Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca^(2+),reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca^(2+)overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

乌头及其炮制品、制剂中剧毒性成分Aconitine等含量测定的研究

本文研究了用改良的异羟肟酸反应—高氯酸铁显色分光光度法,在波长515nm处测定乌头中剧毒的生物碱的含量。本法简便,显色稳定性好,回收率为98.5%。

The aconitine analog bulleyaconitine A exhibits anti-hypersensitivity through direct stimulation of dynorphin A release from spinal microglia in the rat model of neuropathy

Aim Aconitine and its structurally-related diterpenoid alkaloids have been shown to interact differential- ly with neuronal voltage-dependent sodium channels and be responsible for their analgesia and toxicity. Bulleya- conitine A （ BAA or BLA） is an aconitine analog and has been prescribed for the management of pain. The present study aimed to evaluate the inhibitory effects of BAA on pain hypersensitivity and morphine anti-nociceptive toler- ance, and explore whether the release of dynorphin A from spinal microglia was associated with its mechanism of actions. Methods Rat models of neuropathic pain, formalin test and bone cancer pain were used, and spinal dynorphin A level and expression were measured. Sample size of animals was six in each study group. Resultes A single intrathecal or subcutaneous （but not intraventricular or local） injection of BAA blocked spinal nerve liga- tion-induced painful neuropathy, bone cancer-induced pain and formalin-induced hyperalgesia by 60% - 100% with the ED50 values of 94 - 126 ng/rat （intrathecal） and 42 - 59 μg · kg^-1 （ subcutaneous）, respectively. Follow- ing chronic treatment, BAA did not induce either self-tolerance to anti-nociception or cross-tolerance to morphine anti-nociception, and completely prevented morphine tolerance. Spinal BAA anti-nociception, but not neurotoxici- ty, was completely blocked by the specific microglial inhibitor minocycline. In a minocycline-sensitive and lido- BAA stimulated the release of dynorphin A from the spinal cord, and the caine- or ropivacaine-insensitive manner, primary culture of microglia but not from neurons or astrocytes. The blockade effects of BAA on nociception and morphine tolerance were completely blocked by the specific dynorphin A antiserum and/or K-opioid receptor antago- nist. Conclusions Our results demonstrated that BAA eliminated pain hypersensitivity and morphine tolerance through the direct stimulation of dynorphin A release from spinal microglia, which was not dependent on the interac- tions with sodium channels.

Acute myocardial necrosis caused by aconitine poisoning:A case report

BACKGROUND Herbal medicine has a long history of use in the prevention and treatment of disease and is becoming increasingly popular globally.However,there are also widespread concerns about its safety.Among them,the cardiotoxicity of aconitine has been described.CASE SUMMARY We report a case of a 61-year-old male with aconitine poisoning presenting with malignant arrhythmia and severe cardiogenic shock,which was successfully managed with aggressive advanced life support and heart transplantation.CONCLUSION This is the first case wherein in vivo cardiac pathology was obtained,confirming that aconitine caused acute myocardial necrosis.

Effects of Aconitine on [Ca^(2+)] Oscillation in Cultured Myocytes of Neonatal Rats

In order to investigate the effects of aconitine on [Ca2＋] oscillation patterns in cultured myocytes of neonatal rats, fluorescent Ca2＋ indicator Fluo-4 NW and laser scanning confocal micro- scope （LSCM） were used to detect the real-time changes of [Ca2＋] oscillation patterns in the cultured myocytes before and after aconitine （1.0 μmol/L） incubation or antiarrhythmic peptide （AAP） and aconitine co-incubation. The results showed under control conditions, [Ca2＋] oscillations were irregu- lar but relatively stable, occasionally accompanied by small calcium sparks. After incubation of the cultures with aconitine, high frequency [Ca2＋] oscillations emerged in both nuclear and cytoplasmic regions, whereas typical calcium sparks disappeared and the average [Ca2＋] in the cytoplasm of the cardiomyocyte did not change significantly. In AAP-treated cultures, intracellular [Ca2＋] oscillation also changed, with periodic frequency, increased amplitudes and prolonged duration of calcium sparks. These patterns were not altered significantly by subsequent aconitine incubation. The basal value of [Ca2＋] in nuclear region was higher than that in the cytoplasmic region. In the presence or absence of drugs, the [Ca2＋] oscillated synchronously in both the nuclear and cytoplasmic regions of the same cardiomyocyte. It was concluded that although oscillating strenuously at high frequency, the average [Ca2＋] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incuba- tion, compared to the controls. The observations indicate that aconitine induces the changes in [Ca2＋] oscillation frequency other than the Ca2＋ overload.

POSTMORTEM REDISTRIBUTION KINETICS OF ACONITINE IN RABBITS

The postmortem redistribution of aconitine(AC) and its influencing factors by orally ingested Aconitum brachypodum Diels (AbD) in rabbits were studied. The results showed that postmortem AC redistribution did exist, and the diffusion along a concentration gradient was the major influencing factor on it. Change of temperature and incomplete distribution in life also influenced it.Besides those mentioned above, there were other influencing factors. These may be related to postmortem blood movement and toxin released from cells occurring as part of the processes of autolysis and putrefaction.

INFLUENCE OF ETHANOL ON TOXICOKINETICS OF ACONITINE

A reverse-phase High-performance Liquid Chromatography (HPLC) method for the detection of aconitine (AC) in biological samples was used. After orally ingested Aconitum brochypodum Diels (AbD) to rabbits, the toxicokinetics process of AC showed a two-compartment open model.Ka. A and B of which were 1.1629±0. 4053, 0. 6046±0.2574 and 1. 1607±0. 3781 mg/L respectively. The influence of ethanol on this kinetics process was studied. It was indicated that ethanol did not change the model type, but the absorption and distribution of which were improved significantly (P<0. 01), its elimination process was not influenced significantly, the values of Ka. A and B were 2. 4026 ±0. 5376, 1. 205± 0. 5328, 1. 2037 ±0. 4095 mg/L respectively. All these suggest that ethanol increases the toxicity of AbD.

STUDY OF ACONITINE DEGRADATION KINETICS IN RABBIT CORPSES

in order to research on the degradation kinetics rule or aconitine (AC) in rabbit corpses,in this article,through the acute intoxic models set up by orally administrating Aconitum brachypodum Diels (AbD) of absolute lethal dose (ALD) to New Zealand rabbits,the changing rules of aconitine degradation in tissues of rabbit corpses stored at 4℃ refrigirater were studied. The result showed that AC degradation process in rabbit corpses was apparent two-order degradation kinetics. AC degradation kinetics equations in liver and kidney were the half lives of them were 5.66 and 6. 47 days respectively.

Refinement of the Crystal Structure of Aconitine

The crystal of aconitine has been obtained through an unexpected spontaneous change of the solid power of aconitine in air and characterized by single-crystal X-ray diffraction analysis and elemental analysis. Crystallographic data： orthorhombic, space group P2_12_12_1 with a = 17.0745（6）, b = 15.5990（6）, c = 12.2443（6） ？, V = 3261.2（2） ？~3, Z = 4, C_（34）H_（47）NO_（11）, M_r = 645.73, D_c = 1.315 g/cm^3, F（000） = 1384, μ（MoKa） = 0.098 mm^（-1）, R = 0.0605 and wR = 0.1311. Aconitine is diester diterpenoid alkaloid, and its main structure is composed of four six-membered and two five-membered rings. The intramolecular and intermolecular O–H···O and C–H···O hydrogen bonds extend the adjacent molecules into a one-dimensional chain and a two-dimensional framework, which may be the essential reason for the change.

Severe aconite poisoning successfully treated with veno-arterial extracorporeal membrane oxygenation:A case report

BACKGROUND Most species of aconite contain highly toxic aconitines,the oral ingestion of which can be fatal,primarily because they cause ventricular arrhythmias.We describe a case of severe aconite poisoning that was successfully treated through venoarterial extracorporeal membrane oxygenation(VA-ECMO)and in which detailed toxicological analyses of the aconite roots and biological samples were performed using liquid chromatography-tandem mass spectrometry(LC-MS/MS).CASE SUMMARY A 23-year-old male presented to the emergency room with circulatory collapse and ventricular arrhythmia after ingesting approximately half of a root labeled,“Aconitum japonicum Thunb”.Two hours after arrival,VA-ECMO was initiated as circulatory collapse became refractory to antiarrhythmics and vasopressors.Nine hours after arrival,an electrocardiogram revealed a return to sinus rhythm.The patient was weaned off VA-ECMO and the ventilator on hospital days 3 and 5,respectively.On hospital day 15,he was transferred to a psychiatric hospital.The other half of the root and his biological samples were toxicologically analyzed using LC-MS/MS,revealing 244.3 mg/kg of aconitine and 24.7 mg/kg of mesaconitine in the root.Serum on admission contained 1.50 ng/mL of aconitine.Beyond hospital day 2,neither were detected.Urine on admission showed 149.09 ng/mL of aconitine and 3.59 ng/mL of mesaconitine,but these rapidly decreased after hospital day 3.CONCLUSION The key to saving the life of a patient with severe aconite poisoning is to introduce VA-ECMO as soon as possible.

Research progress of aconitine toxicity and forensic analysis of aconitine poisoning

Chinese herbal medicines have been extensively used in China and other countries for cen-turies.Aconitine,a diterpenoid alkaloid extracted from Aconitum plants,has anti-inflamma-tory and analgesic activities,but can also induce severe arrhythmia and neurotoxicity.Aconitine poisoning accidents caused by misuse,suicide,or homicide have been reported in recent years.In China,fatal aconitine poisoning can occasionally happen on account of acci-dental ingestion of some wild plants or consumption of herbal decoction made from the roots of Aconitum plants.However,it is rather difficult for forensic experts to find the specific results in present forensic autopsy of aconitine-induced death.To further clarify its potential risk following the widespread application of aconitine,toxicological characteristics and pharmacokinetics of aconitine are reviewed.Moreover,gastrointestinal,neurological,and car-diovascular symptoms were observed frequently in aconitine poisoning cases.In addition,the review also aims at providing some convincing evidences for forensic experts to identify unexplained death with postmortem examination.

Rapid construction of the unique BCD ring system of tricyclo[6.2.1.0]undecane in the C_(19)-diterpenoid alkaloid aconitine

A model study leading to the preparation of the unique tricyclo [6.2.1.0] undecane BCD ring systems of aconitine is described. The synthesis features an unprecedented diastereoselective oxidative dearomatization/dimerization/retro-DA/IMDA cascade reaction and a highly efficient Wagner-Meerwein rearrangement.

Observation on Effects of Aconitine via Acupoint Injection in Rabbits

Objective： To investigate the ability of the pericardium meridian （PM） to mitigate or enhance the cardiotoxic effects of aconitine injected at specific acupoint and non-acupoint sites in rabbits. Methods： This study consisted of 3 experiments that were designed to test the effects of injection of 30 IJg/kg of aconitine at acupoints on the PM （Test 1）, at non-acupoint sites on the PM （Test 2）, and at acupoints on other meridians and non-meridian sites （Test 3）. in Test 1, 24 rabbits were randomly assigned to receive injections at Quze （PC3）, Tianquan （PC2）, or intramuscularly. In Test 2, 24 rabbits were randomly assigned to receive injections of aconitine at non-acupoint ｜, non-acupoint 11, or intramuscularly. In Test 3, 48 rabbits were randomly assigned to receive injections at Neiguan （PC6）, Sanyinjiao （SP6）, Yangjiao （GB35）, a non-meridian and non-acupoint site （NMNA）, intravenously, and intramuscularly. Electrocardiographs of the rabbits were performed before, during and after injection to determine the incidence of arrhythmia, latency of ventricular rhythm, and recovery rate after aconitine injection. The recovery time index and extent of arrhythmia scores were calculated. Results： In all groups the incidence of arrhythmia was 100%, and the latency of ventricular rhythm was less than 30 min. In Tests 1 and 2, the recovery rates of the Quze and non-acupoint I1 groups were significantly higher than those of the muscular group （P〈0.05）. In Test 3, the recovery time index and extent of arrhythmia scores of the Neiguan group were low. There were no significant differences between the other acupoint groups, or the NMNA group, when compared with the group receiving aconitine intramuscularly. Conclusions： Acupoints or non-acupoints along the PM could reduce the severity of the arrhythmia induced by aconitine in healthy rabbits. Meridians play an important role in Drotecting bodv functions.

Ginsenoside Rg_(1)Reduces Cardiotoxicity While Increases Cardiotonic Effect of Aconitine in vitro

Objective To explore the synergic mechanism of ginsenoside Rg_(1)(Rg_(1))and aconitine(AC)by acting on normal neonatal rat cardiomyocytes(NRCMs)and pentobarbital sodium(PS)-induced damaged NRCMs.Methods The toxic,non-toxic,and effective doses of AC and the most suitable compatibility concentration of Rg_(1) for both normal and damaged NRCMs exposed for 1 h were filtered out by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide,respectively.Then,normal NRCMs or impaired NRCMs were treated with chosen concentrations of AC alone or in combination with Rg_(1) for 1 h,and the cellular activity,cellular ultrastructure,apoptosis,leakage of acid phosphatase(ACP)and lactate dehydrogenase(LDH),intracellular sodium ions[Na^(+)],potassium ions[K^(+)]and calcium ions[Ca^(2+)]levels,and Nav1.5,Kv4.2,and RyR_(2) genes expressions in each group were examined.Results For normal NRCMs,3000µmol/L AC significantly inhibited cell viability(P<0.01),promoted cell apoptosis,and damaged cell structures(P<0.05),while other doses of AC lower than 3000µmol/L and the combinations of AC and Rg_(1) had little toxicity on NRCMs.Compared with AC acting on NRCMs alone,the co-treatment of 3000 and 10µmol/L AC with 1µmol/L Rg_(1) significantly decreased the level of intracellular Ca^(2+)(P<0.01 or P<0.05),and the co-treatment of 3000µmol/L AC with 1µmol/L Rg_(1) significantly decreased the level of intracellular Ca^(2+)via regulating Nav1.5,RyR_(2) expression(P<0.01).For damaged NRCMs,1500µmol/L AC aggravated cell damage(P<0.01),and 0.1 and 0.001µmol/L AC showed moderate protective effect.Compared with AC used alone,the co-treatment of Rg_(1) with AC reduced the cell damage,0.1µmol/L AC with 1µmol/L Rg_(1) significantly inhibited the level of intracellular Na+(P<0.05),1500µmol/L AC with 1µmol/L Rg_(1) significantly inhibited the level of intracellular K+(P<0.01)via regulating Nav1.5,Kv4.2,RyR_(2) expressions in impaired NRCMs.Conclusion Rg_(1) inhibited the cardiotoxicity and enhanced the cardiotonic effect of AC via regulating the ion channels pathway of[Na^(+)],[K^(+)],and[Ca^(2+)].

超高效液相色谱串联四极杆质谱联用分析动物血样中乌头类生物碱

An ultra performance liquid chromatography-tandem mass spectrum method has been developed for the determination of three aconitum alkaloids (aconitine,hypaconitine and mesaconitine).The three alkaloids were analyzed simultaneously with an Waters Acquity BHE C18 column by gradient elution using 0.05% aqueous ammonia-acetonitrile as mobile phase and detected by the MRM mode of the mass spectrum.The recoveries of the SPE method were between 68.79%-95.65% (RSD<7.94%,n=4),and all the alkaloids showed good linearity (r>0.998) in a relatively wide concentration range.The LOD reached 0.0516,0.0640,0.0744 ng/mL of aconitine,hypaconitine and mesaconitine,respectively.The analysis time was within 3 min which could meet the high-throughput detection.The results indicated that contents of alkaloids in animal blood can be well detected;and this method can be used in quality control of aconitum drugs and pharmacokinetics study.

液相色谱-质谱联用测定乌头碱血药浓度及其药代动力学参数的方法学研究

乌头碱(aconitine)是存在于乌头属多种植物中的一种双酯二萜生物碱,具有镇[1]、抗炎[2]、抗癫痫[3]、抗肿瘤[4]、提高免疫功能[5]等多种生理活性,但因其具有强烈的心脏毒性和神经毒性,安全系数小,治疗剂量与中毒剂量接近(小鼠LD501.8mg/kg,op;人绝对致死量为2～5mg)[7],限制了对该化合物作为药用的进一步研究,目前仅将其作为致心率失常模型的工具药使用,尚未见其药代动力学的报道.在本文中首次应用液相色谱-质谱联用法对乌头碱在大鼠体内的药代动力学进行了研究.……

乌头碱的LC-MS定量分析

乌头生物碱各成分毒性差异很大,其中乌头碱的毒性为其它成分的100～2000倍,是引起中毒和死亡的主要原因.乌头生物碱种类多,在煎煮或泡制过程中易水解产生不同水解产物,进入体内后代谢情况又不明,因此采用液相色谱方法对体内检材乌头碱成分仅靠保留时间确定依据不足,定量工作更是无法开展.……

ANTAGONISM OF TOTAL FLAVONES OF GYNOSTEMMA PENTAPHYLLUM (THUNB) MAK TO ANIMAL EXPERIMENTAL ARRHYTHMIA

The action of total flavones of Gynostemma pentaphyllum (Thunb) Mak (TFG) 60 mg/kg i.v. could prevent arrhythmias induced by drugs and myocardial ischemia were investigated. In anesthetized rats and guinea pigs. The results showed that in TFG group the duration of ventricular tachycardia (VT) induced by aconitine in rats was shortened (P<0 01) and the incidence of ventricular fibrillation (VF) and the mortality were decreased (P<0 01) respectively. The arrhythmias induced by i.v. BaCl 2 4 mg/kg in rats were immediately recovered to a normal sinus rhythm and VT induced by i.v. CaCl 2 130 mg/kg in rats was decreased by TFG i.v. TFG i.v. elevated the doses of i.v. strophanthin G to induce ectopic beats (EB), VT, VF and cardiac arrest (CA) in guinea pigs by 89%, 58%, 27%, and 28% (P<0 01) respectively. The incidence of VF induced by coronary artery ligation in rats was decreased (P<0 01), duration of EB, duraton of VT and VF decreased to 45%, 42% and 0% of the NS group respectively. The results suggest that TFG in dosage of 60 mg/kg might be useful for the prevention of VT and VF.