| Radix Aconiti Lateralis Preparata, Radix Aconiti, Radix AconitiKusnezoffii (abbreviate as Aconiti Class) are poisonous Chinese Meteria Medicawhich has aconitine and similar continents.The crude drugs without processingare very poisonous and the reason for causing death is mainly due to heavyarrhythmia.Therefore, they are always to be processed to reduce the toxicity.Thetoxicity has been found to be due to diester diterpenoid alkaloids (DDAs) inaconitum plants which include aconitine, its N-methyl homolog mesaconitineand hypaconitine, etc.However, they are also the main active components.Thetoxicity to the heart is considered to activate the Na+ channel in cadiocyte, topromote the Na+ to inflow quickly, to make the cells depolarize so that thearrhythmia is breaking.The toxicity of Aconiti Class is due to producing area, variety, processingmethods.Some other research in our research group indicated the fingerprint ofRadix Aconiti Lateralis Preparata in different producing area differed greatlywhich showed the content of the alkaloid was widely different.Therefore thetoxicity of this kind of crude drugs is influenced largely due to the difference ofthe alkaloid content.The processing method is also the main way to reduce thetoxicity.The earliest processing way of Aconiti Class crude drugs began inSynopsis of the Golden Chamber.After that, the processing way of Aconiti Classcrude drugs has already developed more than 70 kinds of methods so far, such asinfusing, decocting, steaming, processing with Radix Glycytthizae, processingwith honey, processing with bean curd, processing with the black soya bean,processing with RadiX Glycytthizae and Alumen, processing with RadixGlycytthizae and gleditsiae, processing with Radix Glycytthizae and FlosLonicerae, etc. In modern time, there have been developed the salt cookingmethod, high pressure steaming method, pyretic fumigation etc.The ester bondin diester diterpenoid alkaloids hydrolyzes during the processing of AconitiClass crude drugs.The acetyl loses and single ester diterpenoid alkaloids such asbenzoyl aconitine are produced.The benzoyl hydrolyzes and it becomesaconine.The toxicity of hydrolyzate decreases greatly after hydrolization.Thetoxicity of benzoyl aconitine is 1/200 that of aconitine and the toxicity ofaconine is 1/2000 that of aconitine.The fatty acyl group takes the place of acetylin the C8-OH of DDAs and it becomes sulfatide alkali during the processingwhich can also decrease the toxicity of Aconiti Class crude drugs.Therefore, the toxicity of Aconiti Class crude drugs can be decreased by decreasing the contentof diester diterpenoid alkaloids during processing.Past dynasties doctors thoughtthat the object of processing Aconiti Class crude drugs was to decrease thetoxicity and increase the curative effect.But the processing research and cutcrude drug production pay close attention to decreasing the toxicity, whileincreasing the curative effect do not be paid much attention.For reducing thepoison, the current processing method of Aconiti Class crude drugs use rinsingand soaking at first to lose some heavy poisonous diester diterpenoid alkaloidsand then steaming and decocting to hydrolyse the DDAs to single ester alkaloidsand amine alcoh alkaloids.However, DDAs in Aconiti Class crude drugs are not only the poisonouscomponent, but also the anti-inflammatory and demulcent activecomponent.Therefore, the degree of processing has great relation with the levelof DDAs' hydrolysis, the toxicity and curable effect of Aconiti Class crudedrugs.The active component loss and bio activity weakening due to excessprocessing often cause the amount of Radix Aconiti Lateralis Preparata to haveto be increased to 10-20 times of ordinary usage to cure the disease.But thesame dose cut crude drug often brings toxicity due to not enough processing.Thesafety and efficiency of Aconiti Class crude drugs can not be ensured becausethe non-standard processing and instability quality.Therefore, The Medicationby compatibility is an effective way to reduce toxicity and increase curableeffect of Radix Aconiti Lateralis Preparata.Medication by compatibility is one of the characteristics of TraditionalChinese Medicine.Base on the guidelines of "seven emotions regulating" theoryand "chief, adjuvant, assistant and courier herbs" theory, doctors in history usedcompatibility in the application of Radix Aconiti Lateralis Preparata to reduceits toxicity and increase its effect.It is said in Dietetic Materia Medica: "decoctwith black bean to get rid of the toxicity of Aconitum carmichaelii and RadixAconiti Lateralis Preparata." Collective Notes to the Canon of Materia Medicahas the saying: "Every time Radix Aconiti Lateralis Preparata is used incommon formulae, Radix Glycytthizae, Radix Ginseng, Rhizoma ZingiberisRecens are in compatibility to restrict the toxicity." So traditional compatibilitymethod including slowing the poisonous with the sweet, restricting thescattering with the adducting, restraining the hot with the cold, overcoming therigid with the tender. The crude drugs often in compatibility with Radix AconitiLateralis Preparata are Radix Glycytthizae, Rhizoma Zingiberis, Radix etRhizoma Rhei, Radix Rehmanniae,Radix Paeonia, Sinomeniumacutum,etc.Among them, Radix Glycytthizae is widest used in the formulaes which have Radix Aconiti Lateralis Preparata.Jing-Yue Complete Works says:"Radix Glycytthizae is always used in compatibility with Radix AconitiLateralis Preparata.The reason is as follows.The hastiness of Radix AconitiLateralis Preparata can be slowed by Radix Glycytthizae; The toxicity of RadixAconiti Lateralis Preparata can be dispelled by Radix Glycytthizae; RadixAconiti Lateralis Preparata has the moving character and the heart and spleencan be strengthen by Radix Glycytthizae; Radix Aconiti Lateralis Preparata hasscaterring character and Radix Glycytthizae can harmonize nutrient qi anddefense qi." In other words, the compatibility of Radix Aconiti LateralisPreparata and Radix Glycytthizae can not only warm nourishing yang qi, adjustmiddle jiao and reinforce the deficiency, strengthen the effect of Radix AconitiLateralis Preparata on restoring yang to rescue the patient from collapse, butalso remedy defects and rectify errors, moderate drug property.Radix Glycyrrhizae is one of the clinical crude drugs that are usedextensively in Chinese medicine.The chemical composition of RadixGlycytthizae is made mainly of triterpenoid saponins, flavonoids, amylase, etc.Ithas been proved that the flavonoids of Radix Glycytthizae are the maincomposition of antiarrhythmia.And liquiritin is the main component of theflavonoids in Radix Glycytthizae.It has not been reported that the oppositioneffect of liquritin on aconition.Yhe research studied the effect and mechanism oflicorice decreasing the aconitine toxicity to heart through the study of liquritin.It mostly concentrates on the interaction among different chemicalcomposition about reducing toxicity mechanism of the compatibility betweenRadix Aconiti Lateralis Preparata and Radix Glycytthizae at present, but thecompatibility effect on biological function of poisonous result is seldomstudied.So this research use modern technological means to study thecompatibility effect on biological function of Radix Aconiti Lateralis Preparataand Radix Glycytthizae and the reducing toxicity mechanism.The achievementof this research is meaningful to explain the TCM theory and instruct clinicaldrugs application.First, the research compared and analyzed quantity-time-toxicityrelationship and the effect on heart function of aconitine, mesaconitine andhypaconitine in the integral, organ, cell level.Second, the research studied theeffect of liquiritin on heart function, antagonism to aconitine on toxicity to heartand the effects of aconitine on aconitine strengthening heart function in the threelevels.Third, the mechanism of liquiritin on alleviating the heart of aconitinewas analyzed with the patch clamp technique and the whole cell recording.The experimental results were as follows: Part one: The toxicity to heart and effect on heart function of DDAs(aconitine, mesaconitine and hypaconitine) in Radix Aconiti Lateralis Preparata1 The toxicity to heart of DDAs (aconitine, mesaconitine and hypaconitine)in Radix Aconiti Lateralis Preparata1.1 The toxicity to heart of DDAs (aconitine, mesaconitine andhypaconitine) in Radix Aconiti Lateralis Preparata in rats heart in vivoRegarding rat's electrocardiogram (ECG) change as the index, observe andcompare the toxicity to heart induced by aconitine, mesaconitine andhypaconitine.The result indicated that DDAs had obvious toxicity to heart afterthe slow intravenous injection continuously.It appeared in turn prematureventricualr contraction(PVC), ventricular tachycardia(VT), ventricularflutter(VF), ventricular fibrillation, asystole as well as death as the dose ofaconitine accumulated to 17.5, 42.8, 80.8, 84.5, 106.7μg·kg-1, the dose ofmesaconitine accumulated to 18.1, 25.9, 158.2, 148.0, 265.3μg·kg-1, the dose ofhypaconitine accumulated to 134.1, 2571.4, 4182.3, 6177.4μg·kg-1.Amongthem, the accumulating dose of hypaconitine which caused ventricular flutter(VF), ventricular fibrillation asystole was higher than that of aconitine andmesaconitine (VS aconitine or mesaconitine group, p＜0.01).1.2 The toxicity to heart of DDAs (aconitine, mesaconitine andhypaconitine) in Radix Aconiti Lateralis Preparata on isolated rat heartLangendorff perfusion isolated rat heart was subjected to be observed andcompared the toxicity to heart of aconitine, mesaconitine and hypaconitine.Theresult showed: aconitine, mesaconitine and hypaconitine had obvious toxicity toheart.The isolated rat heart ceased to beat after the DDAs poured into itcontinuously in slow speed.Among them, the accumulating dose of aconitine,mesaconitine and hypaconitine were 18.3μg, 21.0μg, 226.7μg respectively.Andthe accumulating dose of hypaconitine was the maximum among them (comparewith aconitine or mesaconitine group, p＜0.01).1.3 Effects of DDAs (aconitine, mesaconitine and hypaconitine) in RadixAconiti Lateralis Preparata on cells vigor, membrane permeabilityin myocardialcells of suckling ratsRegarding the cells vigor, LDH as the index, the toxicity-time-effectrelationship of aconitine, mesaconitine and hypaconitine was investigated Theresult showed: the cells vigor decreased significantly and the vigor of LDH inthe culture fluid increased significantly while the concentration of aconitine,mesaconitine and hypaconitine improved(aconitine, mesaconitine were 0.5, 2, 8,32μM, hypaconitine are 1, 4, 16, 64μM). So did as the incubation time becamelonger (30,60,120min).Cell vigor reduce 10％～50％among them, LDH vigor rise 5～10 times (VS control group p＜0.05, p＜0.01).2 The effect of DDAs in Radix Aconiti Lateralis Preparata on heartfunction2.1 The effect of DDAs in Radix Aconiti Lateralis Preparata on heartfunction of heart failure rats induced by nimodipineThe experiment chose nimodipine, one of Ca2+ antagon, to make heartfailure model in rats.The Medlab bio information recording system wasused.The three DDAs were injected in the rats through femoral vein.The effectof accumulated dosage aconitine, mesaconitine, hypaconitine was observed onstrengthening the heart function.The result showed: aconitine and mesaconitinecould obviously raise the level of heart rate (HR)(The increasing maximumreached 24％,35％) and shorten intersystolic period(The shortening maximumreached 38％,45％)(compare with model group p＜0.05,p＜0.01) as the dosageincreased.The cardiac muscle shrink performance was obviously strengthenedwhich was shown as Vmax, +dp/dtmax, left ventricular developed pressure(LVDP), LVSP, the product of HR and LVDP-Rate Pressure Product(RPP)rising(compare with model group p＜0.01), the increasing maximum of+dp/dtmax could reach 32％,21％respectively while aconitine and mesaconitineaccumulated to 1.67μg-10μg.And the increasing maximum of Vmax could reach100％,45％respectively.The increasing maximum of LVDP could reach36％,30％respectively.the increasing maximum of RPP could reach 34％,38％respectively.Hypaconitine shared similar effect as aconitine.While hypaconitineaccumulated to 3.33μg～20μg, the increasing maximum of HR could reach 8.5％,the decreasing maximum of intersystolic period could reach 10.3％, theincreasing maximum of+dp/dtmaxcould reach 30％, the increasing maximum ofVmax could reach 67％, the increasing maximum of LVDP could reach 13％, theincreasing maximum of RPP could reach 17％.Aconitine, mesaconitine andhypaconitine could obviously reduce LVDP (compare with model group p＜0.05,p＜0.01), but they had no obvious function on -dp/dtmax.2.3 The effect of DDAs (aconitine, mesaconitine and hypaconitine) inRadix Aconiti Lateralis Preparata on the function in the isolated rat heartLangendorff perfusion isolated rat heart was subjected to observe andcompare the effect of aconitine, mesaconitine and hypaconitine on the heartfunction.The result showed: as the dose increasing, aconitine and mesaconitinecould obviously raise the level of heart rate (HR) in the isolated rat hear(compare with contral group p＜0.05).The HR increased 98％, 40％whileaconitine and mesaconitine accumulated to 11.67μg.But hypaconitine couldobviously decrease the HR of the isolated rat hear (compare with contral group p＜0.05, p＜0.01).The HR decreased 66％while hypaconitine accumulated to40μg,46.67μg.the +dp/dtmax could be increased 14.9％,23.2％,85.7％respectively while Aconitine, mesaconitine and hypaconitine accumulated to5μg, 6.67μg, 26.67μg respectively.The left ventricular developed pressure(LVDP) could also increase 44％,32.5％,157.3％respectively(compare withcontral group p＜0.05, p＜0.01), but reduce gradually afterwards.Mesaconitineand hypaconitine showed a tendency to increase-dp/dtmax as the dose increasedgradually.3 The therapeutic index (TI) of DDAs (aconitine, mesaconitine andhypaconitine) in Radix Aconiti Lateralis Preparata on heart functionCalculating the median toxic dose (TD50) and median lethal dose (LD50) ofaconitine, mesaconitine and hypaconitine with the initial data of 1.1 results, theLD50 of aconitine, mesaconitine and hypaconitine are 334μg·kg-1, 271μg·kg-1,921μg·kg-1 respectively.Calculated the median median effective dose (ED50) ofincreasing RPP or +dp/dtmax of aconitine, mesaconitine and hypaconitine withthe initial data of 2.1 results.Regarding RPP as the index, ED50 of aconitine,mesaconitine and hypaconitine were 5.69μg·kg-1, 3.58μg·kg-1, 7.24μg·kg-1respectively.Regarding +dp/dtmax as the index, ED50 of aconitine, mesaconitineand hypaconitine were 8.49μg·kg-1 7.65μg·kg-1, 18.09μg·kg-1 respectively.WithLD50/ED50 (regarding RPP, +dp/dtmax in rate as the index)to calculate thetherapeutic index (TI) of DDAs on heart function, the result indicated the TI ofaconitine, mesaconitine and hypaconitine were 58.73, 60.62, 127.19respectively while regarding RPP as the index.Regarding +dp/dtmax as theindex, the result indicated the TI of aconitine, mesaconitine and hypaconitinewere 39.34, 28.37, 50.92 respectively.Part two: The antagonism and its mechanism of liquiritin on toxicity toheart induced by aconitine1 The antagonism of liquiritin on toxicity to heart induced by aconitine inmice1.1 The antagonism of liquiritin on mice toxicity induced by aconitineRegarding the mouse's survival rate as the index, the antagonism of thedifferent dosage liquiritin was observed on the toxicity caused by aconitine.Theresult showed: the liquiritin could partly dose-dependent oppose the toxicity ofaconitine, reduce the death rate of the mice, and improve the mouse's toleranceto the aconitine.Among them liquiritin 6, 3, 1.5 mg·kg-1 could make the deathrate induced by aconitine 0.4 mg·kg-1 reduce by 55％, 30％,10％respectivelyand the effects of liquiritin 6, 3 mg·kg-1 were significant (compare with theaconitine group p＜0.01, p＜0.05). 1.2 The antagonism of liquiritin on toxicity to heart of aconitine in ratsRegarding rat's electrocardiogram (ECG) change as the index, theantagonism of liquiritin on toxicity to heart of aconitine in rats wasobserved.The result showed: the liquiritin could obviously improve rat'stolerance amount to aconitine, delay such toxicity to heart as VF, CAasystole,etc induced by aconitine.But it was not obvious to delay the appearanceof VEBS, VT.When the dosage of aconitine was 17.5, 42.8, 80.8, 84.5,106.7μg·kg-1, it appeared in order VEBS, VT, VF,CA, asystole and death.Thetoxicity to heart dosage of aconitine was improved by 1.7％, 55.8％, 930.2％,930.4％, 845.9％respectively after injection of liquiritin 3 mg·kg-1.The toxicityto heart dosage of aconitine was improved by 46.9％, 110.0％, 285.4％, 365.3％,458.8％respectively after injection of liquiritin 1.5 mg·kg-1.The toxicity to heartdosage of aconitine was improved by 12.6％, 0.9％, 274.8％, 301.9％, 319.0％respectively after injection of liquiritin 0.75 mg·kg-1.1.3 The antagonism of liquiritin on toxicity to heart of aconitine in theisolated rat heartLangendorff perfusion isolated rat heart was subjected to observe theantagonism of liquiritin on toxicity to heart of aconitine.The result showed: theisolated rat heart ceased to beat when the aconitine accumulated to 18.3μg.Liquiritin at the perfusing speed of 80, 40, 20μg/h could raise the tolerancequantity of aconitine on isolated rat heart by 342.1％, 198.4％, 177.6％respectively and the asystole was decreased.(Compare with aconitine groupp＜0.05, p＜0.01).1.4 Antagonistic action of liquritin on aconitine's effect on proliferation,dehydrogenase (LDH), frequency and the activities of Na+-K+-ATPase,Ca2+-Mg2+-ATPase in rat's myocardial cellsThe influence of liquiritin in different concentration on aconitine toxicitywas observed with the index of cell vigor, LDH, cell membrane penetrating,Na+-K+-ATPase, Ca2+-Mg2+-ATPase.The results showed: 8μM aconitine hadcertain inhibition on the vigor of the suckling mice myocardium cell.The cellvigor was 79.4％of the control group (compare with control group, p＜0.05);64μM liquiritin was not obvious for inhibition on myocardium cell's vigor, thecell's vigor was 90％of the control group; but they could increase LDH vigorobviously.(Compare with control group p＜0.05, p＜0.01).After the twosubstances mixed in certain proportion (Aconitine: liquiritin was respectively8μM: 1μM, 8μM: 4μM, 8μM: 8μM, 8μM: 16μM, 8μM: 64μM), the inhibitionon myocardium cell's vigor was strengthened (The cells' vigor was 63.3％, 64％,64.3％, 73.6％, 85.7％of the control group respectively).And the effect had the positive correlation with the liquritin ratio.(compare with control group, p＜0.05,p＜0.01), 8μM aconitine, 64μM liquiritin increased the cell membranepenetrating significantly which made the LDH vigor improved 570.1%,558.4%(compare with control group p＜0.05). This indicated that both could doharm to the cadiocyte. After the two substances mixed in certain proportion(Aconitine: liquiritin was respectively 8μM: 1μM, 8μM: 4μM, 8μM: 8μM, 8μM:16μM, 8μM: 64μM), the effects did not strengthen.(LDH vigor raised 499.3%,511.3%, 533.9%, 545.6%, and 601.1% respectively)(Compare with controlgroup p＜0.05, p＜0.01).8μM aconitine, 16μM liquiritin respectively hatching 1hour could not improve the Ca2+-Mg2+-ATPase and Na+-K+ATPase ofmyocardium cell. However, 8μM aconitine, 16μM liquiritin mixed and hatching1 hour could improve Na+-K+ATPase of myocardium cell by about114.4%(compare with control group p＜0.05)2 The effect of liquiritin on aconitine strengthening the heart function2.1 The effect of liquiritin on aconitine strengthening the heart functions ofheart failure rats induced by nimodipine. The experiment chose nimodipine, oneof Ca2+ antagon, to make heart failure model in rats. The Medlab bioinformation recording system was used. The three DDAs were injected in therats through femoral vein. The effect of different dosage liquritin was observedon aconitine strengthening the heart function.The result indicated that liquiritin could obviously increase model animal'sheart rate (HR) (compare with the model group p＜0.05, p＜0.01).However,liquiritin administrated in advance could suppress the increasing of HR causedby aconitine (Compare with aconitine group p＜0.05). With the increasing of dose,Liquiritin, aconitine, and liquiritin + aconitine could obviously strengthen thecardiac muscle shrinks performance which showed as +dp/dtmax, Vmax, Vpm,LVDP, LVSP obviously rising. They could suppress the reducing of RPP, LVDPand the extension of intersystolic period on the model animal (compare with themodel group p＜0.05, p＜0.01). 3mg·kg-1 of liquiritin could obviously improve thefunction of aconitine which showed as the increasing of +dp/dtmax, Vmax,LVDP, RPP and trend shortening of intersystolic period (compare with aconitinep＜0.05, p＜0.01), but not obvious improve VPM function.1.5, 0.75mg·kg-1 ofliquiritin have not been obviously influenced on the function of aconitine.In sum: aconitine and liquiritin could improve cardiac muscle shrinkfunction, increase the HR and myocardium oxygen consumption; thestrengthening heart function of liquiritin 3mg·kg-1 and that of aconitine was incoordination.2.2 The effect of liquiritin on aconitine effecting the heart function in isolated rat heartLangendorff perfusion isolated rat heart was subjected to observe the effectof liquiritin on aconitine affecting the heart function. The result showed: Theliquiritin at the speed of 80μg/h could strengthen the function of aconitineraising +dp/dtmax and -dp/dtmax by 73.8% and 160.4%(Compare withaconitine group p＜0.05). The effect strength of aconitine was increased and theeffect time of acotine was prolonged by liquritin..3 The effect of liquiritin on aconitine effecting TI in heartCalculating the median toxic dose(TD50) and median lethal dose (LD50) ofgroups with the initial data of 2.1 results, the LD50 of aconitine and aconitineafter injection of liquiritin 3, 1.5, 0.75mg·kg-1 were 334μg·kg-1, 1041μg·kg-1,440μg·kg-1, 386μg·kg-1 respectively. The TD50 about the ventricular fibrillationand ventricular flutter of aconitie was improved significantly after usage ofliquritin. And the effect became more significant as the dosage increased. The3mg·kg-1 liquritin could increase the TD50 about the ventricular fibrilIation andventricular flutter of aconitie by 5, 7 times. The 1.5mg·kg-1 liquritin can increasethe TD50 about the ventricular fibrillation and ventricular flutter of aconitie by3.5, 4 times. The 0.75mg·kg-1 liquritin can increase the TD50 about theventricular fibrillation and ventricular flutter of aconitie by 2.5, 3.5 times. Butthe ventricular premature contraction and ventricular rrhythmia was not effectedby the liuritin.Calculating the median median effective dose (ED50) by variation rate ofRPP or +dp/dtmax of groups with the data of 2.1 results, regard RPP or+dp/dtmax as the index, ED50 of aconitine were 5.69μg·kg-1 and 8.49μg·kg-1respectively. But the ED50 of aconitine were 14.51μg·kg-1, 7.26μg·kg-1,7.36μg·kg-1 and 12.91μg·kg-1, 7.79μg·kg-1, 9.7μg·kg-1 respectively after theinjection of liquiritin 3, 1.5, 0.75mg·kg-1. The TI of aconitine (RPP, +dp/dtmaxvariation rate as the index) were 58.73, 39.34 respectively. After liquiritin 3, 1.5,0.75mg·kg-1 injected the TI of aconitine were 71.68, 60.35, 52.88 and 80.59,56.45, 40.1 respectively.4. Intervention effects of liquiritin on the aconitine affecting the cadiocyteion in channel guinea pigsThe effects of aconitine and liquiritin on myocardium Voltage-DependentNa+ channel and L type Ca2+ channel through patch clamp technique wereobserved. The result showed: The liquiritin can inhibit the Voltage-DependentNa+ channel, but was not effected on the L type calcium ion channel.End NotesOur date showed that aconitine, mesaconitine, hypaconitine had the heart toxicity and effect of strengthening the heart function. Among them, the TI ofhypaconitine was biggest. Liquiritin can antagonise the heart toxicity induced byaconitine, improve the function of strengthening the heart of aconitine andincreased the TI of aconitine. The mechanism of liquiritin on toxicity to heartinduced by aconitine is inhibition the Voltage-Dependent Na+ channel.New Findings:1. This research had observed the relationship of dose-effect and dose-toxicity of aconitine, mesaconitine, hypaconitine for the first time, comparingthe TD50, the ED50 and the TI of them.2. Our study definited that Liquiritin was the composition of antagonisingthe heart toxicity induced by aconitine for the first time, and had offered theexperiment basis in terms of biological effect. Our research offered the newexperiment basis to the theory of the compatibility of Aconiti Class and RadixGlycyrrhizae.3. Our research has studied the Liquiritin opposed the toxic mechanism ofrhizome of aconitine in terms of ion channel of myocardium cell membrane forthe first time.
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