weight, macmundong 112 parts by weight, corn oil 224 parts by weight, sweet ginseng 224 parts by weight, acetic acid crude 186 parts by weight, 186 parts by weight, red medicine 89 parts by weight, magnetic insects 75 parts by weight, 89 parts by weight of persimmon fragrance, 45 parts by weight of yellow lotus, 67 parts by weight of Schizandra chinensis and 149 parts by weight of keel.

The pharmaceutical composition preferably comprises the following components:

Ginseng 45 parts by weight, Mcmundong 112 parts by weight, corn oil 224 parts by weight, sweet ginseng 225 parts by weight, acetic acid 186 parts by weight, 186 parts by weight of fresh raw material, 89 parts by weight of red radish, 45 parts by weight of persimmon fragrance, 35 parts by weight of barberry Part, Schisandra chinensis 67 parts by weight, keel 149 parts by weight.

Dosage forms of the pharmaceutical composition are capsules, tablets, medical granules, powders or oral solution forms.

The invention also provides supraventricular arrhythmia, ventricular tachycardia, ventricular flutter, ventricular fibrillation, premature atrial contractions, atrial tachycardia, atrial tachycardia Provided is the use of a pharmaceutical composition in the manufacture of a medicament for the treatment of atrial flutter or atrial fibrillation.

The present invention also provides a method of preparing a pharmaceutical composition comprising the following steps.

(a) add an appropriate amount of 70% ethanol to 45-180 parts by weight of ginseng, perform reflux extraction three times, pool the extraction solution, filter, concentrate and dry; Grounding it into fine powder,

(b) add an appropriate amount of 70% ethanol to 125-450 parts by weight of cornus oil and 125-450 parts by weight of salvia, perform reflux extraction three times, pool the extraction solution, filter, and condense it Concentrating with,

(c) grinding 35-150 parts by weight of worms into fine powder and drying it for later use,

(d) add an appropriate amount of water to 50-200 parts by weight of ganmun-dong, 95-400 parts by weight of acetic acid, 95-400 parts by weight of the above product, 45-200 parts by weight of the white peony, boil twice, and extract the extraction solution, Concentrating it to a coagulum,

(e) pooling the condensate from steps (b) and (d), adding to it the pharmaceutical fine powder from step (c) and the pharmaceutical fine powder from step (a), mixing the powder Treating the homogenized powder with a manufacturing process to obtain a pharmaceutical composition.

The raw material in step (b) further comprises 45-200 parts by weight of fennel fragrance, 35-150 parts by weight of schizandra and 25-90 parts by weight of yellow lotus. The raw material in step (d) further comprises 75-300 parts by weight of keel.

The preparation method of the pharmaceutical composition is preferably the following components, that is, 89 parts by weight of ginseng, 112 parts by weight of Macmundong, 224 parts by weight of corn oil, 224 parts by weight of salvia, 186 parts by weight of acetic acid crude, 186 parts by weight, 89 drops of red pepper Part, 75 parts by weight of insects, 89 parts by weight of saengsonghyang, 45 parts by weight of yellow lotus, 67 parts by weight of Schizandra chinensis, 149 parts by weight of keel.

In the application of the present invention, the dosage form of the pharmaceutical composition is selected from the form of capsules, tablets, medicinal granules, powders or oral solution. To obtain such dosage forms, bulking agents, disintegrants, lubricants, suspending agents, binding agents, sweeteners, Pharmaceutically acceptable adjuvants such as flavoring agents, preservatives and the like are complemented. Extenders include starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose and the like. Disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose (low- substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, and the like. Lubricants include magnesium stearate, sodium dodecyl sulfate, talc, silica and the like. Suspending agents include polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose and the like. Binders include starch pulp, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and the like. Sweeteners include sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, and the like. Flavoring agents include sweeteners and various types of fragrances. Preservatives include nipagins, benzoic acid, sodium benzoate, sorbic acid and salts thereof, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil (eucalyptus oil) and the like.

The pharmaceutical composition according to the present invention can suppress inward rectified potassium currents and transient outward potassium currents in a voltage dependent manner, and can delay rectified potassium currents. The pharmaceutical composition can reduce the excitability of cardiomyocytes and inhibit repolarization of action potentials (APs) in cardiomyocytes by regulating the potassium channels of the cardiomyocyte membranes. It prolongs the action potential duration of cardiomyocytes by regulating potassium channels in the cardiomyocyte membranes.

The invention also provides the use of the pharmaceutical composition in the manufacture of antiarrhythmic drugs.

Arrhythmia includes ventricular arrhythmias, ventricular tachycardia, ventricular fibrillation, ventricular fibrillation, early atrial contraction, atrial tachycardia, atrial flutter or atrial fibrillation.

The pharmaceutical composition according to the present invention can inhibit I _K1 of the myocardial cell membrane and reduce the current density of I _K1 .

The pharmaceutical composition according to the invention can inhibit the transient outward potassium current (I _to ) of the myocardial cell membrane, which can accelerate the inactivation of the current.

The pharmaceutical composition according to the present invention can suppress the inward portion of the inward rectifying potassium current I _K1 (I _K1 current) without affecting the reversal potential and the rectifying characteristics of the channel, and slightly outward the portion of the current. It can be increased, which is useful for stabilizing cell membranes and eliminating early afterdepolarization.

The amount of pharmaceutical composition according to the invention is 3-28 g / day (g / day) (by total weight of active ingredient in the raw material). It may be administered once daily, preferably in divided doses of 2-4.

FIG. 1 shows an ex vivo heart perfusion system (Landgendorff perfusion system).
Figure 2 shows the effect on the I _K1 current of the pharmaceutical composition according to the present invention. A. Control. B. Drugs can inhibit I _K1 at a concentration of 0.5%. C. Effect of the pharmaceutical composition according to the invention on the current density-voltage relationship.
3 shows the characteristics of the current I _to and the effect on the current of the pharmaceutical composition according _to the invention. A. Pre-stimulus (-40 mV, 20 ms) is given first, then outward polarization to +60 mV is performed at intervals of 10 mV and recorded on single rat ventricular myocytes with a clamp time of 400 ms. The current, Vh = -90mV, the stimulated outward current includes two parts, transient and holding current. B. Drugs can inhibit I _to when the concentration is 0.5%. C. Curve of I _to Current-Voltage Relationship. ●: Total peak current, ▲: Holding current, ■: Transient current (■ = ●-▲). D. Plot of current density-voltage of peak of transient current. ●: before drug treatment ▲: After drug treatment, the drug inhibits I _to manifested by a decrease in current density.
4 shows the effect of the pharmaceutical composition according to the invention on the time dependent-inactivation of I _to . A. Before Medication. B. The drug concentration is 0.5% and the inactive part of the I _to current is adapted using a single exponential equation.
5 shows the effect of a pharmaceutical composition according to the invention on I _to steady state inactivation. A. Before Medication. B. Drug concentration is 0.5%. C. Voltage dependence of different parts of outward current deactivation. ●: peak current, ▲: holding current, ■: transient current (■ = ●-▲). D. Steady state inactivation curves of transient outward current I _to before and after drug treatment, ●: before drug treatment, ▲: after drug treatment, the solid line in the plot represents the adaptation using the Boltzman equation.
6 shows the effect of the pharmaceutical composition according to the invention on the recovery of the I _to current after inactivation. A. Control. B. Drug concentration is 0.5%. C. Plot of time-dependent recovery after I _to inactivation. ●: before drug treatment, ▲: after drug treatment, the solid line in the plot indicates adaptation using a single exponential equation.
Figure 7 shows and illustrates the voltage-dependent inhibition of the delayed rectified potassium current (I _K ) of the pharmaceutical composition according to the present invention. A. V _H = -40 mV, stepwise depolarizating to 60 mV for a duration of 5 s, voltage-dependent and time-dependent inactivation of outward current I _K are obtained in single guinea pig ventricular myocytes. B. Drug concentration is 0.5% and current changes for a duration of 5 s at V _T = 50 mV before and after drug treatment. C. Plot of IV relationships before and after drug treatment (current density versus voltage of peak tail current (under various test voltages)).
Figure 8 shows the inhibition of the I _K current activated by the time-dependent method of the pharmaceutical composition according to the present invention. A. V _H = -40 mV and V _T = 50 mV for 500 ms to 5 s increments in 500 ms increments. B. I _K current and tail current (drug concentration: 0.5%) under the same test voltage and time after drug treatment. C. Time-dependent activation plot before and after drug treatment (peak tail current versus activation time under various activation times).

The following example illustrates the preparation of a pharmaceutical composition according to the present invention. These examples do not limit the scope of the invention in any way.

Example 1

The pharmaceutical compositions are prepared as capsules to facilitate the application of such pharmaceutical compositions, including as potassium channel regulators.

Ginseng 89g, ophiopogonis 112g, corni 224g, miltiorrhizae 224g, fried semen ziziphi spinosae 186g, taxilli 186g, rubra 89g, steleophaga 75g.

Manufacturing method:

(a) using 70% ethanol, three times reflux extracting for 3 h for the first extraction, 2 h for the second extraction and the third extraction, pooling the extraction solution, filtering, Recovering, concentrating and drying the ethanol and grounding it to fine powder for later use,

(b) refluxing the cornus oil and salvia with 70% ethanol three times, pooling the extraction solution, filtering, recovering the ethanol and concentrating it to a condensate for later use,

(c) grinding the worms into fine powder and drying for later use,

(d) adding water to the mammundong, acetic acid salts, fresh and red peony, boiling twice, pooling the extraction solution, filtering, and concentrating it to a condensate for later use,

(e) pooling the condensate from steps (b) and (d), to which the pharmaceutical fine powder from step (c) is added, dried and ground to a fine powder, followed by step (a) Adding, mixing, and adding an adjuvant for the preparation of 1000 capsules.

Indications: supraventricular arrhythmia, ventricular tachycardia, atrial flutter.

Dosage: Oral, 2-4 capsules per time, 3 times a day

Example 2

Ginseng 45g, Macmundong 50g, Cornus 125g, Sweet Ginseng 125g, Acetic acid 95g, Sangi 95g, Red Radish 89g, Self-propelled 35g, Sengsong 45g, Rhubarb 25g, Omija 35g, Keel 75g.

Manufacturing method:

(a) Using an appropriate amount of 70% ethanol, ginseng was refluxed three times for 3 h for the first extraction and 2 h for the second and third extractions, the extraction solution was pooled, filtered and ethanol recovered Concentrating, drying, grinding it into fine powder for later use,

(b) three reflux extracts of Cornus, Salvia, Schisandra chinensis, persimmon fragrance, and Rhododendron with an appropriate amount of 70% ethanol, pool the extraction solution, filter, recover ethanol, and coagulate it for later use. Concentrating with water,

(c) grinding the worms into fine powders and drying the powders for later use,

(d) adding the appropriate amount of water to the mungmundong, acetic acid salt, the above-mentioned and the red peony, boiling twice, pooling the extraction solution, and concentrating it to a condensate for later use,

(e) pooling and drying the condensate from steps (b) and (d), wherein the pharmaceutical fine powder from step (c) is added, and grinding it into fine powder, which is then added from step (a) Adding the pharmaceutical fine powder, mixing and formulating it into medical granules by conventional methods.

Example 3

Ginseng 180g, Megmun-dong 200g, Cornus 450g, Salvia 450g, Acetic acid 400g, Sanggi 400g, Red radish 200g, Insects 150g, Sengyang 200g, Rhubarb 90g, Schisandra chinensis 150g, Keel 300g.

Manufacturing method:

(a) Using a suitable amount of 70% ethanol, ginseng was refluxed three times for 3 h for the first extraction, 2 h for the second extraction and the third extraction, pooled the extraction solution, filtered and ethanol Recovering, concentrating, drying and grinding it into fine powder for later use,

(b) three reflux extracts of cornus, salvia, Schisandra chinensis, persimmon fragrance, and yellow lotus, using an appropriate amount of 70% ethanol, pooled the extraction solution, filtered, ethanol recovery, and for future use, Concentrating to a condensate,

(c) grinding the worms into fine powders and drying the powders for later use,

(d) adding the appropriate amount of water to the mungmundong, acetic acid salt, the above-mentioned and the red peony, boiling twice, pooling the extraction solution, and concentrating it to a condensate for later use,

(e) pooling the condensate from steps (b) and (d), adding to it the pharmaceutical fine powder from step (c), drying, grinding it to fine powder, followed by step (a) Adding the pharmaceutical fine powder from), mixing and formulating it into a tablet by conventional methods.

Example 4

Ginseng 60g, Macmundong 135g, Cornus 270g, Salvia ginseng 200g, Acetic acid 150g, Sanggi 150g, Red radish 100g, Self-propelled 100g, Sengyang 95g, Rhubarb 55g, Schisandra chinensis 75g, Keel 145g.

Manufacturing method:

(a) Ginseng was refluxed three times for 1 h in the first extraction and 2 h for the second and third extractions using an appropriate amount of 70% ethanol, the extraction solution was pooled, filtered, and ethanol was recovered, Concentrate, dry and grind it into fine powder for later use,

(b) three reflux extracts of Cornus, Salvia, Schisandra chinensis, persimmon fragrance, and Rhododendron with an appropriate amount of 70% ethanol, pool the extraction solution, filter, recover ethanol, and coagulate it for later use. Concentrating with water,

(c) grinding the worms into fine powders and drying the powders for later use,

(d) adding the appropriate amount of water to the mungmundong, acetic acid salt, the above-mentioned and the red peony, boiling twice, pooling the extraction solution, and concentrating it to a condensate for later use,

(e) pooling the condensate from steps (b) and (d), adding the pharmaceutical fine powder from steps (a) and (c), mixing and formulating it into an oral solution by conventional methods Step to make up.

Example 5

Prescription:

Ginseng 89g, Megmun-dong 112g, Cornus 224g, Sweet Ginseng 224g, Acetic acid 186g, Fresh 186g, Red Radish 89g, Self-propelled 75g, Ginseng flavored 89g, Rhubarb 45g, Omija 67g, Keel 149g.

Manufacturing method:

(a) According to the above formulation, ginseng was refluxed three times for 3 h in the first extraction, 2 h in the second extraction and the third extraction using 70% ethanol, the extraction solution was pooled, filtered and the ethanol was recovered Concentrated, dried and ground to a fine powder for later use,

(b) by refluxing three times reflux of cornus and salvia with 70% ethanol, pooling the extraction solution, filtering, recovering ethanol, and concentrating it to a condensate for later use ,

(c) pulverizing the worms into fine powders by said prescription and drying for later use,

(d) By the above formulation, water is added to the pulmonary dong, acetic acid, the above-mentioned raw and red peony, boiled twice, pooled the extraction solution, filtered, and pooled the filtrate in the extraction solution containing Schizandra et al. For later use, concentrating it into a coagulum,

(e) pooling the condensate from steps (b) and (d), to which the pharmaceutical fine powder from step (c) is added, dried and ground to a fine powder, followed by step (a) Adding, mixing, and filling 1000 capsules with pharmaceutical fine powder from;

Experiment

The following is a pharmacological activation experiment of the pharmaceutical composition according to the present invention.

To illustrate sodium potassium-channel-regulated-activation of the pharmaceutical composition according to the invention, the following animal experiments using powders inside the capsules from Example 2 (hereinafter referred to as “capsule powders according to the invention”) This was done.

1. Materials and Methods

1.1 materials

(1) Reagent: The capsule powder according to the present invention is produced by SHIJIANZHUANG YILING PHARMACEUTICAL CO. Is provided by LTD. Collagenase collagenase collagenase. Pronase E is from Merch. N-2-hydroxyethylpiperazine-N'-2-ethaneinsulfonic acid (HEPES), ethylene glycol bis-tetraacetic acid (EGTA), L-glutamic acid, taurine, aspartate, disodium creatine phosphate, chlorine-Cl, CaCl ₂ , K ₂ ATP is from Sigma. 4-aminopyridine (4-AP) is from Fluka. Other agents are of analytical grade and are provided by Beijing Chemical Reagent.

(2) solutions

Calcium-free Krebs solutions (mmol / L): NaCl 136, KCl 5.4, MgCl ₂ 1.0, NaH ₂ PO ₄ 0.33, HEPES 10, Glucose, adjusted to pH 7.4 with NaOH 10.

② KB solution (mmol / L): KCl 40, KH ₂ PO ₄ 20, MgSO ₄ 3.0, KOH 80, L-glutamic acid 50, Taurine 20, HEPES 10, Glucose 10, EGTA, adjusted to pH 7.4 using KOH 0.5.

③ Perfusate (mmol / L) to detect potassium currents in whole cells: chlorine-Cl 136, MgCl ₂ 1.2, KCl 5.4, NaH ₂ PO ₄ 0.33, CaCl ₂ 1.8, adjusted to pH 7.4 using LiOH CdCl ₂ 0.15, HEPES 10, Glucose 10. Add 1 mmol / L BaCl ₂ to block I _K1 current to detect I _K.

④ electrode filling solution (mmol / L) for detecting potassium current in whole cells: potassium aspartate 120, KCl 20, HEPES 5, MgCl ₂ 1.0, K ₂ -ATP 4, adjusted to pH 7.3 using KOH EGTA 10, disodium creatine phosphate 2. The electrode filling solution used is filtered by a micro filter membrane having a 0.22 μm diameter.

Preparation of the capsule powder solution according to the invention: preparing the capsule powder according to the invention into a 5% solution (w / w) containing KCl free extracellular perfusate, followed by K ⁺ Adjusting the concentration to 5.4 mmol / L, centrifuging the solution, obtaining a supernatant for later use.

(3) Animal: Beijing WEI TONG LI HUA experimental animal technoogy Co. Male SD rats weighing 250-300 g, provided by LTD.

1.2 Method.

1.2.1 Isolation of Single Cardiomyocytes: Single ventricular myocytes are prepared by the acute enzymatic method. Rats were anesthetized with 50 mg / kg of mebumal sodium via intraperitoneal injection and the heart was removed by thoracotony. The fat and pericardium were then removed in a calcium depleted Tyrode solution at 4 ° C. and the aorta was removed to insert a cannula to perform Langendorf perfusion at a rate of 6-8 ml / min ( 1). Perfusion was carried out for 3 minutes using a calcium removal creb solution to remove blood in the coronary artery (perfusion pressure was 20 cmH ₂ O), followed by a low calcium enzyme solution (CaCl ₂ 0.05 mmol / L, 0.4 Low calcium crebes solution containing mg / ml collagenase II and 0.04 mg / ml protease E) was used to perform recycle perfusion. After about 20 minutes, the heart was separated from the Langendorf system and placed in a room temperature (RT) KB solution, the atrial and cardiac base tissues were dissected, and the ventricular tissues smoothed with ophthalmic tweezers. It was torn and slowly pipetted with a wide-tip pipette to detach the myocardial cells from the myocardial tissue to obtain a single ventricular myocyte. Supernatant is filtered by nylon filter (120 mesh / inch) and myocardial cells are stored in tubes filled with KB solution. The temperature of the total perfusion system is maintained at 37 ° C., and all perfusion and KB solutions are saturated with 100% O ₂ for a time greater than 30 minutes. Cells were left at room temperature for approximately 30 minutes and re-claked to 0.25 mmol / L and then stored at room temperature for later use. The separation method of guinea pig cardiomyocytes is substantially the same.

1.2.2 Experiment of Whole Cell Patch Clamp

Prior to the experiment, a small amount of KB suspension with rich cells was pipetted into a 0.5 ml perfusion chamber and left intact for 5-10 minutes. Once the cells were precipitated and attached to the chamber, surface infusion was performed for 5-10 minutes (at a rate of 2 ml / min) with extracellular fluid saturated with 100% O ₂ . Non-constricted cells with clean interfaces, smooth surfaces and clear stripes were selected under an inverted microscope (OLYMPUS IX70). Experiments were performed at room temperature (22-24 ° C). Channel currents were recorded under voltage clamping by standard whole cell patch clamps. The patch clamp amplifier (Axopatch 700B, Axon Co. US) was connected to the computer via A / D and D / A data converters (Digidata 1322, Axon Co. US). Software (pClampex 10.0, Axon Co. US) controlled the capture of the stimulus signal and the voltage and current input signals. Chunk glass tubes (provided by Institute of Physics, Chinese academy of sciences) were manufactured by a four-step microelectrode puller (p-97, Sutter Co. US), with a tip of 2-3 μm. It was polished with a microforge system (MF-830, Narishige Co. Japan) to induce diameter. The resistance of the electrode filling solution was 1.5-3 MPa. After the compensation liquid was charged according to the potential, the electrode tip was moved to the cell surface through a three-dimensional manipulator, which formed a giga seal with a seal resistance greater than 1 G 1. Fast capacity was compensated for and the cell membranes were broken to create a full cell recording mode. Simulations of 10 mV intervals were provided when determining capacity, and the capacity of the film was calculated by Cm = τ · Iss / ΔV. Slow capacity compensation and tandem resistance compensation were adjusted from 80 to 90% to reduce current and clamp errors due to overcharge and discharge. The signal passed through a fourth-order low pass Bessel filter with a cutoff frequency of 1 kHz and a sampling rate of 10 kHz.

During recording, ATP was added to the electrode filling solution to avoid the effects of current reduction and to complete the experiment within 25 minutes after the cell membrane was broken. Based on the protocol, the prepared drug solution was perfused to the cell perfusion chamber by a peristaltic pump at a constant rate of 2 ml / min. The spent solution was then aspirated by a negative pressure suction device. For 5 minutes after cell membrane destruction, the current before drug treatment is recorded, and for 5 minutes after drug treatment, the current after drug treatment is recorded. The acquired data was stored on the computer's hard disk for later measurement and analysis. To eliminate statistical errors between cells, the current value was expressed as current density (pA / pF).

1.2.3 Design and observation index of the current stimulation method: (1) Stimulation method for I _K1 recording: holding potential (Vh) -40 mV, command potential -120 mV to 0 mV, interval 10 mV, clamping duration 400 ms, stimulation frequency 0.2 Hz , The observation index is the change in the constant current density of I _K1 before and after drug treatment (under test voltage -100 mV), and (2) the stimulus mode for recording the I _to current-voltage relationship: holding potential (Vh) -90 mV, Pre-stimulation (-40 mV, 20 ms) followed by a depolarization stimulus of -40 mV to +60 mV is run at an interval of 10 mV, clamping duration of 400 ms and stimulation frequency of 0.2 Hz, with the observed index before and after drug treatment (under test voltage 60 mV). ) and the change in the peak current density of I _to, (3) I _to a steady state stimulating method for recording inactivation: a holding potential of -90mV, pre-was given to 10mV 60mV intervals from the magnetic pole (400ms) is -120mV, Then, clamp at 60mV for 300ms to record the I _to current, and the drug Compare currents before and after treatment and (4) stimulus mode to record time-dependent recovery after I _to inactivation: a dual-pulse stimulus was given, the holding potential was -90 mV, followed by two depolarization stimuli ( 60 mV, duration 200 ms) P1 and P2 are the next increments (Δt), i.e. 1, 3, 5, 7, 10, 15, 20, 30, 40, 60, 90, 120, 150, 180, 210, Given at 250, 300 (ms). The peak current amplitude I corresponding to P2 was recorded. Plots of recovery after steady state inactivation were made as I / Imax vs. Δt. (5) Voltage-dependent inactivation of I _K is determined: V _H = -40 mV, depolarization was carried out gradually from -40 mV to +60 mV at intervals of 10 mV, and the stimulus was 5 s when returning to V _H. Keep the wave width at 2.5 s to record the tail current. (6) Time-dependent inactivation of the I _K tail current (envelope of the tail test): V _H = -40 mV, depolarization was performed at +50 mV followed by a return to V _H , with 500 to 500 ms increments each time. Ten consecutive pulses were given with a wave width of 5000 ms.

1.3 Data Analysis and Statistics

)로서 표현되었고, 약물 치료 전 및 약물 처리 후 그룹에 대해 쌍을 이룬 t-테스트가 수행되었다. 차이는 P<0.05인 경우에 현저하다.Raw current data was detected and collected by Clampfit 10.0 (Axon Co. US), and the collected data was analyzed, adapted and plotted using Origin 6.0 data process software (Microcal Software Co. US). The data from the experiments are mean ± standard deviation (

) And paired t-tests were performed on groups before and after drug treatment. The difference is significant when P <0.05.

2. Results

2.1 Effect of the Pharmaceutical Compositions According to the Invention on I _K1 of Rat Cardiomyocyte Membrane

Rapidly activated inward currents were recorded on rat single ventricular myocytes without apparent inactivation within 400 ms (holding potential (Vh) -40 mV, command potential -120 mV-0 mV, interval 10 mV, clamping duration 400 ms, stimulation frequency 0.2 Hz) ( 2a). This current can be completely blocked by 1 mmol / L BaCl ₂ , indicating that the current was I _K1 . As the depolarization occurred, the inward portion of the I _K1 current decreased and the inversion potential (characteristic of the inward rectifier) was approximately -40 mV. The capsule powder according to the present invention was dissolved by potassium extracellular solution into a 0.5% solution capable of inhibiting I _K1 (see FIG. 2B). Under -100 mV, the average peak current density was -10.78 ± 1.80 (pA / pF) before drug treatment and the average inhibition was 33.1 ± 16.85% (n = 11, P <0.05). The current density under various pulses is plotted as a function of the corresponding film potential to produce an IV plot. Under all command currents, the drug could reduce the current density of I _K1 , while the inversion potential and the curve shape remained substantially unchanged (see FIG. 2C). After drug treatment, the I _K1 current was suppressed under all clamping voltages specified by the rise of the current density-voltage curve without significantly affecting the reversal potential and rectification.

2.2 Effect of the Pharmaceutical Compositions According to the Invention on I _to of Rat Myocardial Cell Membrane

2.2.1 Effect of Weak Compositions According to the Invention on I _to Current-Voltage (IV) Curves

Potassium channel extracellular and intracellular recording device solutions were used, the holding potential (Vh) was -90 mV, first given a pre-stimulus (-40 mV, 20 ms), followed by -40 mV to +60 mV (with an interval of 10 mV) Depolarization stimulation of was given, clamping duration was 400ms and stimulation frequency was 0.2Hz. Outgoing potassium currents recorded in rat ventricular myocytes comprise two parts. One part is a transient current (I _to ) with a sharp "A" shape that rapidly activates and deactivates, becomes active at 0 mV, peaks at approximately 10 ms, and then rapidly deactivates to normal within 200 ms. . Activation and deactivation processes are voltage- and time-dependent. The current can be completely interrupted by 2mM 4-AP, indicating that the current is I _to . The other part is the maintained current I _maintained which is activated at -10 mV and increases as the voltage increases within -10 to 60 mV (see FIGS. 3A, 3C). The current density under various pulses was plotted as a function of the corresponding film potential, producing an IV plot. The transient current, I _to , is obtained by subtracting the holding current from the peak current. The 0.5% capsule powder according to the present invention can suppress the I _to current (see FIG. 3B), the peak current decreases when the test voltage is 0-60 mV, and the IV curve is moving downward. The average current density at 60 mV decreased from -19.82 ± 7.10 (pA / pF) to -10.02 ± 3.93 (pA / pF) with an average inhibition of 50.60 ± 10.77% (n = 6, P <0.05) (Figure 3d). Reference). The inactive portion of I _to is adapted using a single exponential equation to obtain the time constant τ of time-dependent inactivation. The pharmaceutical compositions according to the invention can accelerate the inactivation of the current (see FIG. 4), and the τ values before and after drug treatment are 29.06 ± 4.66 and 21.44 ± 3.12, respectively (n = 6, P <0.05).

2.2.2 Effect of the pharmaceutical composition according to the invention on the steady state inactivation curve

Stimulation method for recording I _to steady state deactivation: holding potential -90 mV, pre-stimulus (400 ms) given at intervals of 10 mV to 60 mV from -120 mV, followed by clamping for 300 ms at 60 mV and recording I _to current The currents before and after drug treatment are compared (FIG. 5A, FIG. 5B). From the obtained current plot, it can be seen that the deactivation of the outward current can be divided into two parts: deactivation of the holding current with a low inert current and deactivation of the peak current with a higher deactivation voltage than this deactivation. Can be observed. The amplitude of I _to depends on the difference between the hold current and the peak current measured at the end of the test pulse (FIG. 5C). The amplitude of each current is normalized and each membrane potential is plotted as a function of the corresponding current to obtain a steady state deactivation curve. Half the inert ring voltage (V _1/2 ) and the slope factor (k) of the deactivation curve are given by Boltzmann equation (I / Imax = 1 / {1 + exp [(Vm-V _1/2 ) / k]}). Obtained according to (Fig. 5d). The 0.5% capsule powder according to the invention results in a left shift of the I _to steady state inactivation curve with increasing groups. V _1/2 before and after drug treatment were -15.67 ± 2.52 mV and -26.45 ± 3.88 mV, respectively, and the k value changed from 3.41 ± 0.67 to 6.38 ± 2.02 (n = 7, P <0.05) (see Fig. 5). ).

2.2.3 Effect of the pharmaceutical composition according to the invention on the recovery curve after I _to inactivation

Given a double pulse stimulus, the holding potential was -90 mV, followed by two depolarization stimuli (60 mV, duration 200 ms), with P1 and P2 following the next incremental interval (Δt), ie 1, 3, 5, 7, 10 , 15, 20, 30, 40, 60, 90, 120, 150, 180, 210, 250, 300 (ms). The peak current amplitude I corresponding to P2 was recorded. Plots of recovery after steady state inactivation were made as I / Imax vs. Δt. The curve is adapted using a single exponential equation to obtain the time constant τ of the recovery curve. When the concentration of drug is 0.5%, recovery time can be extended from 12.86 ± 0.31 to 18.52 ± 3.76 (n = 5, P <0.05) (see FIG. 6).

2.2 Effect of the pharmaceutical composition according to the invention on I _K of guinea pig cardiomyocytes

FIG. 7 shows the recorded outward current I _K activated in voltage- and time-dependent manner in guinea pig single ventricular myocytes (V _H = -40 mV, depolarized to 60 mV at 10 mV intervals for 5 s). The pharmaceutical composition according to the invention inhibits the I _K current in a voltage-dependent manner (FIG. 7C). The observed index is the recorded I _K when V _T = 50 mV, and the current density of the peak tail current decreased to 30.77 ± 1.11% when the drug concentration was 0.5% (n = 5, p <0.05). 8A and 8B show the inhibition of drug on time-dependently activated I _K as well as the I _K tail current activated with time by an envelope stimulation mode.

Now, I _to includes two types: I _to1 (sensitive to 4-aminopyridine and insensitive to intracellular calcium) and I _to2 (calcium-dependent and can be blocked by caffeine and Co ²⁺ ). It is confirmed. In this study, the pipette solution contains a high concentration of calcium complex EGTA (10 mmol / L) and the perfusate contains 0.15 mmol / L calcium blocker CdCl ₂ . Therefore, the transient outward potassium currents reported in this study do not include the I _to2 moiety. We, there is a pharmaceutical composition according to the invention in this study can be remarkably suppressed I _to, active in species (species) the reduction of I _to in particular with a higher I _to (such as a dog, rat and human) It has been observed that it may arise from the effects of state 1 repolarization of the dislocation and prolong APD. In addition, I _to is the main current leading to uneven trans-myocardial wall repolarization, the inhibition of which reduces the non-flat trans-myocardial wall repolarization and reduces the trans-wall reentry micro-circles. By limiting the formation of the trans-wall reentry micro-circle, it will avoid buzzing fluids such as torsades de pointes ventricular tachycardia. Currently, there are no I _to1 channel-targeted drugs present in clinical antiarrhythmic drugs.

In this study, we used guinea pig single cardiomyocytes to observe the effect of the drug on I _K currents. I _K currents in guinea pigs can be separated into two types: rapidly activated and slowly activated, and we use the tail current recorded for 5 s after the test pulse as the observation index, which is the slow activated portion of _K ( I _KS ). Indeed, when the heart rate increases, I _KS plays a major role in repolarization. In this study, we observed that when drugs can inhibit I _K at a concentration of 0.5%, they can induce prolongation of APD and thereby have antiarrhythmic effects during arrhythmia.

Inhibition of the drug on the inward portion of the I _K1 current was also observed in this study to slightly increase the outward portion of the current, while not affecting the reverse potential and the commutation characteristics of the channel. Although there is no significant difference between increases before and after drug treatment, an increase in the outward portion of I _K1 can promote repolarization of the state 3 action potential of the cell, which facilitates cell membrane stabilization and premature postpolarization Eliminates and suppresses arrhythmia caused by trigger mechanism.

Taken together, the 5% solution prepared by the capsule powder according to the present invention can suppress the inward rectified potassium current and the transient outward potassium current and delay the rectified potassium current in a voltage-dependent manner. Thus, it can reduce the excitability of cardiomyocytes, inhibit the process of repolarization of AP in cardiomyocytes, and have antiarrhythmic effects by prolonging APD.

Claims (10)

Ginseng 45-180 parts by weight, 50-200 parts by weight of ophiopononis, 125-450 parts by weight of corni, 125-450 parts by weight of Miltiorrhizae, 95-400 parts by weight of fried semen ziziphi spinosae Treating arrhythmias caused by abnormal potassium channels in cardiomyocytes, comprising 95-400 parts by weight of taxilli, 45-200 parts by weight of rubra, and 35-150 parts by weight of steleophaga. Pharmaceutical compositions for The method of claim 1,
45-200 parts by weight of Rhizoma Nardostachyos, 25-90 parts by weight of Rhizoma Coptidis, 35-150 parts by weight of Schisandrae Sphenanthenae, Os-draconis 75-300 parts by weight A pharmaceutical composition for treating arrhythmias caused by abnormal potassium channels in cardiomyocytes. 3. The method of claim 2,
45-90 parts by weight of ginseng, 112-135 parts by weight of Macmundong, 224-270 parts by weight of cornus oil, 200-225 parts by weight of sweet ginseng, 150-186 parts by weight of acetic acid, 150-186 parts by weight, 89-89 parts by weight, By abnormal potassium channels in the myocardial cells, characterized in that it comprises 35-100 parts by weight of worms, 45-95 parts by weight of saccharin, 45-60 parts by weight of nasturtium, 67-75 parts by weight of Schizandra chinensis and 145-150 parts by weight of keel. Pharmaceutical compositions for treating induced arrhythmias. 3. The method of claim 2,
Ginseng 89 parts by weight, Mcmundong 112 parts by weight, corn oil 224 parts by weight, sweet ginseng 224 parts by weight, acetic acid 186 parts by weight, 186 parts by weight of fresh raw material, 89 parts by weight of red cherries, insects 75 parts by weight, 89 parts by weight of saengsonghyang, 45 parts by weight of yellow lotus Boo, 67 parts by weight of Schizandra chinensis, 149 parts by weight keel, characterized in that the pharmaceutical composition for treating arrhythmia caused by abnormal potassium channels in the cardiomyocytes. 3. The method of claim 2,
Ginseng 45 parts by weight, Mcmundong 112 parts by weight, corn oil 224 parts by weight, sweet ginseng 225 parts by weight, acetic acid 186 parts by weight, 186 parts by weight of the above raw material, 89 parts by weight of red radish, 45 parts by weight of saengsonghyang, 35 parts by weight of barberry Boo, 67 parts by weight of Schizandra chinensis, 149 parts by weight keel, characterized in that the pharmaceutical composition for treating arrhythmia caused by abnormal potassium channels in the cardiomyocytes. 6. The method according to any one of claims 1 to 5,
The dosage form of the pharmaceutical composition is in the form of capsules, tablets, medical granules, powders or oral solutions, to treat arrhythmias caused by abnormal potassium channels in cardiomyocytes. Pharmaceutical composition for. delete A method of preparing a pharmaceutical composition according to claim 1, wherein the method comprises:
(a) add an appropriate amount of 70% ethanol to 45-180 parts by weight of ginseng, reflux extracting three times, pool the extract solution, filter, concentrate and dry it; Grounding to fine powder,
(b) Appropriate amount of 70% ethanol is added to 125-450 parts by weight of cornus oil and 125-450 parts by weight of salvia, followed by three reflux extractions, pooling the extraction solution, filtering and condensing it into a condensate. Thickening step,
(c) grinding 35-150 parts by weight of worms into fine powder and drying the powder for later use,
(d) add an appropriate amount of water to 50-200 parts by weight of ganmundong, 95-400 parts by weight of acetic acid, 95-400 parts by weight of the above raw material, 45-200 parts by weight of the white peony, boil twice, and pool the extract solution Concentrating it into a coagulum, and
(e) pooling the condensate from steps (b) and (d), to which the pharmaceutical fine powder from step (c) and the pharmaceutical fine powder from step (a) are added and mixed And formulating it into the pharmaceutical composition
Method for producing a pharmaceutical composition comprising a. The method of claim 8,
The raw material of step (b) further comprises 45-200 parts by weight of scent, 35-150 parts by weight of Schizandra chinensis, 25-90 parts by weight of sulfur, and the raw material of step (d) further comprises 75-300 parts by weight of keel Method for producing a pharmaceutical composition, characterized in that. The method of claim 9,
Ginseng 89 parts by weight, Mcmundong 112 parts by weight, corn oil 224 parts by weight, sweet ginseng 224 parts by weight, acetic acid 186 parts by weight, 186 parts by weight of fresh raw material, 89 parts by weight of red cherries, insects 75 parts by weight, 89 parts by weight of saengsonghyang, 45 parts by weight of yellow lotus Part, 67 parts by weight of Schizandra chinensis, 149 parts by weight of keel.

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