Clinical poisoning events involving yunaconitine may be highly correlated with metabolism-based interactions: A critical role of CYP3A4.

Clinical poisoning events involving yunaconitine (YAC), a toxic Aconitum alkaloid, occur more and more frequently, and whether the mechanism is correlated with metabolism-based interactions remains unknown. This study aimed to reveal the presumable mechanism by clarifying the metabolic profiles and kinetic-based mechanism of YAC. YAC could be oxidized into 20 metabolites by human liver microsomes, while CYP3A4 have a critical metabolic superiority. Sixteen of the metabolites were primary generated by CYP3A4, and 4 of them were generated only by CYP3A4. The presence of CYP3A inhibitor ketoconazole (KCZ) significantly suppressed the generation of all the 20 metabolites, with 9 of them being suppressed completely (P < 0.05). The plasma exposure (Cmax and AUC0-t values), cardiotoxicity and neurotoxicity of YAC enhanced remarkably in mice when Cyp3a were inhibited (P < 0.05). Moreover, the CYP3A4-based kinetics of YAC is an example of substrate inhibition, and the inhibitory manner of YAC on CYP3A4 was competitive, with Ki value being 1.76 µmol/L. Overall, YAC was a sensitive substrate and moderately competitive inhibitor of CYP3A4. The inhibition on CYP3A4 could sharply increase the in vivo exposure and toxicity of YAC. Thus, clinical poisoning events involving YAC may be highly correlated with CYP3A4-mediated interactions.

Systematic investigation on the distribution of four hidden toxic Aconitum alkaloids in commonly used Aconitum herbs and their acute toxicity.

Yunaconitine (YAC), crassicauline A (CCA), 8-deacetylyunaconitine (DYA), and 8-deacetylcrassicauline A (DCA), as hidden toxic Aconitum alkaloids, are detected in some products of processed Aconitum carmichaelii lateral root and poisoning cases. The distribution and toxicity of these four components in Aconitum herbs should be further systematically studied for medication safety. This study developed a new UHPLC-QQQ-MS/MS method to determine ten Aconitum alkaloids, including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, YAC, CCA, DYA, and DCA, for Aconitum herbs simultaneously. YAC and CCA were founded in some samples of unprocessed A. carmichaelii lateral root (7.04%), A. carmichaelii root (9.43%), A. brachypodum root (6.00%), and A. ouvrardianum root (100%). Four hidden toxic Aconitum alkaloids were detected in processed A. carmichaelii lateral root (2.56%) and A. vilmorinianum root (100%). Four hidden toxic Aconitum alkaloids played significant roles in the classification of Aconitum herbs by OPLS-DA analysis. The acute toxicity test was performed by up-and-down procedure (UDP). The oral administration of the half lethal dose (LD50) of YAC, CCA, DYA, and DCA to female ICR mice was 2.37 mg/kg, 5.60 mg/kg, 60.0 mg/kg, and 753 mg/kg, respectively. The LD50 by intravenous injection was 0.200 mg/kg, 0.980 mg/kg, 7.60 mg/kg, and 34.0 mg/kg, respectively. The LD50 of unprocessed A. carmichaelii lateral root, A. vilmorinianum root, and A. brachypodum root to mice orally was 1.89 g/kg, 0.950 g/kg, and 0.380 g/kg, respectively. Symptoms of Aconitum alkaloid poisoning in mice were decreased activity, fur erect, palpebral edema, vomiting, polypnea, and convulsions. The main change of organs was flatulence. No poisoning or death occurred in mice at the maximum dosage (27.0 g/kg) of A. ouvrardianum root orally. To better control the quality and safety of Aconitum herbs, this study provides favorable support for improving the existing standards to strengthen the supervision of the four hidden toxic Aconitum alkaloids.

Pharmacokinetics of yunaconitine and indaconitine in mouse blood by UPLC-MS/MS.

Yunaconitine and indaconitine are active ingredients from the rhizomes of Aconitum plants. In this study, an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to measure the concentrations of the yunaconitine and indaconitine in mouse blood, and the method was applied in measuring the pharmacokinetics of the two alkaloids after oral and intravenous administration. A UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm particle size) was used for chromatographic separation by gradient elution using acetonitrile-water (0.1% formic acid) as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring (MRM) mode and electrospray ionization (ESI) (positive-ion mode) were used to monitor the transitions of each analyte by tandem mass spectrometry for quantitative analysis. Yunaconitine and indaconitine were administered to the mice orally at 2 mg/kg and intravenously at 0.05 mg/kg. Blood was collected at various time intervals, and the blood samples were processed after collection and analyzed by UPLC-MS/MS. The standard curve generated for each analyte was linear over the concentration range of 0.5-500 ng/mL. The intra-day and inter-day accuracy of yunaconitine and indaconitine were 90%-103% and 86%-106%, respectively, and the precision (RSD, %) was less than 15% for both intra-day and inter-day measurements. The matrix effect ranged from 96% to 109%, and the recovery was higher than 72%. The UPLC-MS/MS method developed herein was successfully applied to measuring the pharmacokinetic parameters of yunaconitine and indaconitine in mice after intravenous and oral administration. The bioavailability of yunaconitine and indaconitine were 27.4% and 25.8%, respectively.

Development of a simple and rapid UPLC-MS/MS method for determination of 8-deacetyl-yunaconitine in rat plasma and its application to pharmacokinetics.

In this paper, UPLC-MS/MS was used to determine 8-deacetyl-yunaconitine in the plasma of rats after oral and intravenous administration. Six rats were orally (po) administered 8-deacetyl-yunaconitine (5 mg/kg), while another six rats were intravenously (iv) administered the drug (0.1 mg/kg). A standard curve of known concentrations of 8-deacetyl-yunaconitine in rat plasma was generated over the range of 0.3-600 ng/mL. The intra-day and inter-day precision of 8-deacetyl-yunaconitine in rat plasma was lower than 15 %, while the accuracy ranged between 97.7 % and 105.5 %. In addition, the matrix effect ranged between 95.3 % and 105.6 %, while the recovery was greater than 82.8 %. This determined method was then applied in measuring the pharmacokinetics of 8-deacetyl-yunaconitine in rats. The AUC(0-t) values were 73.0 ± 24.6 and 1770.0 ± 530.6 ng/mL h for intravenous and oral administration, respectively, and the bioavailability was 48.5 %. The half-life (t1/2) was determined to be 4.5 ± 1.7 h and 3.2 ± 0.7 h for intravenous and oral administration, respectively, indicating that the metabolism of the xenobiotic was quick in the rats.

Application of UPLC-QTOF-MS in Analysis of Non-targeted Urine Metabolomics in Rats with Yunaconitine Poisoning. / 应用UPLC-QTOF-MSåˆ†æžæ»‡ä¹Œå¤´ç¢±ä¸­æ¯’å¤§é¼ å°¿æ¶²éžé¶å‘ä»£è°¢ç»„å­¦.

OBJECTIVES: To explore the possible mechanism of Yunaconitine poisoning by studying the changes of urine metabolic profile in rats chronically poisoned by Yunaconitine via non-targeted metabolomics. METHODS: A rat model of Yunaconitine poisoning was established, and a metabolomics method based on UPLC-QTOF-MS technology was used to obtain the urine metabolic profile. Principal component analysis (PCA), orthogonal projections to latent structures-discriminant analysis (OPLS-DA), variable importance in projection (VIP) value greater than 1, fold change (FC) value greater than 3 or less than 0.33 and P value less than 0.05 were used to screen potential biomarkers related to the toxicity of Yunaconitine. The metabolic pathway analysis was performed through the MetaboAnalyst website and pathological changes of related tissues were observed. RESULTS: Sixteen potential biomarkers including L-isoleucine were screened, which mainly involved six metabolic pathways including the biosynthesis and degradation of valine, leucine and isoleucine, pentose and glucuronate interconversions, and propanoate metabolism, alanine, aspartate and glutamate metabolism, tyrosine metabolism. Pathological studies showed that rat toxic change in nervous system, liver and cardiac caused by Yunaconitine. CONCLUSIONS: Yunaconitine may cause neurotoxicity, hepatotoxicity and cardiotoxicity by affecting amino acid and glucose metabolism.

Study on the Toxicity Mechanism of Yunaconitine-induced Arrhythmia in Rats Based on Calcium Overload / 中国药房

OBJECTIVE：To study the toxicity mechanism of yunacotine-induced arrhythmia in rats. METHODS ：Totally 32 rats were randomly divided by random number table method into normal control group ，yunacotine low-dose and high-dose groups （0.09，0.14 mg/kg），aconitine group （positive control ，0.88 mg/kg），with 8 rats in each group. Administration groups were given the corresponding drugs once a day ，and normal control group was given the constant volume of normal saline ，for consecutive 7 d. After last intragastric administration ，the changes of electrocardiogram （ECG） were observed. The contents of adenosine triphosphate（ATP）in myocardial tissue and Ca 2+ in myocardial cells ，the activities of Na +-K+-ATPase and Ca 2+-Mg2+-ATPase as well as the protein expression of ranolidine receptor 2（RyR2）and Ca 2+-ATPase（SERCA2）in myocardial tissue were determined. RESULTS：Compared with normal control group ，time limit of QRS wave and QTc intervals of rats were prolonged significantly in yunaconitine low-dose group （P＜0.01）. The content of Ca 2 + in myocardial cells ， the ATP contents ， the activities of Ca2+-Mg2+-ATPase and Na +-K+-ATPase as well as the protein expression of SERCA 2 in myocardial tissue were reduced significantly （P＜0.05 or P＜0.01）. The heart rate of rats in yunaconitine high-dose group and aconitine group were increased significantly （P＜ 0.05 or P＜0.01），and time limit of QRS wave and QTc intervals were significantly prolonged （P＜0.01）；the content of Ca 2+ in myocardial cells was increased significantly （P＜0.01）；ATP content ，the activities of Ca 2+-Mg2+-ATPase and Na +-K+-ATPase，and protein expression of RyR 2 and SERCA 2 in myocardial tissue were decreased significantly （P＜0.01）. CONCLUSIONS ： Yunaconitine can induce arrhythmia in rats ，the mechanism of which may be associated with Ca 2+ overload that resulted from reducing the activities of Na +-K+-ATPase and Ca 2+-Mg2+-ATPase and down-regulating the expression of related calcium transporter RyR2 and SERCA 2.

Application of UPLC-QTOF-MS in Analysis of Non-targeted Urine Metabolomics in Rats with Yunaconitine Poisoning / 法医学杂志

OBJECTIVES@#To explore the possible mechanism of Yunaconitine poisoning by studying the changes of urine metabolic profile in rats chronically poisoned by Yunaconitine via non-targeted metabolomics.@*METHODS@#A rat model of Yunaconitine poisoning was established, and a metabolomics method based on UPLC-QTOF-MS technology was used to obtain the urine metabolic profile. Principal component analysis (PCA), orthogonal projections to latent structures-discriminant analysis (OPLS-DA), variable importance in projection (VIP) value greater than 1, fold change (FC) value greater than 3 or less than 0.33 and P value less than 0.05 were used to screen potential biomarkers related to the toxicity of Yunaconitine. The metabolic pathway analysis was performed through the MetaboAnalyst website and pathological changes of related tissues were observed.@*RESULTS@#Sixteen potential biomarkers including L-isoleucine were screened, which mainly involved six metabolic pathways including the biosynthesis and degradation of valine, leucine and isoleucine, pentose and glucuronate interconversions, and propanoate metabolism, alanine, aspartate and glutamate metabolism, tyrosine metabolism. Pathological studies showed that rat toxic change in nervous system, liver and cardiac caused by Yunaconitine.@*CONCLUSIONS@#Yunaconitine may cause neurotoxicity, hepatotoxicity and cardiotoxicity by affecting amino acid and glucose metabolism.

Partial Synthesis of Crassicauline A from Yunaconitine.

Both Aconitum hemsleyanum and Aconitum geniculatun have abundant contents of yunaconitine (1). Yunaconitine (1) has similar skeleton to crassicauline A (3); the only difference between them is that 1 contains a α-hydroxyl group at C-3. Our team attempts to convert 1 into 3 because 3 owns pharmacological activity. There are two steps to achieve the transformation above: firstly, use dehydration reaction to transform yunaconitine (1) into dehydroyunaconitine (2); secondly, use hydrogen reduction to acquire crassicauline A (3). Compared with other methods, this one below is more suitable for production application and more concise; moreover, the cost is lower with higher yield.

StudyoncontentofalkaloidandcardiactoxicitybeforeandafterpreparationofAconitumvilmorinianum / 中草药

Objective To investigate the content changes of two alkaloid before and after the processing of Aconitumvilmorinianum and its effect on cardiotoxicity. MethodsThe UPLC method was used to determine the content of yunaconitine and 8-acetylacetonarine before and after the processing of A.vilmorinianum. The half lethal dose (LD50) of A.vilmorinianum to rats was determined by Bliss method. Rats wereig administrated A.vilmoriniauum and its processed products for 14 d. The chages of II lead electrocardiogram of anesthetized rats and myocardial morphology were observed,and the serum levels of lactate kinase (CK) and lactate dehydrogenase (LDH)were detected. Results The content of yunaconitine before and after the preparation of A.vilmorinianum was 263.96 μg/g and not detected respectively. The content of 8-acetylacetonarine was not detected and 568.47 μg/g respectively. The LD50 of the raw product was crude drug4.2 g/kg, and the maximum dose of the processed product was crude drug18.0 g/kg; In the anesthetized rats of the raw group, ventricular premature beats, ventricular bipolar law, and ventricular fibrillation occurred successively. The processed product group showed bradycardia in the early stage and returned to normal in 60 min; Compared with the blank group, the LDH and CK levels in the raw group were significantly increased (P< 0.05), and only the LDH level was significantly increased in the processed product group (P< 0.05); The gap between myocardial fibers in the raw group was significantly enlarged, and the infiltration of inflammatory cells was obvious. The myocardial fibers of the product group are arranged neatly, and individual inflammatory cells infiltrated in the interstitial. Conclusion At the given dose, A.vilmorinianum has greater cardiotoxicity, and the toxicity of A.vilmorinianum is obviously reduced after processing.

Tissue Distribution of Yunaconitine in Rats by UPLC-MS/MS Method / 昆明医科大学学报

Objective To establish an acute yunaconitine poisoning rat model with a single oral administration and to determine the contents of yunaconitine in rat tissues by UPLC-MS/MS method, then investigate the distribution of yunaconitine in rats. Method The rats were randomly divided into three groups and were intragastrically administered a single dose of 2.2mg/kg,1.1mg/kg,0.7mg/kg yunaconitine, respectively.. The rats were killed 2h later, the stomach tissue, intestine tissue, liver tissue, pancreas tissue, kidney tissue, lung tissue, spleen tissue, heart tissue, bladder tissue, testis tissue, brain tissue and heart blood samples were collected. The contents of yunaconitine in the biological materials were determined by UPLC-MS/MS method after the biological samples extracted by liquid-liquid extraction. Result A rat model of the yunaconitine poisoning was made with a single dose of 1.1mg/kg, the concentrations of yunaconitine displayed in the organs with the following order:stomach, small intestine, liver, pancreas, kidney, lung, spleen, heart, bladder, testis, heart blood and brain. Conclusion Yunaconitine was widely distributed in rats, especially the levels in the stomach, small intestine and liver were the highest. The conclusion provides a basis for the selection of test materials for the poisoning of Aconitum vilmorinianum Kom.

Diterpenoid alkaloids in roots of Aconitum hemsleyanum var. circinatum / 中草药

Objective To study the chemical constituents in the roots of Aconitum hemsleyanum var. circinatum. Methods The constituents were isolated and purified by silica gel chromatography from the roots of Aconitum hemsleyanum var. circinatum, and the structures were identified by spectral analysis (1H-NMR, 13C-NMR, and MS). Results Seventeen compounds were isolated from Aconitum hemsleyanum var. circinatum and characterized as sachaconitine (1), 8-O-methylsachaconitine (2), liljestrandisine (3), talatisamine (4), chasmannine (5), 8-O-methyltalatisamine (6), 14-O-acetyltalatisamine (7), 8-deacetyl-yunaconitine (8), crassicautine (9), crassicaudine (10), crassicauline A (11), vilmorianine C (12), vilmorianine A (13), yunaconitine (14), transconitine B (15), kongboendine (16), and franchetine (17). Conclusion Seventeen compounds are isolated from Aconitum hemsleyanum var. circinatu. Compounds 2-3, 6-7, 9, and 12-16 are isolated from Aconitum hemsleyanum var. circinatum for the first time.

A Preliminary Research on the Efficacy and Toxicity of Yunaconitine and 8-deacetyl- yunaconitine Isolated from the Processed Products of Aconiti Knsnezoffii Radix / 中国中医药信息杂志

Objective To conduct comparative study on the analgesic and anti-inflammatory effects as well as the acute toxicity of yunaconitine and 8-deacetyl-yunaconitine isolated from the processed products of Aconiti Knsnezoffii Radix.Methods The methods of hot plate test and writhing test were used to evaluate the analgesic effect. Anti-inflammation action was observed by the models of auricle swelling caused by dimethylbenzene. LD50 was determined by the method of Bliss.Results Yunaconitine and 8-deacetyl-yunaconitine have analgesia effect on the pain caused by hot-plate, but there were no statistically significant difference. The pain caused by acetic acid had obvious analgesic action. High and low dose of yunaconitine could significantly reduce the number of mice body torsion and extend the incubation period of pain in mice. The effect of 8-deacetyl-yunaconitine was remarkable only in the high dose. Compared with solvent group, there were little differences in inhibiting effect of auricle swelling caused by dimethylbenzene, and anti-inflammatory action was not exact. The poisonousness of yunaconitine was nearly 20 times of 8-deacetyl-yunaconitine.Conclusion Yunaconitine and 8-deacetyl-yunaconitine may be the analgesic medicine for peripheral analgesic effect. The poisonousness of 8-deacetyl-yunaconitine is less than yunaconitine, the effect is remarkable to the pain caused by acetic acid, and the security is high.

Accelerated solvent extraction and pH-zone-refining counter-current chromatographic purification of yunaconitine and 8-deacetylyunaconitine from Aconitum vilmorinianum Kom.

This study aimed to seek an efficient method to extract and purify yunaconitine and 8-deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH-zone-refining counter-current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L9 (3)(4). Then a separation and purification method was established using pH-zone-refining counter-current chromatography with a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8-deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI-MS and (1)H and (13)C NMR spectroscopy.