CN101234169B - Application of Chinese medicine composition in preparing medicament for adjusting cardiac muscle cell potassium ion channel - Google Patents
Application of Chinese medicine composition in preparing medicament for adjusting cardiac muscle cell potassium ion channel Download PDFInfo
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Abstract
The invention provides an application of a Chinese traditional medicine composition in regulation of potassium ion channel in myocardial cells. The Chinese traditional medicine composition inhibits an inward rectifier potassium current, a transient outward potassium current and delays an outward rectifier potassium current in a voltage dependent mode, which prolong the action potential duration (APD) to play a role of anti-arrhythmic effect through lowering the excitability of myocardial cells and inhibiting the repolarization process of the action potential (AP) of the myocardial cells.
Description
Technical field
The present invention relates to application and the application in preparation treatment antiarrhythmic medicament of a kind of Chinese medicine composition in cardiac muscle cell potassium ion channel is regulated.
Background technology
Cardiovascular disease has become the healthy principal disease of harm humans like hypertension, coronary heart disease, arrhythmia etc., and its M & M is high to occupy first place in the world.Serious arrhythmia, like quick type ventricular tachycardia (VT), quiver (VF) in the chamber and heart broken dead (SCD) then is the dead major reason of cardiovascular disease.
The normal blood-pumping function of heart depends on the alternating activity of the contraction and the diastole of myocardium rhythmicity, and formation that myocardial cell membrane is excited and conductive process then are the initiating agents that triggers contraction of cardiac rhythm property and diastole.Formation that cardiac muscle is excited and conduction are the bases that is changed to the myocardial cell transmembrane potential.The variation abnormality of transmembrane potential will cause the rhythm of the heart to change, and be called arrhythmia.Arrhythmia has a strong impact on the blood-pumping function of heart, in time treatment.
Arrhythmia can be divided into sinus arrhythmia, atrial arrhythmia, atrioventricular junction arrhythmia, ventricular arrhythmia and heart block according to the arrhythmia happening part, and atrial arrhythmia comprises: atrial premature beat, atrial tachycardia, atrial flutter, atrial fibrillation; Ventricular arrhythmia comprises: premature ventricular beat, ventricular tachycardia, ventricular flutter, ventricular fibrillation [the 2nd edition P172-212 in Ye Rengao internal medicine People's Health Publisher October in 1984].
At present clinical use and develop in antiarrhythmic drug classify with mechanism of action according to influence to cardiac electrophysiology; Can be divided into four big types: the I class is a sodium channel inhibitor; The II class is a beta-blocker; The III class is the medicine that selectivity prolongs myocardial cell action potential duration, APD (APD) and effective refractory period (ERP), and selectivity blocks the medicine of myocardium potassium-channel, and the IV class is a calcium channel blocker.The I class, the II class, IV class medicine all has the effect that reduces conduction velocity even cause conduction block, and these effects can increase the probability of reciprocal excitation, and bring out arrhythmia.III class medicine does not have obvious influence [the 4th edition the 14th printing P159-171 in Li Duan pharmacology (the 4th edition) People's Health Publisher November in 1999] to self-disciplining.
Patent ZL02146572.X discloses a kind of pharmaceutical composition of treating Coronary heart disease ventricular early throb and preparation method thereof.Unexposed this pharmaceutical composition of this patent is for the regulating action of potassium-channel, and application only limits to the treatment of Coronary heart disease ventricular early throb, and the present invention has done further research on the basis of patented technology once more.Confirm that Chinese medicine composition of the present invention has inhibitory action to potassium-channel, can be applied to various room property, chamber property and supraventricular arrhythmia.
Summary of the invention
The purpose of this invention is to provide the application of a kind of Chinese medicine composition in cardiac muscle cell potassium ion channel is regulated, the mode that this Chinese medicine composition can voltage relies on suppresses the inward rectification potassium current, instantaneous export-oriented potassium current, and Delayed Rectifier Potassium Current.Through irritability that reduces myocardial cell and the process of repolarization that suppresses myocardial cell action potential (AP), over reach current potential time-histories (APD) is brought into play antiarrhythmic effect.
Another object of the present invention provides the method for preparing of the active component in the application of this Chinese medicine composition in cardiac muscle cell potassium ion channel is regulated.
Another object of the present invention provides the application of this Chinese medicine composition in preparation treatment antiarrhythmic medicament.
Based on the foregoing invention purpose, the invention provides the application of a kind of Chinese medicine composition in cardiac muscle cell potassium ion channel is regulated, it is characterized in that this Chinese medicine composition is processed by following bulk drugs:
Radix Ginseng 45-180 part, Radix Ophiopogonis 50-200 part, Fructus Corni 125-450 part, Radix Salviae Miltiorrhizae 125-450 part, Semen Ziziphi Spinosae (parched) 95-400 part, Herba Taxilli 95-400 part, Radix Paeoniae Rubra 45-200 part, Eupolyphaga Seu Steleophaga 35-150 part, Radix Et Rhizoma Nardostachyos 45-200 part, Rhizoma Coptidis 25-90 part, Fructus Schisandrae Sphenantherae 35-150 part, Os Draconis 75-300 part.
The weight ratio of crude drug is preferably in the Chinese medicine composition of the present invention:
89 parts of Radix Ginsengs, 112 parts of Radix Ophiopogonis, 224 parts of Fructus Corni, 224 parts of Radix Salviae Miltiorrhizaes, 186 parts of Semen Ziziphi Spinosae (parched)s, 186 parts of Herba Taxillis, 89 parts of Radix Paeoniae Rubra, 75 parts of Eupolyphaga Seu Steleophagas, 89 parts of Radix Et Rhizoma Nardostachyos, 45 parts of Rhizoma Coptidis, 67 parts of Fructus Schisandrae Sphenantheraes, 149 parts of Os Draconis.
The weight ratio of crude drug also is preferably in the Chinese medicine composition of the present invention:
45 parts of Radix Ginsengs, 112 parts of Radix Ophiopogonis, 224 parts of Fructus Corni, 225 parts of Radix Salviae Miltiorrhizaes, 186 parts of Semen Ziziphi Spinosae (parched)s, 186 parts of Herba Taxillis, 89 parts of Radix Paeoniae Rubra, 45 parts of Radix Et Rhizoma Nardostachyos, 35 parts of Eupolyphaga Seu Steleophagas, 45 parts of Rhizoma Coptidis, 67 parts of Fructus Schisandrae Sphenantheraes, 149 parts of Os Draconis.
The weight ratio of crude drug also is preferably in the Chinese medicine composition of the present invention:
90 parts of Radix Ginsengs, 135 parts of Radix Ophiopogonis, 270 parts of Fructus Corni, 200 parts of Radix Salviae Miltiorrhizaes, 150 parts of Semen Ziziphi Spinosae (parched)s, 150 parts of Herba Taxillis, 100 parts of Radix Paeoniae Rubra, 100 parts of Eupolyphaga Seu Steleophagas, 95 parts of Radix Et Rhizoma Nardostachyos, 60 parts of Rhizoma Coptidis, 75 parts of Fructus Schisandrae Sphenantheraes, 150 parts of Os Draconis.
In the application of the present invention, the active component of this Chinese medicine composition is processed by the following step:
A) people participates in 70% alcohol reflux three times, and merge extractive liquid, filters, and concentrates oven dry, the fine powder that is ground into;
B) Fructus Schisandrae Sphenantherae, Fructus Corni, Radix Salviae Miltiorrhizae, Rhizoma Coptidis, Radix Et Rhizoma Nardostachyos add 70% alcohol reflux 3 times jointly, and merge extractive liquid, filters spissated extractum;
C) fine powder that is broken into of Eupolyph aga sinesis Walker;
D) Radix Ophiopogonis, Semen Ziziphi Spinosae (parched), Herba Taxilli, Radix Paeoniae Rubra, Os Draconis decocte with water 2 times, merge extractive liquid,, spissated extractum.
E) with step b) and d) merging of gained extractum, the fine drug powder of adding step c) gained, oven dry is ground into fine powder, adds step a) gained fine drug powder, and mixing promptly gets this Chinese medicine composition active component.
In the application of the present invention; Said Chinese medicine composition is a kind of in capsule, tablet, electuary, powder or the oral liquid formulations; For above-mentioned dosage form can be realized; Need when these dosage forms of preparation, to add the pharmacy acceptable auxiliary; For example: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic etc.; Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc., and disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc., lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc., and sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence, antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, fixed, the Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods of acetic acid chloroethene.
During the present invention used, the mode that this Chinese medicine composition can voltage relies on suppressed myocardial cell inward rectification potassium current, instantaneous export-oriented potassium current, and Delayed Rectifier Potassium Current.
During the present invention used, this Chinese medicine composition reduced the irritability and the process of repolarization that suppresses myocardial cell AP of myocardial cell through the adjusting to the myocardial cell membrane potassium channel.
During the present invention used, this Chinese medicine composition can prolong the myocardial cell action potential duration, APD through the adjusting to the myocardial cell membrane potassium channel.
Among the present invention, the application of this Chinese medicine composition in preparation treatment antiarrhythmic drug.
During the present invention used, said arrhythmia comprised: supraventricular arrhythmia or ventricular tachycardia or ventricular flutter or ventricular fibrillation.
During the present invention used, said arrhythmia comprised: atrial premature beat or atrial tachycardia or atrial flutter or atrial fibrillation.
Among the present invention, this Chinese medicine composition is to myocardial cell membrane I
K1Inhibitory action is arranged, reduce I
K1Electric current density.
Among the present invention, this Chinese medicine composition is to myocardial cell membrane export-oriented potassium current (I of moment
To) electric current has inhibitory action, can quicken the inactivation of electric current.
Among the present invention, this Chinese medicine composition to interior to rectification potassium current I
K1(I
K1) the interior of electric current have inhibitory action to composition, but do not change the upset current potential and the rectification characteristic of passage, and slightly increase the export-oriented composition of electric current, help stabilizing cell membrane, eliminate early after depolarization.
The consumption of Chinese medicine composition of the present invention by active component crude drug gross weight, is 7-28 gram/day, but takes every day once, preferably divides and takes for 2-4 time.
Description of drawings
Fig. 1 isolated heart perfusion system (Langendorff perfusion system).
Fig. 2 Chinese medicine composition of the present invention is to I
K1Electric current influence A, contrast; When B, drug level 0.5% to I
K1Inhibitory action is arranged; C, Chinese medicine composition of the present invention is to I
K1The influence of electric current density-voltage relationship.
Fig. 3 I
ToCurrent characteristic and Chinese medicine composition of the present invention are to the effect A of electric current; The outward current that on single rat ventricular myocytes, records, Vh=-90mV gives earlier-the preceding stimulation of 40mV 20ms; Again with step 10mV depolarization to+60mV; Clamp down on time 400ms, activated outward current has two kinds of compositions, comprises transient current and keeps electric current.When B, drug level 0.5% the Ito electric current there is inhibitory action.C, the Ito current-voltage relation curve.● be total peak point current, ▲ for keeping electric current, ■ be transient current (■=●-▲).D, the electric current density-voltage curve of transient current peak value.● before medication, ▲ for after the medication, medicine has inhibitory action to Ito, and show as electric current density and reduce.
Fig. 4 Chinese medicine composition of the present invention is to I
ToThe time dependence inactivation influence A, before the administration; B, drug level 0.5%; And to I
ToThe inactivation of electric current partly carries out match with the single index equation.
Fig. 5 Chinese medicine composition of the present invention is to I
ToThe stable state inactivation influence A, before the administration; B, drug level 0.5%; C, the voltage-dependent of outward current heterogeneity inactivation, ● be peak point current; ▲ for keeping electric current; ■ be transient current (■=●-▲) D, the stable state inactivation curve of instantaneous outward current Ito before and after the medication, ● before medication; ▲ for after the medication, solid line is respectively the curve result with the Bolziman equation model among the figure.
Fig. 6 Chinese medicine composition of the present invention is to I
ToThat recovers behind the electric current inactivation influences A, contrast; B gives drug level 0.5%; C, I
ToTime dependence recovery curve behind the inactivation, ● before medication, ▲ for after the medication, solid line is respectively the curve with the single index equation model among the figure.
Fig. 7 Chinese medicine composition of the present invention is to Delayed Rectifier Potassium Current (I
K) the voltage-dependent inhibitory action A of electric current, V
H=-40mV, progressively depolarization continues 5s to 60mV, the single myocyte of guinea-pig ventricular, can draw a voltage and rely on and the activated outward current I of time-dependent
KWhen B, drug level 0.5%, the medication front and back are at V
T=50mV continues the change C of 5s recorded current, before and after the medication, and the I-V relation curve that obtains with the current density versus voltage mapping of the tail current peak value that writes down under each test voltage.
Fig. 8 Chinese medicine composition of the present invention is to the activated I of time dependence
KThe inhibitory action A of electric current, V
H=-40mV, V
T=50mV continues 500ms～5s, increases progressively with 500ms, can record with the stimulation time prolongation the single myocyte of guinea-pig ventricular to increase I gradually
KElectric current and tail current.When B, drug level 0.5%, the I of record under same test voltage and time
KElectric current and tail current.C is before and after the medication, with the tail current peak value time dependence activating curve that mapping obtains to activationary time of record under the different activationary times.
The specific embodiment
Following embodiment is used to illustrate the preparation of Chinese medicine composition of the present invention, but it can not constitute any restriction to scope of the present invention.
Embodiment 1
Application for the ease of the potassium-channel regulating action of this Chinese medicine composition is prepared as capsule with this Chinese medicine composition
Prescription:
Radix Ginseng 89g 112g Radix Ophiopogonis Fructus Corni 224g Radix Salviae Miltiorrhizae 224g
Semen Ziziphi Spinosae (parched) 186g Herba Taxilli 186g Radix Paeoniae Rubra 89g Eupolyphaga Seu Steleophaga 75g
Radix Et Rhizoma Nardostachyos 89g Rhizoma Coptidis 45g Fructus Schisandrae Sphenantherae 67g Os Draconis 149g
Method for preparing:
A) in the above-mentioned prescription, Radix Ginseng is with 70% alcohol reflux three times, and 3 hours for the first time, each 2 hours later on, merge extractive liquid, filtered, and reclaims ethanol, concentrates, and oven dry is ground into fine powder, and is subsequent use;
B) in the above-mentioned prescription, Fructus Schisandrae Sphenantherae, Fructus Corni, Radix Salviae Miltiorrhizae, Rhizoma Coptidis, Radix Et Rhizoma Nardostachyos are with 70% alcohol reflux three times, and merge extractive liquid, filters, and reclaims ethanol, concentrate extractum, subsequent use;
C) in the above-mentioned prescription, Eupolyph aga sinesis Walker is broken into fine powder, and is subsequent use;
D) in the above-mentioned prescription, Radix Ophiopogonis, Semen Ziziphi Spinosae (parched), Herba Taxilli, Radix Paeoniae Rubra, Os Draconis decocte with water secondary, merge extractive liquid, filters, the extracting solution of filtrating and Fructus Schisandrae Sphenantherae etc. merges, concentrate extractum, subsequent use;
E) with step b) and d) merging of gained extractum, the fine drug powder that adds the step c) gained is dried, and is ground into fine powder, adds step a) gained fine drug powder, mixing, 1000 capsules of packing into.
Indication: be used for supraventricular arrhythmia, ventricular tachycardia, atrial flutter.
Usage and consumption: oral.One time 2～4,3 times on the one.
Test Example
Chinese medicine composition pharmacological activity test of the present invention.
For the activity of the regulating action of illustrating Chinese medicine composition of the present invention sodium channel, use the capsule 's content dry powder (to call capsule dry powder of the present invention in the following text) that makes by the foregoing description 1 method to carry out following animal experiment.
1. material and method
1.1. material
1) reagent capsule dry powder of the present invention is provided by Yiling Pharmaceutical Co., Ltd, Shijiazhuang.II Collagen Type VI enzyme (collagenase II) is the Gibico Company products.Chain protease E is the Merck Company products.N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid (HEPES), ethylene glycol-two tetraacethyls (EGTA), glutamic acid (L-Glutamic acid), taurine (Taurine), aspartic acid, phosphagen disodium, choline chloride (Choline-Cl), CaCl
2, K
2ATP is the Sigma Company products.4-aminopyridine (4-AP) is the Fluka Company products.All the other reagent are homemade analytical pure, and chemical reagents corporation provides by Beijing.
2) solution
1. there are not calcium tyrode's solution (mmol/L): NaCl 136, KCl 5.4, MgCl
21.0, NaH
2PO4 0.33, HEPES 10, Glucose 10 transfer pH complete 7.4 with NaOH.
2. KB liquid (mmol/L): KCl 40, KH
2PO4 20, MgSO
43.0, KOH 80, L-glutamic acid 50, taurine 20, HEPES 10, Glucose 10, EGTA 0.5, transfer pH to 7.4 with KOH.
3. write down the perfusate (mmol/L) of full cell potassium current: Choline-Cl 136, MgCl
21.2, KCl 5.4, NaH
2PO4 0.33, CaCl
21.8, CdCl
20.15 HEPES 10, Glucose 10 transfer pH to 7.4 with LiOH.Record I
KThe time add 1mmol/LBaCl
2With blocking-up I
K1Electric current.
4. write down the interior liquid (mmol/L) of electrode of full cell potassium current: Aspartic acid potassium 120, KCl 20, HEPES 5, MgCl
21.0, K
2-ATP 4, EGTA 10, phosphagen disodium 2 are transferred pH to 7.3 with KOH.Liquid all passes through diameter 0.22 μ m filtering with microporous membrane in the electrode used therein.
The preparation of capsule dry powder solution 5. of the present invention: with capsule dry powder of the present invention, the extracellular perfusate with no KCl is mixed with mass percent concentration 5% solution earlier, adjusts K+ concentration again to 5.4mmol/L, and the centrifuging and taking upper solution is subsequent use.
3) animal male SD rat, body weight 250-300g.Provide by Beijing Vital River Experimental Animals Technology Co., Ltd..
1.2 method
1.2.1. the separation of single myocardial cell prepares the ventricular muscles individual cells with acute enzymatic isolation method.Get rat with 50mg/kg pentobarbital sodium intraperitoneal injection of anesthesia after, open breast and take out heart, in 4 ℃ of no calcium Tyrode liquid, remove fat and pericardium, separation aorta and intubate are carried out Langendorff cardiac perfusion (see figure 1), speed is 6～8ml/min.With no calcium Zinciodati Comp solution perfusion 3min (perfusion pressure is the 20cm water column), clean the arteria coronaria inner blood earlier, reuse hangs down calcium enzymolysis solution (CaCl
2For the low calcium tyrode's solution of 0.05mmol/L, contain II Collagen Type VI enzyme 0.4mg/ml, protease E 0.04mg/ml) the circulation perfusion; After about 20 minutes, heart is taken off from the Langendorff device, insert in the room temperature KB liquid; Cut off the tissue of atrium and basilar part, hold the ophthalmology tweezer, slowly blow and beat with the suction pipe of thick opening with ventricular organization's tearing gently; Myocardial cell is broken away from from cardiac muscular tissue; Obtain single ventricular muscle cell, supernatant filters with 120 purpose nylon drainage screens, and myocardial cell is collected in the test tube that contains KB liquid.Whole perfusion system temperature remains on 37 ℃, and all perfusates and KB liquid all pass to 100% O
2Saturated more than 30 minutes.Cell is at room temperature left standstill about half an hour multiple calcium to 0.25mmol/L, and room temperature preservation is subsequent use.The separation method of guinea pig myocardium cell is basic identical.
1.2.2 draw a little KB suspension that is rich in cell in the perfusion groove of 0.5ml before the experiment of full cell patch pincers experimental record, leave standstill 5～10min, treat cell sink to the bottom attach wall after, use 100%O
2Saturated extracellular fluid carries out perifusion 5～10min, the about 2ml/min of flow velocity.Select clear-cut margin down at inverted microscope (OLYMPUS IX70), smooth surface, band is clear, shrinkage-free cell.Experiment is carried out under 22～24 ℃ of room temperatures.The recording method of the full cell patch pincers of employing standard, recording channel electric current under the voltage clamp pattern.Patch clamp amplifier (Axopatch 700B, U.S. Axon company) is connected with computer through A/D and D/A data converter (Digidata 1322, U.S. Axon company).The collection of stimulus signal and voltage, current input signal is controlled by software (pClampex 10.0, U.S. Axon company).Glass fiber embryonic tube (Inst. of Physics, CAS provides) draws appearance (p-97, U.S. sutter company) through microelectrode and is drawn by 4 steps and form, through polishing appearance (MF-830, Japanese NARISHIGE company) polishing rear tip diameter 2～3 μ m.Charge that to go into water resistance behind the liquid in the electrode be 1.5～3M Ω, behind the compensation liquid junction potential, regulate Three dimensional steerable device (MHW-3, Japanese NARISHIGE company) and make eletrode tip shift to cell surface, form high resistant sealing-in (giga seal), sealing-in resistance is more than the 1G Ω.Compensate fast electric capacity and inhale the broken cell film and form full cell record pattern.Electric capacity gives when measuring-stimulation of 10mV step, and V asks membrane capacitance with Cm=τ Iss/ Δ.Regulate the compensation 80%～90% of slow capacitance compensation and series resistance to reduce instantaneous charging and discharging currents and clamper error.Signal is the quadravalence Bezier low pass filter of 1kHz through cut-off frequency, and sample rate is 10kHz.
When record, for avoiding the relaxation phenomenon influence of electric current, add ATP in the liquid in the electrode, and the control experiment is accomplished in 25 minutes behind the cell rupture of membranes.According to experimental design, with the drug solution for preparing, distinguish perfusion with the speed of 2ml/min to cell perfusion groove with the constant current peristaltic pump, use whole negative pressure suction device that waste liquid is absorbed from the perfusion groove simultaneously.Recording method is to stablize the preceding current value of 5min record administration, the current value behind the administration 5min under the record drug effect behind the rupture of membranes.Sampling back data-storing supplies to measure and analyze and use in computer hard disc.For eliminating the iuntercellular error, current value is represented with electric current density (pA/pF).
1.2.3 each galvanism scheme is set and observation index 1) record I
K1The time stimulation protocol: keep current potential (Vh)-40mV, command potential-120mV～0mV, step 10mV clamps down on time 400ms, stimulus frequency 0.2Hz; I with test voltage-100mV
K1The variation of steady-state current density before and after medication is as observation index; 2) record I
ToStimulation protocol during current-voltage correlation: keep current potential (Vh)-90mV, give earlier-the preceding stimulation of 40mV 20ms, give again-40mV～+ depolarization of 60mV stimulates, and step 10mV clamps down on time 400ms, stimulus frequency 0.2Hz; I with test voltage 60mV
ToThe variation of peak current density before and after medication is as observation index; 3) record I
ToThe stimulation protocol of stable state inactivation: keep voltage-90mV, give 400ms preceding stimulation, clamp down on then at 60mV 300ms record I from-120mV to 60mV step 10mV
ToElectric current, and relatively change before and after the medication; 4) record I
ToThe stimulation programs that time dependence recovers behind the inactivation: adopt dipulse to stimulate, keep voltage-90mV, successively giving two depolarizations then stimulates P1 to the depolarization that 60mV continues 200ms, and P2, its interval Δ t increase successively and be made as 1,3 respectively; 5,7,10,15,20; 30,40,60,90,120; 150,180,210,250,300 (ms).The corresponding peak current I of record P2.With I/Imax Δ t is made recovery curve behind the stable state inactivation.5) voltage-dependent of measuring Ik activates: V
H=-40mV, from-40mV gradually depolarization to+60mV, step 10mV, stimulus wave is wide to be 5s, gets back to V
HContinue 2.5s record tail current.6) time dependence of measuring the Ik tail current activates (envelope of tails test): V
H=-40mV, depolarization is back to V to+50mV
H, continuous 10 pulses, the wide 500～5000ms that is respectively of each impulse stimulation ripple does not wait, and increases progressively gradually with the 500ms amplitude.
1.3 data analysis and statistics
Primary current The data Clampfit 10.0 (U.S. Axon company) measures collection, uses Origin 6.0 data processing softwares (U.S. Microcal Software company) the back data are analyzed to gathering, match and mapping.(x ± s) expression adopts paired t-test to experimental data before and after the administration, P＜0.05 is thought has significant difference with mean ± standard deviation.
2. result
2.1 Chinese medicine composition of the present invention is to the effect of rat myocardial cell film IK1
Keep current potential (Vh)-40mV, command potential-120mV～0mV, step 10mV clamps down on time 400ms, and stimulus frequency 0.2Hz can record the inward electric current of a quick active, no obvious inactivation (seeing Fig. 2 A) in 400ms on the single ventricular muscle cell of rat.This electric current can be by 1mmol/L BaCl
2Blocking-up shows that this electric current is I fully
K1I
K1Reduce with depolarization to composition in the electric current, reversal potential pact-40mV has the inward rectification characteristic.Dissolve 0.5% solution that Chinese medicinal composition capsules dry powder of the present invention is mixed with the outer liquid of potassium, to I
K1Inhibitory action (seeing Fig. 2 B) is arranged.The preceding average peak electric current density of medication is-10.78 ± 1.80 (pA/pF) during-100mV, and the average peak electric current density is-7.18 ± 2.05 (pA/pF) after the medication, and average suppression ratio is 33.1 ± 16.85% (n=11, P＜0.05).With electric current density amplitude under each pulse to the corresponding membrane current potential map the I-V curve.Medicine all reduces I on each command voltage level
K1Electric current density, but do not change the shape (seeing Fig. 2 C) of reversal potential and curve basically.After the administration, clamp down on that in difference the IK1 electric current is suppressed, show as on electric current density-voltage curve and move, but upset current potential and rectification property are not had appreciable impact.
2.2 Chinese medicine composition of the present invention is to rat myocardial cell film I
ToEffect
2.2.1 Chinese medicine composition of the present invention is to I
ToThe influence of current-voltage (I-V) curve
The inside and outside liquid of service recorder cell potassium channel is kept current potential (Vh)-90mV, gives earlier-the preceding stimulation of 40mV20ms, give again-40mV～+ depolarization of 60mV stimulates, and step 10mV clamps down on time 400ms, stimulus frequency 0.2Hz.Record export-oriented potassium current in rat ventricular myocytes and comprise two kinds of compositions: a kind of is the transient current (I of quick active and deactivation
To), electric current is " A " type, and peak shape is sharp-pointed, begins to activate from 0mV, and electric current peaks about 10ms, and rapid afterwards inactivation is in 200ms Nei Jibenda stable state.It activates and inactivation all is voltage and time dependence.This electric current can be blocked by the 4-AP of 2mM fully, shows that this electric current is I
ToAnother kind of composition is follow current (I
Maintained), begin to activate from-10mV, in-10～60mV scope, increase with voltage and (to see Fig. 3 A, C).With electric current density amplitude under each pulse to the corresponding membrane current potential map the I-V curve.Deduct with the peak point current amplitude and to keep electric current and obtain transient current, be i.e. I
To0.5% capsule dry powder solution of the present invention is to I
ToElectric current has inhibitory action (seeing Fig. 3 B), under 0～60mV test voltage, shows as the decline of peak point current, and the I-V curve moves down.Average current density at 60mV is reduced to-10.02 ± 3.93 (pA/pF) from-19.82 ± 7.10 (pA/pF), and average suppression ratio is 50.60 ± 10.77% (n=6, P＜0.05) (seeing Fig. 3 D).To I
ToThe inactivation of electric current partly carries out match with the single index equation, can obtain the time constant (τ) of time dependence inactivation.Chinese medicine composition of the present invention can quicken the inactivation (see figure 4) of electric current, and the τZhi before and after the medication is respectively 29.06 ± 4.66 and 21.44 ± 3.12 (n=6, P＜0.05).
2.2.2 Chinese medicine composition of the present invention is to I
ToThe influence of stable state inactivation curve
Record I
ToThe stimulation protocol of stable state inactivation: keep voltage-90mV, give 400ms preceding stimulation, clamp down on then at 60mV 300ms record I from-120mV to 60mV step 10mV
ToElectric current, and relatively change before and after the medication (Fig. 5 A, B).The inactivation that from the map of current of drawing, can see outward current is divided into two processes, at first is the inactivation of keeping electric current, and inactivation voltage is lower, is the inactivation of peak current then, and inactivation voltage will be higher than the former.I
ToAmplitude by peak current and test pulse terminal measure keep the difference decision (Fig. 5 C) between electric current.Each current amplitude of standardization, with relative electric current to each transmembrane potential map the stable state inactivation curve. and with Boltzmann equation I/Imax=1/{1+exp ((Vm-V
1/2)/k) } half inactivation voltage (V is obtained in match
1/2) and inactivation curve slope factor (k) (Fig. 5 D).0.5% capsule dry powder solution of the present invention can make I
ToThe stable state inactivation curve moves to left, and it is big that slope becomes.V1/2 before and after the medication is respectively-15.67 ± 2.52mV and-26.45 ± 3.88mV, and k value becomes 6.38 ± 2.02 (n=7, P＜0.05) (see figure 5) from 3.41 ± 0.67.
2.2.3 Chinese medicine composition of the present invention is to I
ToThe influence of recovery curve behind the inactivation
The employing dipulse stimulates, and keeps voltage-90mV, and successively giving two depolarizations then stimulates P1 to the depolarization that 60mV continues 200ms, and P2, its interval Δ t increase successively and be made as 1,3,5 respectively; 7,10,15,20,30,40,60; 90,120,150,180,210,250,300 (ms).The corresponding peak current amplitude I of record P2.With I/Imax Δ t is made recovery curve behind the stable state inactivation.With the be restored time constant (τ) of curve of single index equation model curve.Can be when drug level 0.5% so that extend to 18.52 ± 3.76 (n=5, P＜0.05) (see figure 6) from 12.86 ± 0.31 recovery time.
2.2 Chinese medicine composition of the present invention is to the myocyte I of guinea-pig ventricular
kEffect
Fig. 7 A is at V
H=-40mV, step 10mV progressively depolarization continue 5s to 60mV, and the single myocyte of guinea-pig ventricular, the voltage of record relies on and the activated outward current I of time-dependent
KChinese medicine composition of the present invention is to I
KElectric current has voltage-dependent inhibitory action (Fig. 7 C).With V
TThe I of=50mV record
KBe observation index, when drug level 0.5%, can make tail current peak current density decline 30.77 ± 1.11% (n=5, p＜0.05).Fig. 8 A, B are the activated in time I with envelope stimulation protocol record
KTail current, and medicine is to the activated I of time dependence
kInhibitory action.
The I that finds at present
ToComprise two types, a kind of for responsive to the insensitive I of cellular calcium to 4 amido pyridines
To1, another kind relies on for calcium and can be by caffeine and Co
2+The I of blocking-up
To2In this research, contain the calcium chelating agent EGTA (10mmol/L) of high concentration in the electrode solution, contain 0.15mmolL in the perfusate
-1Calcium blockers CdCl
2, thereby the moment extroversion potassium current of record does not comprise Ito in this research
2Composition.We observe Chinese medicine composition of the present invention to I in test
ToThe obvious suppression effect is arranged, reduce I
ToCan be through influencing 1 phase multipole of action potential, especially dog, rat and the mankind etc. contains I
ToBigger kind, and APD is prolonged.In addition, I
ToAlso be the main electric current that causes striding myocardial wall multipole inhomogeneity, suppress this electric current wall multipole inhomogeneity is striden in minimizing, reduce and to stride the turn back formation of microcircuit of wall, avoid ARR generations such as torsade de pointes.Also do not have special in the antiarrhythmic drug of clinical practice at present to I
To1The medicine of passage.
In this research, we have observed medicine to I with the single ventricular muscle cell of Cavia porcellus
KThe effect of electric current.The I of Cavia porcellus
KElectric current has quick active and slowly activates two kinds of compositions, and the tail current that we write down after 5 seconds with test pulse is as observation index, and this has mainly represented I
KIn slow activated composition (I
KS), when the rhythm of the heart is accelerated, be I in fact
KSIn process of repolarization, play a major role.We see in test drug level be 0.5% o'clock to I
KInhibitory action is arranged, and this will cause that APD prolongs, and helps medicine and when tachycardia, brings into play antiarrhythmic effect.
In this test, also observe medicine to I
K1The interior of electric current has inhibitory action to composition, but do not change the upset current potential and the rectification characteristic of passage, slightly increases the export-oriented composition of electric current.Though compare this effect there was no significant difference before and after the medication, increase I
K1The export-oriented composition of electric current possibly make action potentials of cells 3 additions reply immediately the utmost point, helps stabilizing cell membrane, eliminates early after depolarization, and the arrhythmia that trigger mechanism is caused has inhibitory action.
In sum, with 5% solution of capsule dry powder of the present invention preparation, the mode that can voltage relies on suppresses the inward rectification potassium current, instantaneous export-oriented potassium current, and Delayed Rectifier Potassium Current.Through irritability that reduces myocardial cell and the process of repolarization that suppresses myocardial cell AP, prolong APD and bring into play antiarrhythmic effect.
Claims (8)
1. the application of Chinese medicine composition in the myocardium potassium-channel dysfunctional antiarrhythmic medicament of preparation treatment, this Chinese medicine composition is processed by following bulk drugs:
Radix Ginseng 45-180 part, Radix Ophiopogonis 50-200 part, Fructus Corni 125-450 part, Radix Salviae Miltiorrhizae 125-450 part, Semen Ziziphi Spinosae (parched) 95-400 part, Herba Taxilli 95-400 part, Radix Paeoniae Rubra 45-200 part, Eupolyphaga Seu Steleophaga 35-150 part, Radix Et Rhizoma Nardostachyos 45-200 part, Rhizoma Coptidis 25-90 part, Fructus Schisandrae Sphenantherae 35-150 part, Os Draconis 75-300 part.
2. application as claimed in claim 1 is characterized in that, this Chinese medicine composition is processed by following bulk drugs:
89 parts of Radix Ginsengs, 112 parts of Radix Ophiopogonis, 224 parts of Fructus Corni, 224 parts of Radix Salviae Miltiorrhizaes, 186 parts of Semen Ziziphi Spinosae (parched)s, 186 parts of Herba Taxillis, 89 parts of Radix Paeoniae Rubra, 75 parts of Eupolyphaga Seu Steleophagas, 89 parts of Radix Et Rhizoma Nardostachyos, 45 parts of Rhizoma Coptidis, 67 parts of Fructus Schisandrae Sphenantheraes, 149 parts of Os Draconis.
3. application as claimed in claim 1 is characterized in that, this Chinese medicine composition is processed by following bulk drugs:
45 parts of Radix Ginsengs, 112 parts of Radix Ophiopogonis, 224 parts of Fructus Corni, 225 parts of Radix Salviae Miltiorrhizaes, 186 parts of Semen Ziziphi Spinosae (parched)s, 186 parts of Herba Taxillis, 89 parts of Radix Paeoniae Rubra, 45 parts of Radix Et Rhizoma Nardostachyos, 35 parts of Eupolyphaga Seu Steleophagas, 45 parts of Rhizoma Coptidis, 67 parts of Fructus Schisandrae Sphenantheraes, 149 parts of Os Draconis.
4. application as claimed in claim 1 is characterized in that, this Chinese medicine composition is processed by following bulk drugs:
90 parts of Radix Ginsengs, 135 parts of Radix Ophiopogonis, 270 parts of Fructus Corni, 200 parts of Radix Salviae Miltiorrhizaes, 150 parts of Semen Ziziphi Spinosae (parched)s, 150 parts of Herba Taxillis, 100 parts of Radix Paeoniae Rubra, 100 parts of Eupolyphaga Seu Steleophagas, 95 parts of Radix Et Rhizoma Nardostachyos, 60 parts of Rhizoma Coptidis, 75 parts of Fructus Schisandrae Sphenantheraes, 150 parts of Os Draconis.
5. like each described application among the claim 1-4, it is characterized in that the active component of this Chinese medicine composition is processed by the following step:
A) people participates in 70% alcohol reflux three times, and merge extractive liquid, filters, and concentrates oven dry, the fine powder that is ground into;
B) Fructus Schisandrae Sphenantherae, Fructus Corni, Radix Salviae Miltiorrhizae, Rhizoma Coptidis, Radix Et Rhizoma Nardostachyos add 70% alcohol reflux 3 times jointly, and merge extractive liquid, filters spissated extractum;
C) fine powder that is broken into of Eupolyph aga sinesis Walker;
D) Radix Ophiopogonis, Semen Ziziphi Spinosae (parched), Herba Taxilli, Radix Paeoniae Rubra, Os Draconis decocte with water 2 times, merge extractive liquid,, spissated extractum;
E) with step b) and d) merging of gained extractum, the fine drug powder of adding step c) gained, oven dry is ground into fine powder, adds step a) gained fine drug powder, and mixing promptly gets this Chinese medicine composition active component.
6. like each described application among the claim 1-4, it is characterized in that said Chinese medicine composition is a kind of in capsule, tablet, electuary, powder or the oral liquid formulations.
7. like each described application among the claim 1-4, it is characterized in that this Chinese medicine composition suppresses myocardial cell membrane inward rectification potassium current, instantaneous export-oriented potassium current, and Delayed Rectifier Potassium Current with the mode that voltage relies on; This Chinese medicine composition reduces the irritability and the process of repolarization that suppresses the myocardial cell membrane action potential of myocardial cell through the adjusting to the myocardial cell membrane potassium channel; Prolong the myocardial cell action potential duration, APD.
8. like each described application among the claim 1-4, it is characterized in that said arrhythmia is supraventricular arrhythmia or ventricular tachycardia or ventricular flutter or ventricular fibrillation.
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| CN101683458B (en) * | 2008-09-23 | 2012-09-05 | 河北以岭医药研究院有限公司 | Application of a traditional Chinese medicine composition in preparation of medicament for treating vertigo |
| CN104997819B (en) * | 2015-08-11 | 2018-10-12 | 河北工业大学 | Storax and application of the cell bath mixture in terms of inhibiting Kir2.1 outward currents |
| CN105055467B (en) * | 2015-08-11 | 2019-05-03 | 河北工业大学 | Storax and cell bath mixture and its application in terms of inhibiting Kir2.1 inward electric current |
| CN105738458A (en) * | 2016-02-22 | 2016-07-06 | 中山大学中山眼科中心 | Measuring method for cell potential of bioengineering retina nerve scaffold |
| CN105943605A (en) * | 2016-06-29 | 2016-09-21 | 山东省科学院生物研究所 | Application of Danhong injection as terfenadine heart adverse effect antagonist |
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