Cytotoxic Effects of Mesaconitine, the Aconitum carmichaelii Debx Bioactive Compound, on HBEC-5i Human Brain Microvascular Endothelial Cells: Role of Ca2+ Signaling-Mediated Pathway.

Mesaconitine, one of Aconitum carmichaelii Debx bioactive compounds, was shown to evoke Ca2+ homeostasis and its related physiological effects in endothelial cell types. However, the effect of mesaconitine on Ca2+ signaling and cell viability in human brain microvascular endothelial cells is unclear. This study focused on exploring whether mesaconitine changed cytosolic Ca2+ concentrations ([Ca2+]i), affected cell viability, and established the relationship between Ca2+ signaling and viability in HBEC-5i human brain microvascular endothelial cells. In HBEC-5i cells, cell viability was measured by the cell proliferation reagent (WST-1). [Ca2+]i was measured by the Ca2+-sensitive fluorescent dye fura-2. Mesaconitine (10-100 µM) concentration dependently induced [Ca2+]i rises. Ca2+ removal reduced the signal by approximately 25%. Mesaconitine (40-100 µM) caused cytotoxicity in HBEC-5i cells. This cytotoxic response was significantly reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-containing medium, mesaconitine-induced Ca2+ entry was inhibited by 25% by modulators of store-operated Ca2+ channels and protein kinase C (PKC). Furthermore, mesaconitine also induced Mn2+ influx suggesting of Ca2+ entry. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished mesaconitine-evoked [Ca2+]i rises. Conversely, treatment with mesaconitine abolished thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished mesaconitine-induced [Ca2+]i rises. In sum, mesaconitine caused cytotoxicity that was triggered by preceding [Ca2+]i rises. Furthermore, mesaconitine induced [Ca2+]i rises by evoking Ca2+ entry via PKC-sensitive store-operated Ca2+ channels and PLC-dependent Ca2+ release from the endoplasmic reticulum. It suggests that Ca2+ signaling have a potential cytotoxic effect on mesaconitine-treated human brain microvascular endothelial cells.

Pharmacokinetic effects of ginsenoside Rg1 on aconitine, benzoylaconine and aconine by UHPLC-MS/MS.

Ginseng and aconite are well-known couplet medicinals. Ginsenoside Rg1 is the main active ingredient in ginseng, and aconitine (AC), benzoylaconine (BAC) and aconine (ACN) are three representative alkaloids in aconite, which belong to the diester alkaloids, monoester alkaloids and alkanolamine alkaloids respectively. The aim of this study was to investigate the pharmacokinetic effects of ginsenoside Rg1 on the three types of alkaloids and to provide evidences for their compatibility mechanism. In this study, the ginsenoside Rg1 was simultaneously intragastrically administered to rats with AC, BAC and ACN, respectively, and the rat plasma was collected at different time points. The plasma drug concentrations of the three types of alkaloids were determined by UHPLC-MS/MS, and the pharmacokinetic parameters were calculated. The results indicated that the peak concentration and area under the concentration-time curve of BAC were significantly increased (P < 0.05), those for AC were decreased (P < 0.05), and the values for ACN did not change after pretreatment with ginsenoside Rg1. It was inferred that ginsenoside Rg1 may affect the absorption and metabolism of AC and BAC and then change their pharmacokinetic parameters. Subsequently, their absorption and metabolism were further investigated using the Caco-2 cell monolayer and rat liver microsomes in vitro. The Caco-2 cell monolayer absorption assay indicated that ginsenoside Rg1 could promote the absorption of AC and BAC, and the rat liver microsomes metabolism assay indicated that ginsenoside Rg1 accelerated the metabolism of AC and did not affect the other two alkaloids. All of the results indicated that ginsenoside Rg1 may reduce the toxicity of aconite and improve its efficacy by promoting the absorption of BAC and accelerating the metabolism of AC. These results could provide evidence for the compatibility mechanism of the traditional Chinese herbal formula Shenfu Decoction.

Lappaconitine trifluoroacetate contained polyvinyl alcohol nanofibrous membranes: Characterization, biological activities and transdermal application.

Lappaconitine (LA), a potent analgesic drug extracted from the root of natural aconitum species, has been clinically used for years because of its effectiveness and non-addictive properties. However, it is mainly limited in oral and intravenous administration in the form of Lappaconitine Hydrobromide (LAH). In this work, Lappaconitine trifluoroacetate (LAF), a new derivative of LA, was successfully obtained by introducing organofluorine group to LA. This new compound had a lower toxicity (LD50 of 21.14 mg·kg-1), improved analgesic effect and longer half-life (T1/2 of 2.24 h) when compared with LAH. Moreover, in vitro transdermal permeation (Jss of 206.82 µg·cm-2·h-1) of LAF was 30.54% higher than that of LAH, means that LAF can be conveniently used for transdermal drug delivery (TDD). Therefore, drug membranes with PVA solution (10 wt%) containing LAF in various amounts were fabricated by electrospinning. The in vitro release tests confirmed that up to 81.43% of LAF in the PVA/LAF nanofibrous membranes could be released in 72 h, accompanied by significant analgesic effect when compared with the blank control group. In conclusion, the prepared LAF-loaded membrane is a novel formulation for the treatment of chronic and long-term pain.

Effects of liquorice on pharmacokinetics of aconitine in rats.

Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated. Pharmacokinetics of AC after oral administration of AC at 1.5 mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299 g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively. The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161 ± 37.8 to 58.8 ± 8.97 and 44.7 ± 8.20 ng/mL*h, respectively. Similarly, Cmax decreased from 26.2 ± 5.19 to 11.8 ± 1.15 and 6.86 ± 0.600 ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control. In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp.

Antiviral activity of aconite alkaloids from Aconitum carmichaelii Debx.

Four diterpenoid alkaloids, namely, (a) hypaconitine, (b) songorine, (c) mesaconitine and (d) aconitine, were isolated from the ethanol root extract of Aconitum carmichaelii Debx. The antiviral activities of these alkaloids against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) were evaluated. Antiviral activity test in vivo showed that compounds a and c, which were C19-diterpenoid alkaloids, showed inactivation efficacy values of 82.4 and 85.6% against TMV at 500 µg/mL, respectively. By contrast, compound c presented inactivation activity of 52.1% against CMV at 500 µg/mL, which was almost equal to that of the commercial Ningnanmycin (87.1% inactivation activity against TMV and 53.8% inactivation activity against CMV). C19-Diterpenoid alkaloids displayed moderate to high antiviral activity against TMV and CMV at 500 µg/mL, dosage plays an important role in antiviral activities. This paper is the first report on the evolution of aconite diterpenoid alkaloids for antiviral activity against CMV.

Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo.

In this study, solid lipid nanoparticles were formulated for transdermal delivery of aconitine to improve its safety and permeability. Aconitine-loaded solid lipid nanoparticles were formulated as an oil-in-water microemulsion. Drug encapsulation efficiencies for these formulations were higher than 85%, and correlated positively with levels of surfactant and oil matrix. The size of the solid lipid nanoparticles was increased with an increase of the oil matrix, and reduction of the surfactant levels. Compared with an ethanol tincture, all the tested solid lipid nanoparticle formulations achieved improved transdermal fluxes and drug deposition in skin in vitro. Real-time monitoring of drug distribution in rat dermis using in vivo microdialysis showed that aconitine concentration was markedly higher following application of solid lipid nanoparticles, compared to tincture, throughout the experimental period. A regional comparison of rat skin found that application of solid lipid nanoparticles to the scapular region resulted in higher AUC(0-t) and C(max), compared to those achieved with application to the abdomen or chest (p < 0.05). In contrast, the application to the chest resulted in the lowest AUC(0-t) and C(max). Together with findings of a structural study of the skin, these results indicated that the drug accumulated more readily in thicker skin regions, and to a lesser extent in well-perfused skin, because of drug transfer to capillaries. The superior transdermal permeability of aconitine-loaded solid lipid nanoparticles contributed to stronger anti-inflammatory and analgesic effects on mouse in vivo models of pain than the tincture (p < 0.05). In vitro and in vivo studies indicated that smaller particle sizes of solid lipid nanoparticles enhanced the transdermal permeability of aconitine, which can promote drug efficacy, reduce administration time, and improve medication safety.

Comparative pharmacokinetics of hypaconitine after oral administration of pure hypaconitine, Aconitum carmichaelii extract and Sini Decoction to rats.

Hypaconitine (HC) is one of the main aconitum alkaloids in Aconitum carmichaelii (AC), which is considered to be effective on cardiovascular disease, although it also has high toxicity. Sini Decoction (SND), composed of Aconitum carmichaelii, Glycyrrhiza uralensis and Zingiber officinale, is a traditional Chinese multi-herbal formula for recuperating the depleted yang. The aim of this study was to compare the pharmacokinetics of HC in rat plasma after oral administration of HC, AC extract and SND, and investigate the effect of other two herbal ingredients on absorption, metabolism and elimination of HC. A sensitive and specific LC-MS/MS method was developed to determine HC in rat plasma. Eighteen male Sprague-Dawley rats were randomly assigned to three groups: HC, AC and SND group. Plasma concentrations of HC were determined at designated points after oral administration, and main pharmacokinetic parameters were estimated. It was found that there was obvious difference (p < 0.05) on the pharmacokinetic parameters among three groups. Compared with AC group, Tmax, Cmax, k, AUC(0-24) and AUC(0-∞) decreased in SND group, while t1/2 and MRT had been lengthened, which indicated that the ingredients in other two herbs could influence the pharmacokinetic behavior of HC.

Pharmacokinetic comparisons of benzoylmesaconine in rats using ultra-performance liquid chromatography-tandem mass spectrometry after administration of pure benzoylmesaconine and Wutou decoction.

Wutou decoction is widely used in China because of its therapeutic effect on rheumatoid arthritis. Benzoylmesaconine (BMA), the most abundant component of Wutou decoction, was used as the marker compound for the pharmacokinetic study of Wutou decoction. The aim of the present study was to compare the pharmacokinetics of BMA in rats after oral administration of pure BMA and Wutou decoction. Pure BMA (5 mg/kg) and Wutou decoction (0.54 g/kg, equivalent to 5 mg/kg BMA) were orally administered to rats with blood samples collected over 10 h. Quantification of BMA in rat plasma was achieved using sensitive and validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Specifically, the half-life (T1/2) and mean residence time values of pure BMA were 228.3 ± 117.0 min and 155.0 ± 33.2 min, respectively, whereas those of BMA in Wutou decoction were decreased to 61.8 ± 35.1 min and 55.8 ± 16.4 min, respectively. The area under the curve (AUC) of BMA after administration of Wutou decoction was significantly decreased (five-fold) compared with that of pure BMA. The results indicate that the elimination of BMA in rats after the administration of Wutou decoction was significantly faster compared with that of pure BMA.

Metabonomics study of the effects of pretreatment with glycyrrhetinic acid on mesaconitine-induced toxicity in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii Debx. (Fuzi), a commonly use traditional Chinese medicine (TCM), has often been used in combination with Rhizoma Glycyrrhizae (Gancao) to reduce its toxicity due to diester diterpenoid alkaloids aconitine, mesaconitine, and hypaconitine. However, the mechanism of detoxication is still unclear. Glycyrrhetinic acid (GA) is the metabolite of glycyrrhizinic acid (GL), the major component of Gancao. In present study, the effect of GA on the changes of metabolic profiles induced by mesaconitine was investigated using NMR-based metabolomic approaches. MATERIALS AND METHODS: Fifteen male Wistar rats were divided into a control group, a group administered mesaconitine alone, and a group administered mesaconitine with one pretreatment with GA. Their urine samples were used for NMR spectroscopic metabolic profiling. Statistical analyses such as orthogonal projections to latent structures-discriminant analysis (OPLS-DA), t-test, hierarchical cluster, and pathway analysis were used to detect the effects of pretreatment with GA on mesaconitine-induced toxicity. RESULTS: The OPLS-DA score plots showed the metabolic profiles of GA-pretreated rats apparently approach to those of normal rats compared to mesaconitine-induced rats. From the t-test and boxplot results, the concentrations of leucine/isoleucine, lactate, acetate, succinate, trimethylamine (TMA), dimethylglycine (DMG), 2-oxo-glutarate, creatinine/creatine, glycine, hippurate, tyrosine and benzoate were significantly changed in metabolic profiles of mesaconitine-induced rats. The disturbed metabolic pathways include amino acid biosynthesis and metabolism. CONCLUSIONS: GA-pretreatment can mitigate the metabolic changes caused by mesaconitine-treatment on rats, indicating that prophylaxis with GA could reduce the toxicity of mesaconitine at the metabolic level.

The exposure of highly toxic aconitine does not significantly impact the activity and expression of cytochrome P450 3A in rats determined by a novel ultra performance liquid chromatography-tandem mass spectrometric method of a specific probe buspirone.

Aconitum species are widely used to treat rheumatism, cardiovascular diseases, and tumors in China and other Asian countries. The herbs are always used with drugs such as paclitaxel. Aconitine (AC) is one of the main bioactive/high-toxic alkaloids of Aconitum roots. AC is metabolized by cytochrome P450 (CYP) 3A. However, whether AC inhibits/induces CYP3A, which causes drug-drug interaction (DDI) is unclear. Our study aims to explore the potent effects of AC, as a marker component of Aconitum, on CYP3A using the probe buspirone in rats. The effects of oral AC on pharmacokinetics of buspirone were evaluated. CYP3A activity and protein levels in rat liver microsomes pretreated with oral AC were also measured using in vitro buspirone metabolism and Western blot. Buspirone and its major metabolites 1-(2-pyrimidinyl)piperazine and 6'-hydroxybuspirone were determined using a newly validated UPLC-MS/MS method. Single dose and 7-day AC administration at 0.125mg/kg had no effect on CYP3A activity since no change in the formation of 1-(2-pyrimidinyl)piperazine and 6'-hydroxybuspirone. CYP3A activity and protein levels in liver microsomes were also not affected by 7-day AC pretreatment at 0.125mg/kg. Therefore, AC neither inhibits nor induces CYP3A in rats, indicating AC does not cause CYP3A-related DDI in the liver.

Cardioprotective effect of grape-seed proanthocyanidins on doxorubicin-induced cardiac toxicity in rats.

CONTEXT: Doxorubicin (Dox) is an anthracycline antibiotic used as anticancer agent. However, its use is limited due to its cardiotoxicity which is mainly attributed to accumulation of reactive oxygen species. OBJECTIVE: This study was conducted to assess whether the antioxidant, proanthocyanidins (Pro) can ameliorate Dox-induced cardiotoxicity in rats. MATERIALS AND METHODS: Male Sprague-Dawely rats were divided into four groups. Group I was control. Group II received Pro (70 mg/kg, orally) once daily for 10 days. Group III received doxorubicin 15 mg/kg i.p. as a single dose on the 7th day and Group IV animals were treated with Pro once daily for 10 days and Dox on the 7th day. The parameters of study were serum biomarkers, cardiac tissue antioxidant status, ECG, and effect on aconitine-induced cardiotoxicity. RESULTS: Cardiac toxicity of doxorubicin was manifested as a significant increase in heart rate, elevation of the ST segment, prolongation of the QT interval and an increase in T wave amplitude. In addition, Dox enhanced aconitine-induced cardiotoxicity by a significant decrease in the aconitine dose producing ventricular tachycardia (VT). Administration of Pro significantly suppressed Dox-induced ECG changes and normalized the aconitine dose producing VT. The toxicity of Dox was also confirmed biochemically by significant elevation of serum CK-MB and LDH activities as well as myocardial MDA and GSH contents and decrease in serum catalase and myocardial SOD activities. Administration of Pro significantly suppressed these biochemical changes. DISCUSSION AND CONCLUSION: These results suggest that proanthocyanidins might be a potential cardioprotective agent against Dox-induced cardiotoxicity due to its antioxidant properties.

[Antiarrhythmic therapy of paroxysmal tachycardias and extrasystoles in patients with sinus node dysfunction].

Antiarrhythmic therapy of patients with disturbed automatism of the sinus node and impaired atrioventricular conductance may be complicated by hemodynamically significant bradycardias and contraindications for implantation of a cardiac electrical stimulator This study aimed at estimating effect of antiarrhythmic therapy with allapinin on the function of sinus and atrioventricular nodes. It included 20 patients (mean age 37.5+-2.3 years) with disturbed cardiac rhythm and sinus node dysfunction treated with allapinin (37.5 - 50 mg/d per os). This therapy had well apparent antiarrhythmic effect manifest as improvement of supraventricular and ventricular ectopic activities in the absence of negative influence on the function of sinus and atrioventricular nodes.

Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs). Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles. Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster" from Augustin et al, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Pharmacokinetics of aconitine as the targeted marker of Fuzi (Aconitum carmichaeli) following single and multiple oral administrations of Fuzi extracts in rat by UPLC/MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzi, which is the processed lateral roots of Aconitum Carmichaeli. Debx and is widely distributed over the southwest provinces of China, is recognised for its anti-inflammatory and analgesic effects. AIM OF THE STUDY: The pharmacokinetic properties of Fuzi are inadequately understood. Aconitine, the primary highly toxic ingredient of Fuzi, is well known as the target marker of Fuzi. The purpose of the present study is to investigate the pharmacokinetic behaviours of aconitine in vivo following single and multiple administrations of processed Fuzi extracts and to compare the pharmacokinetic characteristics of aconitine after administrations of pure aconitine or Fuzi extracts as well as compare the difference at single dose and multiple doses. The in vitro aconitine protein binding in plasma through equilibrium dialysis was also examined. METHODS: A high performance liquid chromatography (HPLC) method was developed for the determination of aconitine in Fuzi crude extracts and a fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) was developed to investigate the pharmacokinetic behaviour of aconitine as the targeted marker of Fuzi. RESULTS: The absolute bioavailability (F %) after the administration of 0.5 mg/kg aconitine and Fuzi extract (0.118 mg/kg aconitine) in rat was 8.24±2.52% and 4.72±2.66%, respectively. Aconitine absorption was very fast at the t(max) 30.08±9.73 min for pure aconitine and 58.00±21.68 min for Fuzi extract administration. Aconitine was also eliminated rapidly with a short half-life (i.v., 80.98±6.40 min) and a low rate of protein bounding (23.9-31.9%). No significance was observed on all the pharmacokinetics parameters following the single and multiple doses of pure aconitine (ANOVA, p>0.05). However, the absorption of aconitine after multiple administrations of Fuzi extract was much faster than that of a single dose (t(max): 58.00±21.68 vs. 20.00±8.66 min, p<0.05), and the area under the plasma concentration-time curve (AUC) was higher than that of a single dose. CONCLUSIONS: The pharmacokinetic behaviour of processed Fuzi was determined in this paper. The aconitine has low bioavailability. No variation in the pharmacokinetic behaviours of pure aconitine was observed after single and multiple administrations. In contrast, multiple administrations of processed Fuzi extract could result in variations in its pharmacokinetic behaviour in AUC and t(max) indicating that multiple dose might increase the bioavailability of aconitine, which may result in its toxicity. In addition, aconitine has a low protein bounding (23.9-31.9%), resulting in its rapid elimination.

Paeoniflorin reduced acute toxicity of aconitine in rats is associated with the pharmacokinetic alteration of aconitine.

ETHNOPHARMACOLOGICAL RELEVANCE: To investigate the influence of paeoniflorin (major bioactive component of Paeonia lactiflora Pall.) on the pharmacokinetic behavior of aconitine (major toxic and bioactive component of Aconitum carmichaeli Debx.) and potential detoxifying effect of paeoniflorin on the acute toxicity of aconitine, which may provide in depth understanding to the toxicity reduction effect of Paeonia lactiflora to Aconitum carmichaeli. MATERIALS AND METHODS: Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was employed to determine the plasma content of aconitine. Aconitine was administrated by oral to SD rats at the dosage of 200 µg/kg with or without paeoniflorin given by intraperitoneal injection at the dosage of 20 mg/kg. Plasma samples were collected for determination and analysis of pharmacokinetic parameters of aconitine. The LD(50) of aconitine and acute animal death induced by aconitine were examined when aconitine was given alone or jointly with paeoniflorin in ICR mice. RESULTS: A sensitive, accurate, precise, reliable and repeatable UHPLC-MS/MS method was successfully established for determination of the plasma content of aconitine in 12.5 µL plasma sample. The lower limit of quantification of aconitine was 0.01 ng/mL. Compared with the SD rats that were orally administrated with aconitine alone, the rats received aconitine and co-administrated with paeoniflorin by peritoneal injection showed a remarkably lower C(max) (5.69 ng/mL vs 9.66 ng/mL, P<0.05) and delayed T(max) (70 min vs 46 min, P<0.05), as well as a trend of reduction in AUC(0-t) (1082.75 ng-min/mL vs 1650.27 ng-min/mL, P=0.395). The LD(50) values of aconitine coadministered with 120 or 240 mg/kg of paeoniflorin were obviously increased to 2.30 and 2.15 mg/kg against 1.80 mg/kg of aconitine by oral administration alone. Mice treated with paeoniflorin (240 mg/kg) and aconitine (1.8 mg/kg) together revealed a significant decreased death rate than that received aconitine treatment alone (15% vs 50%, P<0.05). CONCLUSIONS: The acute oral toxicity of aconitine in rats was significantly reduced by paeoniflorin; this might result from the alterations of pharmacokinetic behavior of aconitine in the animals by coadministration of paeoniflorin.

[Difference of hypaconitine concentration in serum between cold-deficiency and normal mice].

OBJECTIVE: To investigate the difference of hypaconitine concentration in serum between normal and cold-deficiency mice after administration of aconite decoction. To analyze how the toxic dose of aconite decoction correlate to the metabolic environment. METHOD: Prepared cold-deficiency mice model, treated normal and cold-deficiency mice with aconite decoction for 14 days continuously, and then detected hypaconitine concentration in serum by HPLC along with survival ratio of mice on the first, seventh and fourteenth day. RESULT: After administration of aconite decoction for 14 days, the hypaconitine concentration in serum of cold-deficiency mice is close to that in normal mice. It showed aconite decoction has the ability of regulating metabolism environment, the hypaconitine concentration in serum of normal mice was higher on the seventh and fourteenth day than that on first day. It showed that aconite decoction can disturb metabolism environment of normal mice. It was also been observed that the range of variation of hypaconitine concentration in cold-deficiency mice was minor than that in normal mice during the fourteen days' administration. CONCLUSION: The difference of serum concentration in normal and cold-deficiency mice showed that there were different metabolic environments in two mice models, and the metabolic environment changed during administration. These results showed that the different toxic doses of aconite decoction were partially due to the different metabolic environments.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry method for the rapid simultaneous quantification of aconitine, mesaconitine, and hypaconitine in rat plasma after oral administration of Sini decoction.

A rapid, sensitive, and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed and validated for simultaneous determination of aconitine (AC), mesaconitine (MA), and hypaconitine (HA), the three toxic constituents from Sini decoction (SND) in rat plasma. After the addition of citalopram as the internal standard (IS), plasma samples were basified with 100 microL 10% ammonium hydroxide, and then extracted with 1 mL ethyl acetate. Chromatographic separation was performed on a CN column (250 mm x 4.6 mm, 5 microm) with a mobile phase of methanol/40 mM ammonium acetate/formic acid (950:45:5, v/v/v) at the flow rate of 1.0 mL/min. Analytes were determined in a triple-quadrupole mass spectrometer in the selected reaction-monitoring (SRM) mode using electrospray source with positive mode. The method was validated over the concentration ranges of 0.01-10 ng/mL for AC, MA, and HA. The variation coefficients were always < 15% for both intraday and interday precision for each analyte. Mean accuracies were also within +/-15%. The method was proved to be sensitive, rapid, specific, accurate, and reproducible. It has been successfully applied to the pharmacokinetics study on rats after oral administration of SND.

Aconite poisoning.

INTRODUCTION: Aconitine and related alkaloids found in the Aconitum species are highly toxic cardiotoxins and neurotoxins. The wild plant (especially the roots and root tubers) is extremely toxic. Severe aconite poisoning can occur after accidental ingestion of the wild plant or consumption of an herbal decoction made from aconite roots. In traditional Chinese medicine, aconite roots are used only after processing to reduce the toxic alkaloid content. Soaking and boiling during processing or decoction preparation will hydrolyze aconite alkaloids into less toxic and non-toxic derivatives. However, the use of a larger than recommended dose and inadequate processing increases the risk of poisoning. METHODS: A Medline search (1963-February 2009) was conducted. Key articles with information on the use of aconite roots in traditional medicine, active (toxic) ingredients, mechanisms of toxicity, toxicokinetics of Aconitum alkaloids, and clinical features and management of aconite poisoning were reviewed. MECHANISMS OF TOXICITY: The cardiotoxicity and neurotoxicity of aconitine and related alkaloids are due to their actions on the voltage-sensitive sodium channels of the cell membranes of excitable tissues, including the myocardium, nerves, and muscles. Aconitine and mesaconitine bind with high affinity to the open state of the voltage-sensitive sodium channels at site 2, thereby causing a persistent activation of the sodium channels, which become refractory to excitation. The electrophysiological mechanism of arrhythmia induction is triggered activity due to delayed after-depolarization and early after-depolarization. The arrhythmogenic properties of aconitine are in part due to its cholinolytic (anticholinergic) effects mediated by the vagus nerve. Aconitine has a positive inotropic effect by prolonging sodium influx during the action potential. It has hypotensive and bradycardic actions due to activation of the ventromedial nucleus of the hypothalamus. Through its action on voltage-sensitive sodium channels in the axons, aconitine blocks neuromuscular transmission by decreasing the evoked quantal release of acetylcholine. Aconitine, mesaconitine, and hypaconitine can induce strong contractions of the ileum through acetylcholine release from the postganglionic cholinergic nerves. CLINICAL FEATURES: Patients present predominantly with a combination of neurological, cardiovascular, and gastrointestinal features. The neurological features can be sensory (paresthesia and numbness of face, perioral area, and the four limbs), motor (muscle weakness in the four limbs), or both. The cardiovascular features include hypotension, chest pain, palpitations, bradycardia, sinus tachycardia, ventricular ectopics, ventricular tachycardia, and ventricular fibrillation. The gastrointestinal features include nausea, vomiting, abdominal pain, and diarrhea. The main causes of death are refractory ventricular arrhythmias and asystole and the overall in-hospital mortality is 5.5%. MANAGEMENT: Management of aconite poisoning is supportive, including immediate attention to the vital functions and close monitoring of blood pressure and cardiac rhythm. Inotropic therapy is required if hypotension persists and atropine should be used to treat bradycardia. Aconite-induced ventricular arrhythmias are often refractory to direct current cardioversion and antiarrhythmic drugs. Available clinical evidence suggests that amiodarone and flecainide are reasonable first-line treatment. In refractory cases of ventricular arrhythmias and cardiogenic shock, it is most important to maintain systemic blood flow, blood pressure, and tissue oxygenation by the early use of cardiopulmonary bypass. The role of charcoal hemoperfusion to remove circulating aconitine alkaloids is not established. CONCLUSIONS: Aconite roots contain aconitine, mesaconitine, hypaconitine, and other Aconitum alkaloids, which are known cardiotoxins and neurotoxins. Patients present predominantly with neurological, cardiovascular, and gastrointestinal features. Management is supportive; the early use of cardiopulmonary bypass is recommended if ventricular arrhythmias and cardiogenic shock are refractory to first-line treatment.

The toxicity of aconitine, emodin on ICC cell and the antagonist effect of the compatibility.

The study was to investigate the effect and action mechanism of aconitine and emodin on the function of the interstitial cells of Cajal (ICC) cultured in vitro. ICC cells were treated with aconitine (0.05-8%) and emodin (0.001-2%) in single or combined synchronous experiments and the effect of emodin on aconitine was evaluated using cell viability as end-point. Both the two compounds had toxicity on ICC cell. The cell membrane integrity impairment caused by the exposure lead to the efflux of intracellular ionic ([Na+], [Ca2+] and [K+]) and the deactivation of the Na+-K+-ATPase. The ionic disturbance caused the interruption of the cellular breathing chain and resulted in anaerobic metabolism increase and the glycogen massive decomposition, at last the energy metabolism in the cells was obstructed. But the antagonist effect existed when the two compounds were exposed to ICC cells together. The compatibility (aonitine:emodin as 2:1), can significantly reversed the toxicity of aconition. In addition, synergistic effects were never observed in the range of concentrations considered. Although emodin can defer the aconition's toxicity on ICC cell, the impairment can't be totally inhibited by the compatibility with time went on. The results of our work represent a starting point to generate novel information on the interactions between aconitine and emodin in vitro, as well as a new relevant experimental approach useful to investigate the Herb compatibility with aconite and rhubarb and reference for the clinic.

Study of neurotoxic effects and underlying mechanisms of aconitine on cerebral cortex neuron cells.

The study investigated the neurotoxiceffects and underlying mechanisms of aconitine on cerebral cortex neuron cells prepared from neonatal SD rats. The uniform design and MTT method were applied to study the effect of aconitine with different concentrations at scheduled time. The influence of aconitine at the maximal toxicity concentration was observed using optical microscope and electron microscope. The influences of aconitine on neuron cells membrane, neuron cells' inner balance, energy metabolism and neurotransmitters were observed to investigate the action mechanisms of aconitine. The results indicated that the maximal toxicity-concentration was 2% and the critical time were 30 s, 1 min and 20 min respectively. The effects of aconitine on neuron cells' morphology included cells synapse's fracture, cells membrane fragment, mitochondria's swell, cytoplasmic vacuoles, nuclear chromatin's condensation and accumulation. The stability of biomembrane, the internal milieu and the energy metabolism were also disturbed with the increase of activity of LDH and concentration of neurotransmitters (acetylcholine, opioid, catecholamine and SP) in culture medium, the increase of the activity of ACP and [Na+], [Ca2+] concentration, and the decrease of Na(+)-K(+)-ATP, [K+], [Mg2+] and glycogen concentration in the cells. Toxic mechanisms of aconitine damaging neuron cells may be because it inhibited the activity of Na(+)-K(+)-ATP, influenced the concentrations of [Na+], [K+], [Ca2+], [Mg2+] and neurotransmitters in the cells, which resulted in the injuries of cells' morphology and function.