Postmortem Diffusion of Aconitum Alkaloids and Their Metabolites in Rabbits.

OBJECTIVES: To explore the postmortem diffusion rule of Aconitum alkaloids and their metabolites in poisoned rabbits, and to provide a reference for identifying the antemortem poisoning or postmortem poisoning of Aconitum alkaloids. METHODS: Twenty-four rabbits were sacrificed by tracheal clamps. After 1 hour, the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration. Then, they were placed supine and stored at 25 ℃. The biological samples from 3 randomly selected rabbits were collected including heart blood, peripheral blood, urine, heart, liver, spleen, lung and kidney tissues at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h after intragastric administration, respectively. Aconitum alkaloids and their metabolites in the biological samples were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: At 4 h after intragastric administration, Aconitum alkaloids and their metabolites could be detected in heart blood, peripheral blood and major organs, and the contents of them changed dynamically with the preservation time. The contents of Aconitum alkaloids and their metabolites were higher in the spleen, liver and lung, especially in the spleen which was closer to the stomach. The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration. In contrast, the contents of Aconitum alkaloids and their metabolites in kidney were all lower. Aconitum alkaloids and their metabolites were not detected in urine. CONCLUSIONS: Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits, diffusing from high-content organs (stomach) to other major organs and tissues as well as the heart blood. The main mechanism is the dispersion along the concentration gradient, while urine is not affected by postmortem diffusion, which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

Inhibitory Effects of Nobiletin on Voltage-Gated Na+ Channel in Rat Ventricular Myocytes Based on Electrophysiological Analysis and Molecular Docking Method.

Nobiletin (NOB) has attracted much attention owing to its outstanding bioactivities. This study aimed to investigate its anti-arrhythmic effect through electrophysiological and molecular docking studies. We assessed the anti-arrhythmic effects of NOB using aconitine-induced ventricular arrhythmia in a rat model and the electrophysiological effects of NOB on rat cardiomyocytes utilizing whole-cell patch-clamp techniques. Moreover, we investigated the binding characters of NOB with rNav1.5, rNav1.5/QQQ, and hNaV1.5 via docking analysis, comparing them with amiodarone and aconitine. NOB pretreatment delayed susceptibility to ventricular premature and ventricular tachycardia and decreased the incidence of fatal ventricular fibrillation. Whole-cell patch-clamp assays demonstrated that the peak current density of the voltage-gated Na+ channel current was reversibly reduced by NOB in a concentration-dependent manner. The steady-state activation and recovery curves were shifted in the positive direction along the voltage axis, and the steady-state inactivation curve was shifted in the negative direction along the voltage axis, as shown by gating kinetics. The molecular docking study showed NOB formed a π-π stacking interaction with rNav1.5 and rNav1.5/QQQ upon Phe-1762, which is the homolog to Phe-1760 in hNaV1.5 and plays an important role in antiarrhythmic action This study reveals that NOB may act as a class I sodium channel anti-arrhythmia agent.

A Proteomics- and Metabolomics-Based Study Revealed That Disorder of Palmitic Acid Metabolism by Aconitine Induces Cardiac Injury.

Currently, research on cardiac injury by aconitine focuses on its effect to directly interfere with the function of cardiac ion channels. Further, abnormal lipid metabolism could cause cardiac injury via inflammatory signaling pathway. In our preliminary study, we discovered that aconitine could alter the metabolism processes of various substances, including palmitic acid. Inspired by these studies, we investigated how elevation of palmitic acid by aconitine causes cardiac injury. Aconitine induced cardiac injury in rats (0.32 mg/kg, d = 7), and the cardiac injury was confirmed by electrocardiogram and serum biochemical study. The proteomic and metabolomic results showed that the palmitic acid level increases in heart tissue, and the NOD-like receptor (NLR) signaling pathway showed a strong effect of cardiac injury. The palmitic acid results in cell viability decline and activates NLR signaling in vitro. The shRNA-mediated knockdown of NLRP3 and NOD1/2 attenuates palmitic acid-induced inhibitory effect on cells and inhibited activation of the NLR signaling pathway. Collectively, this study reveals that aconitine provoked palmitic acid elevation could aggravate cardiac injury via the NLR signaling pathway. This study suggests that drug triggered disorder of the metabolism process could evoke cardiac injury and could propose a new strategy to study drug cardiac injury.

Bmal1 regulates circadian expression of cytochrome P450 3a11 and drug metabolism in mice.

Metabolism is a major defense mechanism of the body against xenobiotic threats. Here we unravel a critical role of Bmal1 for circadian clock-controlled Cyp3a11 expression and xenobiotic metabolism. Bmal1 deficiency decreases the mRNA, protein and microsomal activity of Cyp3a11, and blunts their circadian rhythms in mice. A screen for Cyp3a11 regulators identifies two circadian genes Dbp and Hnf4α as potential regulatory mediators. Cell-based experiments confirm that Dbp and Hnf4α activate Cyp3a11 transcription by their binding to a D-box and a DR1 element in the Cyp3a11 promoter, respectively. Bmal1 binds to the P1 distal promoter to regulate Hnf4α transcriptionally. Cellular regulation of Cyp3a11 by Bmal1 is Dbp- and Hnf4α-dependent. Bmal1 deficiency sensitizes mice to toxicities of drugs such as aconitine and triptolide (and blunts circadian toxicity rhythmicities) due to elevated drug exposure. In summary, Bmal1 connects circadian clock and Cyp3a11 metabolism, thereby impacting drug detoxification as a function of daily time.

Circadian Cyp3a11 metabolism contributes to chronotoxicity of hypaconitine in mice.

Hypaconitine is an active and highly toxic constituent derived from Aconitum species. Here we aimed to determine the chronotoxicity of hypaconitine in mice, and to investigate a potential role of metabolism in hypaconitine chronotoxicity. Cardiac toxicity was assessed by measuring CK (creatine kinase) and LDH (lactate dehydrogenase) levels after hypaconitine administration to wild-type and Bmal1-/- (a clock disrupted model) mice at different times of day. The mRNA and protein levels of Cyp3a11 in mouse livers were determined by qPCR and western blotting, respectively. In vitro metabolism was assessed using liver microsomes. Pharmacokinetic study of hypaconitine was performed with wild-type mice. We observed injection time-dependent toxicity (i.e., a more severe toxicity during the light phase than the dark phase) for hypaconitine in mice. The chronotoxicity was attributed to a difference in systemic exposure of hypaconitine caused by time of day-dependent metabolism. Furthermore, circadian metabolism of hypaconitine was accounted for by the diurnal expression of Cyp3a11, a major enzyme for hypaconitine detoxification in the liver. Moreover, Bmal1 ablation in mice abolished the daily rhythm of Cyp3a11 expression and abrogated the time-dependency of hypaconitine toxicity. In conclusion, circadian Cyp3a11 metabolism contributed to chronotoxicity of hypaconitine in mice. This metabolism-based chronotoxicity would facilitate the formulation of best timing for drug administration.

Antagonizing α7 nicotinic receptors with methyllycaconitine (MLA) potentiates receptor activity and memory acquisition.

α7 nicotinic acetylcholine receptors (α7nAChRs) have been targeted to improve cognition in different neurological and psychiatric disorders. Nevertheless, no α7nAChR activating ligand has been clinically approved. Here, we investigated the effects of antagonizing α7nAChRs using the selective antagonist methyllycaconitine (MLA) on receptor activity in vitro and cognitive functioning in vivo. Picomolar concentrations of MLA significantly potentiated receptor responses in electrophysiological experiments mimicking the in vivo situation. Furthermore, microdialysis studies showed that MLA administration substantially increased hippocampal glutamate efflux which is related to memory processes. Accordingly, pre-tetanus administration of low MLA concentrations produced longer lasting potentiation (long-term potentiation, LTP) in studies examining hippocampal plasticity. Moreover, low doses of MLA improved acquisition, but not consolidation memory processes in rats. While the focus to enhance cognition by modulating α7nAChRs lies on agonists and positive modulators, antagonists at low doses should provide a novel approach to improve cognition in neurological and psychiatric disorders.

Probing the transcriptome of Aconitum carmichaelii reveals the candidate genes associated with the biosynthesis of the toxic aconitine-type C19-diterpenoid alkaloids.

Aconitum carmichaelii has long been used as a traditional Chinese medicine, and its processed lateral roots are known commonly as fuzi. Aconitine-type C19-diterpenoid alkaloids accumulating in the lateral roots are some of the main toxicants of this species, yet their biosynthesis remains largely unresolved. As a first step towards understanding the biosynthesis of aconitine-type C19-diterpenoid alkaloids, we performed de novo transcriptome assembly and analysis of rootstocks and leaf tissues of Aconitum carmichaelii by next-generation sequencing. A total of 525 unigene candidates were identified as involved in the formation of C19-diterpenoid alkaloids, including those encoding enzymes in the early steps of diterpenoid alkaloids scaffold biosynthetic pathway, such as ent-copalyl diphosphate synthases, ent-kaurene synthases, kaurene oxidases, cyclases, and key aminotransferases. Furthermore, candidates responsible for decorating of diterpenoid alkaloid skeletons were discovered from transcriptome sequencing of fuzi, such as monooxygenases, methyltransferase, and BAHD acyltransferases. In addition, 645 differentially expressed genes encoding transcription factors potentially related to diterpenoid alkaloids accumulation underground were documented. Subsequent modular domain structure phylogenetics and differential expression analysis led to the identification of BAHD acyltransferases possibly involved in the formation of acetyl and benzoyl esters of diterpenoid alkaloids, associated with the acute toxicity of fuzi. The transcriptome data provide the foundation for future research into the molecular basis for aconitine-type C19-diterpenoid alkaloids biosynthesis in A. carmichaelii.

De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids.

Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.

Induction of P-glycoprotein expression and activity by Aconitum alkaloids: Implication for clinical drug-drug interactions.

The Aconitum species, which mainly contain bioactive Aconitum alkaloids, are frequently administered concomitantly with other herbal medicines or chemical drugs in clinics. The potential risk of drug-drug interactions (DDIs) arising from co-administration of Aconitum alkaloids and other drugs against specific targets such as P-glycoprotein (P-gp) must be evaluated. This study focused on the effects of three representative Aconitum alkaloids: aconitine (AC), benzoylaconine (BAC), and aconine, on the expression and activity of P-gp. We observed that Aconitum alkaloids increased P-gp expression in LS174T and Caco-2 cells in the order AC > BAC > aconine. Nuclear receptors were involved in the induction of P-gp. AC and BAC increased the P-gp transport activity. Strikingly, intracellular ATP levels and mitochondrial mass also increased. Furthermore, exposure to AC decreased the toxicity of vincristine and doxorubicin towards the cells. In vivo, AC significantly up-regulated the P-gp protein levels in the jejunum, ileum, and colon of FVB mice, and protected them against acute AC toxicity. Taken together, the findings of our in vitro and in vivo experiments indicate that AC can induce P-gp expression, and that co-administration of AC with P-gp substrate drugs may cause DDIs. Our findings have important implications for Aconitum therapy in clinics.

Biological activities and pharmacokinetics of aconitine, benzoylaconine, and aconine after oral administration in rats.

Aconitine (AC), benzoylaconine (BAC), and aconine (ACN) are three representative alkaloids in Aconitum tubers. Knowing that the drug disposal process in vivo is closely related to the toxicity and efficacy of a drug, it is important to classify the disposal properties of these alkaloids. In this study, the pharmacokinetics of the three alkaloids was investigated. The results showed that the three alkaloids could be quickly absorbed, especially BAC, whose Tmax was 0.31 ± 0.17 h. Their Cmax was 10.99, 3.99, and 4.29 ng·mL(-1) respectively, indicating that AC had better absorption than BAC and ACN. Subsequently, we further investigated their absorption mechanism using the Caco-2 cell monolayer model in vitro. The results showed that they were poorly absorbed, and the absorption of AC and BAC was inhibited by P-gp, while the absorption of ACN was in a form of passive diffusion. The t1/2 of AC, BAC and ACN was 1.41, 9.49, and 3.32 h, respectively, indicating that the metabolic or excretion rate of AC was quicker than that of BAC and ACN. Therefore, their metabolic stability was further investigated by using rat liver microsomes in vitro, which showed that AC was easier to be metabolized than BAC and ACN. The excretion experiments showed that AC and ACN were primarily excreted in urine, while BAC was excreted in faeces. In addition, the results of tissue distribution experiments showed that the three alkaloids distributed throughout all the organs, although the distribution rate of AC was slower than that of BAC and ACN. Copyright © 2015 John Wiley & Sons, Ltd.

Biomolecular recognition of antagonists by α7 nicotinic acetylcholine receptor: Antagonistic mechanism and structure-activity relationships studies.

As the key constituent of ligand-gated ion channels in the central nervous system, nicotinic acetylcholine receptors (nAChRs) and neurodegenerative diseases are strongly coupled in the human species. In recently years the developments of selective agonists by using nAChRs as the drug target have made a large progress, but the studies of selective antagonists are severely lacked. Currently these antagonists rest mainly on the extraction of partly natural products from some animals and plants; however, the production of these crude substances is quite restricted, and artificial synthesis of nAChR antagonists is still one of the completely new research fields. In the context of this manuscript, our primary objective was to comprehensively analyze the recognition patterns and the critical interaction descriptors between target α7 nAChR and a series of the novel compounds with potentially antagonistic activity by means of virtual screening, molecular docking and molecular dynamics simulation, and meanwhile these recognition reactions were also compared with the biointeraction of α7 nAChR with a commercially natural antagonist - methyllycaconitine. The results suggested clearly that there are relatively obvious differences of molecular structures between synthetic antagonists and methyllycaconitine, while the two systems have similar recognition modes on the whole. The interaction energy and the crucially noncovalent forces of the α7 nAChR-antagonists are ascertained according to the method of Molecular Mechanics/Generalized Born Surface Area. Several amino acid residues, such as B/Tyr-93, B/Lys-143, B/Trp-147, B/Tyr-188, B/Tyr-195, A/Trp-55 and A/Leu-118 played a major role in the α7 nAChR-antagonist recognition processes, in particular, residues B/Tyr-93, B/Trp-147 and B/Tyr-188 are the most important. These outcomes tally satisfactorily with the discussions of amino acid mutations. Based on the explorations of three-dimensional quantitative structure-activity relationships, the structure-antagonistic activity relationships of antagonists and the characteristics of α7 nAChR-ligand recognitions were received a reasonable summary as well. These attempts emerged herein would not only provide helpful guidance for the design of α7 nAChR antagonists, but shed new light on the subsequent researches in antagonistic mechanism.

Comparative metabolism of Lappaconitine in rat and human liver microsomes and in vivo of rat using ultra high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry.

Lappaconitine (LAP), a non-addictive potent analgesic drug, is broadly used to treat cancer and postoperative pain in many countries, and it also has antibiotic activity against Pseudomonas aeruginosa and Salmonella Typhi. Despite its widespread usage and potential for expanded use, its metabolism was poorly investigated. In this work, the metabolic fate of LAP in liver microsomes of the rat and human was compared, and after oral administration, the metabolites in the rat were investigated using ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS). As a result, a total of 51 metabolites were identified, including 48 metabolites that were reported here for the first time. Based on accurate MS/MS spectra and the known structure of LAP, the metabolites structures and their fragment ions were readily characterized. The biotransformations of LAP in vitro and in vivo were shown to involve hydroxylation, N-deacetylation, O-demethylation, N-deethylation, and hydrolysis. Furthermore, the results indicated a quantitative species difference in the metabolites for LAP between the rat and human. However, 16-DMLAP, DAL and 5'-OH-DAL were the main in vitro and in vivo metabolites. This work provides the LAP metabolite profiles in rat and human, which will help better understand the pharmacological and toxicological activities of LAP.

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC-MS(n) combined with specific inhibitors.

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti-inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC-MS(n). In this research, the 12 metabolites of BLA were identified according to the results of HPLC-MS(n) data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC-MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small.

Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain.

We determined the X-ray crystal structures of the extracellular domain (ECD) of the monomeric state of human neuronal α9 nicotinic acetylcholine receptor (nAChR) and of its complexes with the antagonists methyllycaconitine and α-bungarotoxin at resolutions of 1.8 Å, 1.7 Å and 2.7 Å, respectively. The structure of the monomeric α9 ECD superimposed well with the structures of homologous proteins in pentameric assemblies, denoting native folding, despite the absence of a complementary subunit and transmembrane domain. The interaction motifs of both antagonists were similar to those in the complexes with homologous pentameric proteins, thus highlighting the major contribution of the principal side of α9 ECD to their binding. The structures revealed a functionally important ß7-ß10 strand interaction in α9-containing nAChRs, involving their unique Thr147, a hydration pocket similar to that of mouse α1 ECD and a membrane-facing network coordinated by the invariant Arg210.

An in vitro and in vivo comparison of solid and liquid-oil cores in transdermal aconitine nanocarriers.

This study compared transdermal aconitine delivery using solid lipid nanoparticles (SLN) and microemulsion (ME) vehicles. Aconitine-loaded SLN and ME were formulated with the same surfactant, cosurfactant, and water content, with an equal amount of oil matrix (ATO 888 for SLN and ethyl oleate for ME). These nanosized formulations (70-90 nm) showed suitable pH values and satisfactory skin tissue biocompatibility. SLN contained a higher concentration of smaller nanoparticles, compared with that in ME. Neither of the nanocarriers penetrated across excised skin in their intact form. In vitro transdermal delivery studies found that transdermal aconitine flux was lower from SLN than from ME (p < 0.05), but skin aconitine deposition was higher using SLN (p < 0.05). Fluorescence-activated cell sorting indicated that in vitro uptake of fluorescently labeled SLN by human immortalized keratinocyte (HaCaT) cells was greater than that of ME, indicating that a transcellular pathway may contribute to cutaneous drug absorption more effectively from SLN. In vivo studies found that these formulations could loosen stratum corneum layers and increase skin surface crannies, which may also enhance transdermal aconitine delivery. SLN produced a more sustained aconitine release, indicating that compared with ME, this transdermal delivery vehicle may reduce the toxicity of this drug.

[To the mechanisms of antiarrhythmic action of Allapinine].

Allapinine (lappaconitine hydrobromide) is a drug for the treatment of cardiac arrhythmias, it shows IC class antiarrhythmics properties. Its action mechanism is associated with blockade of Na(+)-channels with subsequent inhibition of the depolarization rate and, consequently, of the slowing and reducing the excitability of the cardiac conduction system. At the moment, it is not established, what factors are associated with side effects of Allapinine, and therefore it seems important to study the molecular mechanisms of its action. The target genes were identified in a rat model of aconitine-induced arrhythmia using a commercial kit "Rat Neuroscience Ion Channels & Transporters RT2 Profiler PCR Array" (SABioscienses). Comparison of the expression of 84 genes in the experimental (aconitine arrhythmias/Allapinine) and control (aconitine arrhythmias/saline) animals revealed changes in the mRNA level of 18 genes. It has been shown an increase in mRNA levels of genes encoding various types of K(+)-channels (kcna6, kcnj1, kcnj4, kcnq2, kcnq4), Ca(2+)-channel (cacna 1g), vesicular acetylcholine transporter (slc 18a3). Decrease in the mRNA level was observed for genes encoding the Na(+)-channel (scn8a), K(+)-channels (kcne 1, kcns 1), membrane transporters (atp4a, slc6a9). Taken together, it appears that the effect of Allapinine on aconitine--induced arrhythmias is due to modulation of genes encoding Na(+)-, K(+)-, Ca(2+)-channels, conducting ionic currents (I(Na), I(to), I(Ks), I(K1), I(CaT)), which are involved in the formation of different phases of the action potential. The effect of the drug on the mRNA levels of genes encoding the acetylcholine and glycine transporters, suggesting the participation of these neurotransmitters in the mechanisms of anti-arrhythmic properties of the Allapinine.

Transcellular transport of aconitine across human intestinal Caco-2 cells.

Aconitine (AC) is a highly toxic compound present in plants of the genus Aconitum. The transcellular transport mechanism of AC was investigated using Caco-2 cells. The flux of AC was time- and concentration-dependent in both apical-to-basolateral and the reverse direction. The efflux of AC was more than two-fold that in the opposite direction. The influx of AC was temperature-, pH- and Na(+)-dependent. Glucose markedly decreased the absorption of AC. However, the efflux of AC was temperature- and pH-dependent, but Na(+)-independent. Cyclosporin A and verapamil, both inhibitors of P-glycoprotein (P-gp), significantly decreased the efflux of AC. In addition, MK-571, an inhibitor of multidrug resistance-associated protein 2 (MRP2), exhibited the same trend but to a lesser extent. These results indicate that both the influx and efflux of AC across Caco-2 monolayers were through an active process. A pH-dependent carrier-mediated transport system was the major absorption mechanism and a sodium-dependent glucose transporter may be involved. The active efflux of AC across Caco-2 cells was mediated mainly by ABC-transporter P-gp. It is involved in reducing the toxicity of AC to organisms and is the major reasons for the poor absorption of AC in vivo.

Metabolite profile analysis of aconitine in rabbit stomach after oral administration by liquid chromatography/electrospray ionization/multiple-stage tandem mass spectrometry.

1. Aconitine (AC), an active and highly toxic constituent extracted from aconitum plants, is well known for its excellent effects against rheumatism and rheumatoid arthritis. The metabolism of AC in liver and intestine has been previously reported. However, little is known about the metabolism of AC in stomach. In this study, the metabolite profiling of AC in stomachs of rabbit and rat was performed by liquid chromatography/electrospray ionization/multiple-stage tandem mass spectrometry (LC/ESI/MS(n)), for the first time. 2. The samples were purified by liquid-liquid extraction, separated using an Agilent extended C18 column following a linear gradient elution and then detected by ESI/MS(n) in positive ion mode. Metabolites were identified by comparing their protonated molecules, fragmentation patterns and chromatographic behaviors with those of standard compounds and data from authorized literature works. 3. In conclusion, 14 metabolites were identified in animal stomach after oral administration of AC. The presentation of a large amount of metabolites of AC in stomach suggested that, for aconitum alkaloids, the stomach might play an important role in their metabolism.

Cladosporium cladosporioides XJ-AC03, an aconitine-producing endophytic fungus isolated from Aconitum leucostomum.

The endophytic fungus XJ-AC03, which was isolated from the healthy roots of Aconitum leucostomum, produced aconitine when grown in potato dextrose agar (PDA) medium. The presence of aconitine was confirmed by the chromatographic and spectroscopic analyses. The yield of aconitine was recorded as 236.4 µg/g by high performance liquid chromatography (HPLC). The mass spectrometry was shown to be identical to authentic aconitine. Further analysis with nuclear magnetic resonance (NMR) spectroscopy to show the chemical structure of the fungal aconitine indicated that the fungal aconitine produced an NMR spectrum identical to that of authentic aconitine. Strain XJ-AC03 was identified as Cladosporium cladosporioides by its characteristic culture morphology and ITS rDNA sequence analysis.