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3-Acetylaconitine (AAC) is a natural compound with strong anti-inflammatory and analgesic properties, but the severely narrow safety range limited its clinical application. Two dissolvable microneedle (MN) systems, AAC-ORP-MN and AAC-MN, loaded with AAC liposomes (AAC-Lips) either mixed with or without Ostrea rivularis polysaccharide (ORP) were developed. The size, zeta potential and encapsulation efficiency of AAC-Lips were 112.9 ± 0.5 nm, -31.3 ± 0.5 mV and 95.7 ± 1.2 %. AAC-ORP-MN demonstrated higher mechanical strength and faster initial drug release rate compared to AAC-MN. The results in the spared nerve injury (SNI) model showed that AAC-ORP-MN and AAC-MN both could significantly increase the mechanical pain threshold on the operated side of the rats, and gradually decrease the difference between the equilibrium pain thresholds of both feet, but AAC-ORP-MN showed a faster onset of action. In addition, AAC-ORP-MN significantly reduced inflammatory factors and improved pathological damage in the spinal cord better than AAC-MN, which might be due to the anti-inflammatory activity of ORP. Overall, the above results supported that AAC-ORP-MN showed promise as a clinical option for analgesia, which had anti-inflammatory and analgesic properties, meanwhile it could effectively deliver poorly water-soluble AAC and improve its safety and availability., Competing Interests: Declaration of competing interest The authors state that they have no potential competing financial interests or personal relationships with other people or organizations that might unduly influence their work in this paper., (Copyright © 2024. Published by Elsevier B.V.)

3-Acetylaconitine (AAC) is a commercially analgesic drug for arthritis, however, due to its narrow safety range, its clinical application is limited. The objective of the study was to investigate the combined effect of Cucumaria frondosa polysaccharide (CFP) and AAC in microneedles on knee osteoarthritis (KOA). The characterization of microneedles was conducted and a rat model of monosodium iodoacetate (MIA)-induced KOA was established to evaluate the anti-inflammatory and analgesic activity in this study. The inhibitory effects on cartilage degradation and synovial inflammation were determined by morphological observation of the knee joint, pathological and imaging results. The von Frey pain threshold and hind limb weight bearing test were used to evaluate the effect of microneedles on pain relief. The results showed that microneedles had the mechanical strength to penetrate the skin and dissolved well in the skin for drug delivery within 30 min after patch administration. Furthermore, AAC-MN and CFP-AAC-MN both played roles in relieving pain, reducing cartilage degeneration and inhibiting synovial inflammation in KOA. In summary, CFP combined with AAC loading into microneedles exhibited higher skin penetration and more advantages in inhibiting cartilage damage and inflammation compared to AAC alone., Competing Interests: Declaration of competing interest The authors state that they have no potential competing financial interests or personal relationships with other people or organizations that might unduly influence their work in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Bulleyaconitine A (BLA) is a promising candidate for treating rheumatoid arthritis (RA) with diverse pharmacological activities, including anti-inflammatory, analgesic and bone repair. Herein, the long-acting bulleyaconitine A microspheres (BLA-MS) were developed to treat RA comprehensively by forming drug reservoirs in joint cavities. The BLA-MS were prepared by emulsion/solvent evaporation method. The particle size and distribution were assessed by SEM. The crystalline state was investigated by DSC and PXRD. The drug loading (DL), encapsulation efficiency (EE) and cumulative release in vitro were determined by HPLC. The DL and EE were 23.93 ± 0.38 % and 95.73 ± 1.56 % respectively, and the cumulative release was up to 69 days with a stable release curve. The pharmacodynamic results in collagen induced arthritis (CIA) rats showed a noticeable reduction in paw thickness (5.66 ± 0.32 mm), and the decreasing expression level of PGE 2 , TNF-α and IL-6 which diminished the infiltration of inflammatory cells, thereby alleviating the progression of erosion and repairing the damaged bones (BV/TV (Bone Volume / Total Volume): 81.97 %, BS/BV (Bone Surface / Bone Volume): 6.08 mm -1 ). In conclusion, intra-articular injection of BLA-MS should have a promising application in the treatment of RA and may achieve clinical transformation in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aconitine linoleate (1) is a lipo-diterpenoid alkaloid, isolated from Aconitum sinchiangense W. T. Wang. The study aimed at investigating the anti-proliferative efficacy and the underlying mechanisms of 1 against MCF-7 and MCF-7/ADR cells, as well as obvious the safety evaluation in vivo. The cytotoxic activities of 1 were measured in vitro. Also, we investigated the latent mechanism of 1 by cell cycle analysis in MCF-7/ADR cells and topo I and topo IIα inhibition assay. Molecular docking is done by Discovery Studio 3.5 and Autodock vina 1.1.2. Finally, the acute toxicity of 1 was detected on mice. 1 exhibited significant antitumor activity against both MCF-7 and MCF-7/ADR cells, with IC 50 values of 7.58 and 7.02 μM, which is 2.38 times and 5.05 times more active, respectively than etoposide in both cell lines, and being 9.63 times more active than Adriamycin in MCF-7/ADR cell lines. The molecular docking and the topo inhibition test found that it is a selective inhibitor of topoisomerase IIα. Moreover, activation of the damage response pathway of the DNA leads to cell cycle arrest at the G 0 G 1 phase. Furthermore, the in vivo acute toxicity of 1 in mice displayed lower toxicity than aconitine, with LD 50 of 2.2 × 10 5  nmol/kg and only slight pathological changes in liver and lung tissue, 489 times safer than aconitine. In conclusion, compared with aconitine, 1 has more significant anti-proliferative activity against MCF-7 and MCF-7/ADR cells and greatly reduces in vivo toxicity, which suggests this kind of lipo-alkaloids is powerful and promising antitumor compounds for breast cancer., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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

The aim was to investigate the role of the α7nAChR-mediated cholinergic anti-inflammatory pathway in vagal nerve regulated atrial fibrillation (AF).18 beagles (standard dogs for testing) were used in this study, and the effective refractory period (ERP) of atrium and pulmonary veins and AF inducibility were measured hourly during rapid atrial pacing at 800 beats/minute for 6 hours in all beagles. After cessation of 3 hours of RAP, the low-level vagal nerve stimulation (LL-VNS) group (n = 6) was given LL-VNS and injection of salinne (0.5 mL/GP) into four GPs, the methyllycaconitine (MLA, the antagonist of α7nAChR) group (n = 6) was given LL-VNS and injection of MLA into four GPs, and the Control group (n = 6) was given saline into four GPs and the right cervical vagal nerve was exposed without stimulation. Then, the levels of the tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), acetylcholine (ACh), STAT3, and NF-κB proteins were measured. During the first 3 hours of RAP, the ERPs gradually decreased while the dispersion of ERPs (dERPs) and AF inducibility gradually increased in all three groups. During the last 3 hours of 6 hours' RAP in this study, the ERPs in the LL-VNS group were higher, while the dERPs and AF inducibility were significantly lower when compared with the Control and MLA groups at the same time points. The levels of ACh in the serum and atrium in the LL-VNS and MLA groups were higher than in the Control group, and the levels of TNF-α and IL-6 were higher in the Control and MLA groups than in the LL-VNS group. The concentrations of STAT3 in RA and LA tissues were higher in the LL-VNS group while those of NF-κB were lower.In conclusion, the cholinergic anti-inflammatory pathway mediated by α7nACh plays an important role in low-level vagal nerve-regulated AF.

Acute pancreatitis (AP) is associated with impaired acinar cell autophagic flux, intracellular zymogen activation, cell necrosis and inflammation. Activation of the cholinergic system of vagus nerve has been shown to attenuate AP, but the effect of organ-intrinsic cholinergic system on pancreatitis remains unknown. In this study, we aim to examine the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation within the pancreas during AP. In vivo, AP was induced by caerulein plus LPS or ethanol plus palmitoleic acid in mice. In vitro, pancreatic acini were isolated and subjected to cholecystokinin (CCK) stimulation. Mice or acini were pre-treated with PNU-282987 (selective α7nAChR agonist) or methyllycaconitine citrate salt (selective α7nAChR antagonist). Pancreatitis severity, acinar cell injury, autophagic flux, and transcription factor EB (TFEB) pathway were analyzed. Both caerulein plus LPS in vivo and CCK in vitro led to an up-regulation of α7nAChR, indicating activation of pancreas-intrinsic α7nAChR signaling during AP. PNU-282987 decreased acinar cell injury, trypsinogen activation and pancreatitis severity. Conversely, methyllycaconitine citrate salt increased acinar cell injury and aggravated AP. Moreover, activation of α7nAChR by PNU-282987 promoted autophagic flux as indicated by reduced p62, increased LysoTracker staining and decreased number of autolysosomes with undegraded contents. Furthermore, PNU-282987 treatment significantly increased TFEB activity in pancreatic acinar cells. α7nAChR activation also attenuated pancreatic inflammation and NF-κB activation. Our results showed that activation of α7nAChR protected against experimental pancreatitis through enhancing TFEB-mediated acinar cell autophagy, suggesting that activation of pancreas-intrinsic α7nAChR may serve as an endogenous protective mechanism during AP., (Copyright © 2020. Published by Elsevier B.V.)

Rheumatoid arthritis (RA) is a kind of systemic autoimmune disease, and patients with RA usually suffer serious pain, resulting in low quality of life. The development of drug delivery systems (DDSs) provides a valid approach for RA therapy via inhibiting the secretion of inflammatory cytokines from macrophages. As a prevailing drug nanocarrier with distinctive superiority, polymeric nanoparticles (NPs) have attracted much attention in recent years. However, low biocompatibility and limited exploitation of drug with high efficiency are still the main challenges in RA treatment. To overcome the limitations, we prepared a biocompatible copolymer methoxy-poly(ethylene glycol)-poly(lactide-co-glycolide) (mPEG-PLGA). Moreover, benzoylaconitine (BAC) with superior anti-inflammatory effect was selected as model drug. It was isolated from Aconitum kusnezoffii Reichb and encapsulated into mPEG-PLGA NPs (NP/BAC) to increase the bioavailablity of BAC. The NPs exhibited high cytocompatibility for activated macrophages and well compatibility with red blood cells. Furthermore, the anti-inflammatory property of NP/BAC was testified by substantially inhibiting secretion of pro-inflammatory cytokines. The TNF-α and IL-1β cytokines of NP/BAC group reduced 70 % and 66 % compared with that of activated macrophages. Especially, NP/BAC reduced the overexpression of NF-κB p65 to inhibit NF-κB signaling pathway, which was a critical regulator of inflammatory responses. NP/BAC also showed efficient in vivo anti-inflammatory effect with high ear (69.8 %) and paw (87.1 %) swelling suppressing rate. These results revealed the anti-inflammatory mechanism of NP/BAC and proved it was a suitable DDS to suppress inflammation, providing a promising strategy for RA therapy and research of Aconitum kusnezoffii Reichb., Competing Interests: Declaration of Competing Interest No conflict of interest exits in the submission of this manuscript, and the manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that this work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Visceral pain is one of the most common types of pain and particularly in the abdomen is associated with gastrointestinal diseases. Bulleyaconitine A (BAA), isolated from Aconitum bulleyanum , is prescribed in China to treat chronic pain. The present study is aimed at evaluating the mechanisms underlying BAA visceral antinociception., Methods: The rat model of chronic visceral hypersensitivity was set up by colonic perfusion of 2,4,6-trinitrobenzene sulfonic acid (TNBS) on postnatal day 10 with coapplication of heterotypic intermittent chronic stress (HeICS)., Results: The rat model of chronic visceral hypersensitivity exhibited remarkable abdominal withdrawal responses and mechanical hyperalgesia in hind paws, which were dose-dependently attenuated by single subcutaneous of administration of BAA (30 and 90  μ g/kg). Pretreatment with the microglial inhibitor minocycline, dynorphin A antiserum, and κ -opioid receptor antagonist totally blocked BAA-induced visceral antinociception and mechanical antihyperalgesia. Spontaneous excitatory postsynaptic currents (sEPSCs) in spinal dorsal horn lamina II neurons were recorded by using whole-cell patch clamp. Its frequency (but not amplitude) from TNBS-treated rats was remarkably higher than that from naïve rats. BAA (1  μ M) significantly reduced the frequency of sEPSCs from TNBS-treated rats but not naïve rats. BAA-inhibited spinal synaptic plasticity was blocked by minocycline, the dynorphin A antiserum, and κ -opioid receptor antagonist. Dynorphin A also inhibited spinal synaptic plasticity in a κ -opioid receptor-dependent manner., Conclusions: These results suggest that BAA produces visceral antinociception by stimulating spinal microglial release of dynorphin A, which activates presynaptic κ -opioid receptors in afferent neurons and inhibits spinal synaptic plasticity, highlighting a novel interaction mode between microglia and neurons., Competing Interests: The authors declare that there are no competing interests in this work., (Copyright © 2020 Sheng-Nan Huang et al.)

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., (© 2020 John Wiley & Sons, Ltd.)

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 (LD 50 of 21.14 mg·kg -1 ), improved analgesic effect and longer half-life (T 1/2 of 2.24 h) when compared with LAH. Moreover, in vitro transdermal permeation (J ss 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nicotine, a potent parasympathomimetic alkaloid, manifests anti-inflammatory properties by activating nicotinic acetylcholine receptors (nAChRs). In this study, we evaluated the effects of nicotine on concanavalin A (ConA)-induced autoimmune hepatitis. Nicotine (0.5 and 1 mg/kg) was intraperitoneally administered to BALB/c mice and mice were intravenously injected with ConA (15 mg/kg) to induce hepatitis. The results showed that nicotine treatment ameliorated pathological lesions in livers and significantly suppressed the expression of pro-inflammatory cytokines in the livers. Such effects were mediated by inhibiting the nuclear factor-kappa B (NF-κB) signaling in livers. Interestingly, nicotine inhibited the ConA-induced inflammatory response in primary cultured Kupffer cells (KCs) but did not alter the proliferation of splenocytes. The protective effects of nicotine against ConA-induced hepatitis were abolished in KC-depleted mice, indicating the requirement of KCs in this process. Additionally, the expression of α7-nAChR on KCs was dramatically increased by nicotine treatment, and the protective effects of nicotine on ConA-induced liver injury were significantly suppressed by treatment with methyllycaconitine (MLA), a specific α7-nAChR antagonist. Consistently, in primary cultured KCs, the activation of NF-κB signaling was also regulated by nicotine treatment. This study suggests that nicotine increases α7-nAChR-mediated cholinergic activity in KCs resulting in decrease of ConA-induced autoimmune hepatitis through inhibiting NF-κB signaling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nanostructured lipid carriers (NLCs) can enhance the safe transdermal delivery system of drugs. Moreover, dissolving microneedles (MNs) can enhance the permeability and controlled drug release. In this study, NLCs were formulated as a suitable vehicle for aconitine (ACO) delivery to effectively inhibit the inflammation of fibroblast-like synoviocytes isolated from a rat model of adjuvant-induced arthritis (AA-FLS). To improve drug delivery, the ACO-loaded NLCs (ACO-NLCs) were embedded in polyvinylpyrrolidone-based dissolving MNs fabricated by an ultraviolet cross-linking method. The nanoparticles maintained good physical stability in the dissolving MNs. The insertion capabilities of the ACO-NLCs-MNs were determined by observing histological sections of the skin after insertion, and scanning electron microscopy was used to observe the changes in the MNs over time. In vivo microdialysis showed that the NLCs-MNs enhanced the transdermal delivery of ACO through disrupting the barrier function of the stratum corneum (SC) and releasing the drug continuously. The ACO-NLCs-MNs showed a significant inhibitory effect on the paw swelling and inflammation in AA model rats. Moreover, this dual approach involving NLCs-loaded dissolving MNs formed a drug reservoir and effectively improved the ACO-induced arrhythmia. These results indicate that NLCs-containing MNs could be promising systems for the effective transdermal delivery and controlled local administration of ACO., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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, C max 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.

Talatisamine, as the efficacy ingredient of Aconitum, was known as a novel specific blocker for the delayed rectifier K + channels in rat hippocampal neurons. In this study, a rapid, selective and reproducible UPLC-MS/MS separation method was established and fully validated for the quantitative determination of talatisamine levels in ICR (Institute of Cancer Research) mouse blood. A total of 24 healthy male ICR mice were divided into four groups that was administered talatisamine via intravenous at a dose of 1 mg/kg and oral administration of three doses (2, 4, 8 mg/kg). All blood samples were protein precipitate by using acetonitrile with an internal standard (IS) deltaline. The effective chromatographic separation was carried out through an UPLC BEH C18 analytical column (2.1 mm × 50 mm, 1.7 μm) with an initial mobile phase that consisted of acetonitrile and 10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. Also, an electrospray ionization (ESI) was applied to quantify the talatisamine in the positive ions mode. The method validation demonstrated good linearity over the range of 1-1000 ng/mL (r 2  ≥ 0.9993) for talatisamine in mouse blood with a lower limit of quantification (LLOQ) at 1 ng/mL. The accuracy values of the method were within 89.4% to 113.3%, and the matrix effects were between 103.2% and 106.3%. The mean extraction recoveries for talatisamine obtained from four concentrations of QC blood samples were exceeded 71.7%, and the relative standard deviation (RSD) both of intra- and inter-day precision values for replicate quality control samples did not exceed 15% respectively for all analytes during the assay validation. This method was successfully applied to the evaluation of the pharmacokinetic of talatisamine, regardless of intragastric or intravenous administration in mice. Based on the pharmacokinetics data, the bioavailability of talatisamine in mice was >65.0% after oral administration, exhibiting an excellent oral absorption., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Lappaconitine (LA) has been widely used for postoperative and cancer pain control. LA exhibits excellent analgesic activity with a longer effective time than common local anesthetics such as tetracaine and bupivacaine. However, the mechanisms underlying the featured analgesic activity of LA remain largely unknown. Here, we report that LA is an inhibitor of voltage-gated sodium channel 1.7 (Nav1.7) stably expressed in human embryonic kidney (HEK293) cells. LA inhibited Nav1.7 in a voltage-dependent manner with an IC 50 value (with 95% confidence limits) of 27.67 (15.68-39.66) µmol/L when the cell was clamped at -70 mV. In comparison with the quick and reversible inhibition of Nav1.7 by tetracaine and bupivacaine, the inhibitory effect of LA was rather slow and irreversible. It took more than 10 min to achieve steady-state inhibition when LA (300 µmol/L) was administered. Unlike tetracaine and bupivacaine, LA affected neither the voltage-dependent activation nor the inactivation of the channels. Five residues in domain III and domain IV have been reported to be critical for the effects of the two local anesthetics on Nav channels. But our mutant study revealed that only two residues (F1737, N1742) located in domain IV were necessary for the inhibitory activity of LA. The slow onset, irreversibility, and lack of influence on channel activation and inactivation accompanied with the different molecular determinants suggest that LA may inhibit Nav1.7 channels in a manner different from local anesthetics. These results may help to understand the featured analgesic activity of LA, thus benefiting its application in the clinic and future drug development.

Lappaconitine is extracted from Aconitum sinomontanum Nakai, which belongs to the Ranunculaceae. Lappaconitine is as a diterpenoid alkaloid used as a nonaddictive analgesic. To assure the rational use of the drug, ultrahigh-pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was conducted to determine lappaconitine in mouse blood and its application to pharmacokinetics. In this study, khasianine was used as internet standard (IS). A UPLC BEH C18 column was used for chromatographic separation and the mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid). The flow rate of was 0.4 mL/min. Quantitative detection was performed in a multiple reaction monitoring (MRM) mode using an electrospray ionization source in positive mode. Twenty-four mice were randomly divided into four groups, three of which received 2, 4, and 8 mg/kg lappaconitine by intragastric administration, while the other group received 1 mg/kg lappaconitine by intravenous administration. After 0.0833, 0.5, 1, 1.5, 2, 3, 4, and 8 h, blood samples were collected and acetonitrile was used for protein precipitation. A linear calibration relationship (R 2 = 0.9979) in the range of 0.1-500 ng/mL in mouse blood indicated good results. The lower limit of quantitation was 0.1 ng/mL and the limit of detection was 0.04 ng/mL. The intra-day and inter-day precision were below 13% and 14%, respectively. The accuracy was 90.1-107.2%, and the recovery exceeded 81.1%. The matrix effect ranged between 102.1 and 108.8%. The absolute bioavailability of lappaconitine was 2.0%. UPLC-MS/MS achieved high sensitivity, speed, and selectivity. Methodological verification indicated this method as suitable for determination of lappaconitine in mouse blood.

Objectives: Alpha7 nicotinic acetylcholine receptor (α7-nAChR), an emerging pharmacological target for a variety of medical conditions, is expressed in the most mammalian tissues with different effects. So, this study was designed to investigate the expression, localization and effect of α7-nAChR in rat corpus cavernosum (CC)., Methods & Key Findings: Reverse transcription polymerase chain reaction (RT-PCR) revealed that α7-nAChR was expressed in rat CC and double immunofluorescence studies demonstrated the presence of α7-nAChR in corporal neurons. The rat CC segments were mounted in organ bath chambers and contracted with phenylephrine (0.1 μm -300 μm) to investigate the relaxation effect of electrical field stimulation (EFS,10 Hz) assessed in the presence of guanethidine (adrenergic blocker, 5 μm) and atropine (muscarinic cholinergic blocker, 1 μm) to obtain non-adrenergic non-cholinergic (NANC) response. Cumulative administration of nicotine significantly potentiated the EFS-induced NANC relaxation (-log EC50 = 7.5 ± 0.057). Whereas, the potentiated NANC relaxation of nicotine was significantly inhibited with different concentrations of methyllycaconitine citrate (α7-nAChR antagonist, P < 0.05) in preincubated strips. L-NAME (non-specific nitric oxide synthase inhibitor, 1 μm) completely blocked the neurogenic relaxation induced by EFS plus nicotine., Conclusion: To conclude α7-nAChR is expressed in rat CC and modulates the neurogenic relaxation response to nicotine., (© 2017 Royal Pharmaceutical Society.)

Objective: To explore the effects of lappaconitine (LA) on pain and inflammatory response of severely burned rats and the mechanism. Methods: Forty SD rats were divided into healthy+ normal saline group, sham injury+ normal saline group, pure burn group, burn+ LA group, and healthy+ LA group according to the random number table (the same dividing method below), with 8 rats in each group. Rats in pure burn and burn+ LA groups were inflicted with about 32% total body surface area deep partial-thickness scald (hereinafter referred to as burn) on the back and right hind. Rats in sham injury+ normal saline group were sham injured. Rats in burn+ LA group were intraperitoneally injected with 1 g/L LA solution in the dosage of 4 mL/kg at 2.0 h before injury and post injury hour (PIH) 0 (immediately), 24.0, 48.0, and 72.0. Rats in healthy+ LA group were intraperitoneally injected with LA solution in the same dose at the same time points as above, and rats in healthy+ normal saline and sham injury+ normal saline groups were intraperitoneally injected with normal saline in the dose of 4 mL/kg at the same time points as above. At 1.5 h before injury and PIH 12.5, 24.5, 36.5, 48.5, and 72.5, the paw withdrawal mechanical threshold (PWMT) of injured rats was detected, and their pain behaviors were observed. The same observation and detection were conducted in rats without injury in the two groups at the same time points as above. Another 32 SD rats were divided into normal saline group, trinitrophenyl (TNP)-ATP group, minocyline group, pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) group, with 8 rats in each group, and all the rats were inflicted with the same burn injury as above. At PIH 48.0, rats in normal saline group were intrathecally injected with 10 μL normal saline; rats in TNP-ATP group were intrathecally injected with 10 μL TNP-ATP in the concentration of 30 nmol/μL; rats in minocyline group were intrathecally injected with 10 μL minocyline in the concentration of 5 g/L; rats in PPADS group were intrathecally injected with 10 μL PPADS in the concentration of 10 nmol/μL. The PWMT of rats was detected at 0.5 h before injection and 0.5 h after. At PIH 72.5, the tissue in the dorsal horn of spinal cord of rats in sham injury+ normal saline, pure burn, and burn+ LA groups was harvested to observe the co-expression of P2X(4) receptor and OX42 receptor with immunofluorescent staining and to observe the expression of P2X(4) receptor and count the positive cells with immunohistochemical staining. The venous blood was harvested for determination of serum content of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) with enzyme-linked immunosorbent assay. The same observation and determination were conducted in rats without injury in the two groups at the same time point as above. Data were processed with one-way analysis of variance, analysis of variance for repeated measurement, SNK test, paired t test, and Bonferroni correction. Results: (1) There were no abnormal activity in rats of healthy+ normal saline, sham injury+ normal saline, healthy+ LA groups at all time points. Until PIH 72.5, rats in pure burn group were in poor mental state; red and swollen manifestation and blister were observed in burn wounds on the back and right hind; imbalance in gait, lick, bite, and scratch were observed occasionally. Fewer behaviors such as lick, bite, and limp were observed in rats in burn+ LA group than in pure burn group, and the red and swollen manifestation in wounds of rats in burn+ LA group dissipated faster than that in pure burn group. (2) At 1.5 h before injury, there were no significant differences in the PWMT values of rats in healthy+ normal saline, sham injury+ normal saline, pure burn, burn+ LA, and healthy+ LA groups ( F =0.106, P >0.05). PWMT values of rats in pure burn group were significantly lower than those in the other 4 groups at all post injury time points (with P values below 0.05). PWMT values of rats in burn+ LA group were significantly lower than those in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups at all post injury time points (with P values below 0.05). (3) At 0.5 h before injection, PWMT values of rats in normal saline, TNP-ATP, PPADS, and minocyline groups were close, respectively 15.3±0.8, 15.1±1.0, 15.3±0.9, and 15.6±1.1 ( F =0.343, P >0.05). At 0.5 h after injection, PWMT values of rats in normal saline group and PPADS group were respectively 15.2±1.2 and 14.8±1.0, which were significantly lower than 20.8±1.4 and 26.3±1.0 in TNP-ATP group and minocyline group respectively (with P values below 0.05). PWMT values of rats in normal saline and PPADS groups were similar before and after injection (with t values respectively 0.073 and -0.772, P values above 0.05), while those of rats in TNP-ATP and minocyline groups were higher after injection than before injection (with t values respectively -10.180 and -20.813, P values below 0.01). (4) At PIH 72.5, co-expression of P2X(4) receptor and OX42 receptor was observed in a few microglias of rats in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups, while co-expression of P2X(4) receptor and OX42 receptor was observed in a large number of microglias of rats in pure burn and burn+ LA groups. At PIH 72.5, more P2X(4) receptor positive cells were observed in rats in pure burn group than in the other 4 groups (with P values below 0.05), and more P2X(4) receptor positive cells were observed in rats in burn+ LA group than in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups (with P values below 0.05). (5) At PIH 72.5, the serum content of TNF-α and IL-1β of rats in pure burn group was significantly higher than that in the other 4 groups (with P values below 0.001). The serum content of TNF-α and IL-1β of rats in burn+ LA group was significantly lower than that in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups (with P values below 0.001). Conclusions: LA has significant analgesic effects on severely burned rats, and it can ameliorate the excessive inflammational situation. The mechanism may be related to its inhibition of expression of P2X(4) receptor in microglias in the dorsal horn of spinal cord and reduction in the release of inflammatory factors TNF-α and IL-1β.

1. A model of aconitine-induced bradycardia and hypotension, which is similar to aconitine poisoning in humans, was constructed in conscious rats by oral administration. 2. Blood pressure (BP) and heart rate (HR) of Sprague-Dawley rats were measured using a volume pressure recording (VPR) system. The pharmacokinetics of toxic doses of aconitine and its metabolites were analyzed using UPLC-MS/MS. 3. The HR was significantly decreased by 29% at 2 h after oral administration of 200 μg/kg aconitine. When the dose was increased to 400 μg/kg, systolic BP and diastolic BP were significantly decreased by 11% and 12% at 2 h after the administration, except when bradycardia occurred at 2 h and 4 h. The drug concentration-time curve showed a double-peak phenomenon in rats administered a 400 μg/kg dose. The AUC 0-12 h value in the 400 μg/kg group significantly increased 0.8-fold compared to the 200 μg/kg group. Moreover, a high plasma concentration of 16-O-demethyaconitine was found in the rats that received two toxic doses. 4. In conclusion, bradycardia and hypotension are induced in conscious rats by a toxic dose of aconitine (400 μg/kg), and there was no significant difference in dose-normalized AUC 0-12 h values between oral administrations of 200 μg/kg and that of 400 μg/kg. However, the dose-normalized C max and AUC 0-12 h values in 200 μg/kg and 400 μg/kg groups were significantly smaller than those in 100 μg/kg group. The metabolites of aconitine, 16-O-demethyaconitine, and benzoylaconitine may also contribute to the hypotensive response.

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., (Copyright © 2015 John Wiley & Sons, Ltd.)

Sustained neuroinflammation contributes to the pathogenesis of postoperative cognitive dysfunction. Dexmedetomidine, a selective α-2 adrenergic receptor agonist, exhibits a protective role in the brain. This study investigated whether dexmedetomidine pretreatment attenuates neuroinflammation induced by tibial fracture in rats, as well as the mechanism by which dexmedetomidine provides its neuroprotection. In our study, we observed that tibial fracture significantly increased the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and the expression of nuclear factor-kappa B (NF-κB) in the hippocampus. Overexpression of microglial (CD11b) and astrocytic (GFAP) responses to injury were observed in the hippocampus. Dexmedetomidine pretreatment significantly suppressed the inflammatory responses, as evidenced by lower TNF-α and IL-1β levels, significantly inhibited NF-κB activity, and alleviated overexpression of microglia and astrocytes in the hippocampus. However, pretreatment with dexmedetomidine failed to attenuate cytokine responses and activity of NF-κB, CD11b and GFAP after vagotomy or treatment with methyllycaconitine, an α-7 nicotinic acetylcholine receptor (α7nAChR) antagonist. These results suggest that pretreatment with dexmedetomidine may attenuate neuroinflammation caused by tibial fracture in rats through vagal-dependent and α7nAChR-dependent mechanisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

One-step self-assembly was used to prepare pH-sensitive lappaconitine-loaded low-molecular-weight heparin (LMWH-LA) and to demonstrate that the sulfur group promotes dissolution and has synergistic effect on the analgesic property of lappaconitine (LA). The LMWH-LA was characterized in terms of releasing behavior, pH-sensitivity, analgesic activity and anticoagulation property. The drug loading level of LA in low-molecular-weight heparin (LMWH) reached 24.3% (w/w). The LA, self-assembled in LMWH, released faster in an acidic environment than that in neutral or alkaline environments. Analgesic experiments showed that the LMWH-LA had earlier onset time and longer duration than the LA. Compared with LMWH, the LMWH-LA can reduce clotting time more effectively. These results suggest that the LMWH is a good template and has great potential to achieve synergistic effect of LA. In addition, similar macromolecular structure can be used as a new natural polymeric carrier for loading hydrophobic alkaloids., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aggression is frequently comorbid with neuropsychiatric conditions and is a predictor of worse outcomes, yet current pharmacotherapies are insufficient and have debilitating side effects, precluding broad use. Multiple models of aggression across species suggest that the nicotinic acetylcholine receptor (nAChR) agonist nicotine has anti-aggressive (serenic) properties. Here we demonstrate dose-dependent serenic effects of acute nicotine administration in three distinct mouse strains: C57BL/6, BALB/c, and CD1. While acute nicotine administration (0.25mg/kg) modestly reduced solitary homecage locomotion, this could not account for nicotine's serenic effects since social encounters eliminated the hypolocomotor effect, and nicotine did not alter social interaction times. Pretreatment with the homomeric (α7 subunit) nAChR antagonist methyllycaconitine (5mg/kg), but not the heteromeric (β2 or β4 subunit-containing) nAChR antagonist dihydro-β-erythroidine (DHβE, 3mg/kg), blocked the serenic effects of nicotine. By contrast, pretreatment with DHβE blocked the effect of acute nicotine administration on locomotion, uncoupling nicotine's serenic and hypolocomotor effects. Finally, the α7 nAChR partial agonist GTS-21 reduced aggression in C57BL/6 mice. These results support the idea that acute nicotine administration has serenic effects and provide evidence for specificity of this effect distinct from effects on locomotion. Furthermore, pharmacological studies suggest that activation of α7 nAChRs underlies the serenic effects of nicotine. Further studies of nAChRs could enhance understanding of the neurobiology of aggression and may lead to the development of novel, more specific treatments for pathological aggression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Lipid-based nanosystems have great potential for transdermal drug delivery. In this study, nanostructured lipid carriers (NLCs) for short-acting alkaloids lappacontine (LA) and ranaconitine (RAN) isolated from Aconitum sinomontanum (AAS) at 69.47 and 9.16% (w/w) yields, respectively, were prepared to enhance percutaneous permeation. Optimized NLC formulations were evaluated using uniform design experiments. Microstructure and in vitro/in vivo transdermal delivery characteristics of AAS-loaded NLCs and solid lipid nanoparticles (SLNs) were compared. Cellular uptake of fluorescence-labeled nanoparticles was probed using laser scanning confocal microscopy and fluorescence-activated cell sorting. Nanoparticle integrity during transdermal delivery and effects on the skin surface were also investigated., Results: NLC formulations were less cytotoxic than the AAS solution in HaCaT and CCC-ESF cells. Moreover, coumarin-6-labeled NLCs showed biocompatibility with HaCaT and CCC-ESF cells, and their cellular uptake was strongly affected by cholesterol and lipid rafts. Significantly greater cumulative amounts of NLC-associated LA and RAN than SLN-associated alkaloids penetrated the rat skin in vitro. In vivo microdialysis showed higher area under the concentration-time curve (AUC)0-t for AAS-NLC-associated LA and RAN than for AAS-SLN-associated alkaloids., Conclusions: NLC formulations could be good transdermal systems for increasing biocompatibility and decreasing cytotoxicity of AAS. AAS-NLCs showed higher percutaneous permeation than the other preparations. These findings suggest that NLCs could be promising transdermal delivery vehicles for AAS.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

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.

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., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

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.

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Enhanced late inward Na current (INa-L) modulates action potential duration (APD) and plays a key role in the genesis of early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) and triggered activity., Objective: The purpose of this study was to define the influence of selective block of INa-L on EAD- and DAD-mediated triggered ventricular tachycardia (VT) and ventricular fibrillation (VF) in intact hearts using (GS967), a selective and potent (IC50 = 0.13 ± 0.01 μM) blocker of INa-L., Methods: VT/VF were induced either by local aconitine injection (50 μg) in the left ventricular muscle of adult (3-4 months) male rats (N = 21) or by arterial perfusion of 0.1 mM hydrogen peroxide (H2O2) in aged male rats (24-26 months, N = 16). The left ventricular epicardial surface of the isolated-perfused hearts was optically mapped using fluorescent voltage-sensitive dye, and microelectrode recordings of action potentials were made adjacent to the aconitine injection site. The suppressive and preventive effects of GS967 (1 μM) against EAD/DAD-mediated VT/VF were then determined., Results: Aconitine induced VT in all 13 hearts studied. Activation map (N = 6) showed that the VT was initiated by a focal activity arising from the aconitine injection site (cycle length [CL] 84 ± 12) that degenerated to VF (CL 52 ± 8 ms) within a few seconds. VF was maintained by multifocal activity with occasional incomplete reentrant wavefronts. Administration of GS967 suppressed the VT/VF in 10 of 13 hearts (P < .001). Preexposure to GS967 for 15 minutes before aconitine injection prevented initiation of VT/VF in 5 of 8 additional hearts (P < .02). VF reoccurred within 10 minutes on washout of GS967. Microelectrode recordings (N = 7) showed that VT/VF was initiated by EAD- and DAD-mediated triggered activity at CL of 86 ± 14 ms (NS from VT CL) that preceded the VF. GS967 shortened APD, flattened the slope of the dynamic APD restitution curve, and reduced APD dispersion from 42 ± 12 ms to 8 ± 3 ms (P < .01). H2O2 perfusion in eight fibrotic aged hearts promoted EAD-mediated focal VT/VF, which was suppressed by GS967 in five hearts (P < .02)., Conclusion: The selective INa-L blocker GS967 effectively suppresses and prevents aconitine and oxidative stress-induced EADs, DADs, and focal VT/VF. Suppression of EADs, DADs, and reduction of APD dispersion make GS967 a potentially useful antiarrhythmic drug in conditions of enhanced INa-L., (Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Objective: To assess and compare the safety and efficacy of allapinine and quinidine bisulphate in the treatment of patients with persistent atrial fibrillation after cardioversion. Design--Prospective, randomised, open study. Patients--73 consecutive patients (men only, mean age 44 ± 11 years) with persisnent atrial fibrillation and flutter. Interventions--37 patients were randomised to allapinine (ALP) (1.5 mg/kg/d), 36 to quinidine bisulphate (QUIN) (15 mg/kg/d) with subsequent successful pharmacological or electrical cardioversion. Main outcome measures--Recurrence of atrial fibrillation or side effects., Results: In the ALP group 15 of the 37 patients developed atrial fibrillation up to 12 month of follow-up, while in the QUIN group 20 patients developed atrial fibrillation and 5 experienced significant side effects. Relative risk (RR) (ALP vs QUIN) 0.58 (95% CI 0.37-0.91, p < 0.02). The number needed to treat (NNT) was (-3.48) (14.2-1.97 harm). When 5 patients with significant side effects were excluded from the analysis RR was 0.62 (95% CI 0.39-1.0, p = 0.052) and NNT--(-4.1) (122.7-2.1 harm) but power of the study was too low--67%., Conclusion: Allapinine is as effective as quinidine bisulphate in the long term treatment of patients with persistent atrial fibrillation after successful cardioversion but causes significantly less side effects.

Unlabelled: Allapinin, class 1C antiarrhythmic drug, is highly effective in treatment of patients with ventricular premature beats (VPB). The purpose of work was retrospective assessment of efficacy and safety of allapinin in patients with benign ventricular arrhythmias. 73 patients with VPB and no structural heart disease were selected from a database. In short course allapininin in dose of 75-150 mg per os daily decreased the number of VPB for more than 90% in 46,6% of patients. In 34,4% cases tolerable drug side effects were observed. Among patients with VPB burden of 10% and higher allapinin reduced this quantity below the indicated value in 76% of cases with tolerable drug side effects in 38,6% of cases. In long treatment study antiarrhythmic effect of allapinin persisted and no other side effects of the drug were documented., Conclusion: Allapinin is highly effective in treatment of patients with VPB without structural heart disease.

We previously reported that nicotine (NIC)-induced analgesia was elicited in part by activation of the endogenous opioid system. Moreover, it is well known that NIC has physical-dependence liability, but its mechanism is unclear. Therefore, we examined whether physical dependence on NIC was mediated by activation of the endogenous opioid system in ICR mice. We evaluated increased serum corticosterone (SCS) as an indicator of NIC withdrawal, as it is a quantitative indicator of naloxone (opioid receptor antagonist, NLX)-precipitated morphine withdrawal in mice. In this study, NLX precipitated an SCS increase in mice receiving repeated NIC, by a dose-dependent mechanism, and correlated with the dose and number of days of repeated NIC administration. When an opioid receptor antagonist (naltrexone) was concomitantly administered with repeated NIC, the NLX-precipitated SCS increase was not elicited. Concomitant administration of the α7 nicotinic acetylcholine receptor (nAChR) antagonist (methyllycaconitine) with repeated NIC, but not the α4β2 nAChR antagonist (dihydro-β-erythroidine), did not elicit an SCS increase by NLX. Thus, a physical dependence on NIC was in part mediated by the activation of the endogenous opioid system, located downstream of α7 nAChR.

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.

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.

This study was conducted to determine the pharmacokinetic characteristics of bulleyaconitine A (BLA) after oral gavage and intravenous administration of BLA at a single dose of 0.04, 0.12, 0.36 mg/kg (oral) or 0.02 mg/kg (i.v.) in male Sprague-Dawley rats. Plasma concentration profiles were analysed using a non-compartmental pharmacokinetic method. Following i.v. 0.02 mg/kg and oral administration 0.04, 0.12 or 0.36 mg/kg, the geometric mean Cmax values were 19.97, 2.11, 5.11 and 11.47 ng/ml, respectively; the corresponding geometric mean AUC(0-t) values were 10.50, 3.19, 9.59 and 18.10 ng x h/ml, respectively. The median Tmax values were 0.033, 0.167, 0.167 and 0.167 h, respectively. The terminal elimination half-lives (t1/2) were 1.23, 2.48, 1.93 and 2.17h, respectively. The results showed that Cmax and AUC(0-t) increased with increasing doses of BLA. The increase in exposure with increasing dose was lower than expected under conditions of strict proportionality.

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.

Tachyarrhythmias (TA) - dangerous postoperative complications of coronary artery bypass grafting, threatening the lives of patients and found, according to different authors, in 13-40% of cases. VFS - an antiarrhythmic drug that belongs to a class 1C, is effective in the treatment and prevention of a variety of cardiac arrhythmias. The aim of the work was to study the clinical efficacy of VFS CHD patients with a history of tachyarrhythmias during the perioperative period of coronary bypass surgery, as well as its comparison with other antiarrhythmic drug (amiodarone). Clinical efficacy was evaluated in 218 patients with coronary heart disease at baseline with a history of tachyarrhythmia (paroxysmal atrial fibrillation or premature ventricular high grade (IV and V class on classification of Lown B. and Wolf M. in the modification of Ryan M.). Shown that the VFS is more effective than amiodarone, both in paroxysmal atrial fibrillation and ventricular arrhythmias when high gradation.

Simultaneous determination of four aconitine analogues (ACs) (AC; aconitine, HA; hypaconitine, JA; jesaconitine, MA; mesaconitine) in leaves and roots of wild Aconitum plants (Aconitum japonicum THUNBERG, Aconitum okuyamae Nakai) was carried out to elucidate the relation between toxicity to mice and ACs content determind by liquid chromatography tandem mass spectrometry. The total amounts of ACs in leaves, roots, petals and nectaries of Aconitum japonicum collected at Sagae-shi Tashiro were 5.9 μg/g, 928.1 μg/g, 46.1 μg/g, and 69.8 μg/g, respectively. Despite the high contents in nectary, commercial honey contained no ACs. Extract of wild Aconitum japonicum roots which contained ACs (2.69 mg/g) was administered to 5 mice orally at 1.0 g/kg (fresh root equivalent), and 2 mice died. On the other hand, 3 of 5 mice died after being given the standard AC (3.0 mg/kg, p.o.). These findings confirmed good coincidence between toxicity and quantitative values. Mice given extract of Aconitum okuyamae root (100 g/kg, p.o.) without ACs showed no toxic symptoms. Residual ACs in Aconitum leaves were examined after boiling. The remaining percentage of ACs in leaves after 0.5 minutes boiling was 31.6%, and the amount in the boiling water was 54.5%. MA is converted into benzoylmesaconine by hydrolysis (by boiling). Therefore food poisoning caused by Aconitum plants is explained by detection of benzoylmesaconine formed during food preparation.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of bulleyaconitine A (BLA) concentration in rat plasma. An aliquot of 100μL plasma sample was prepared by liquid-liquid extraction with diethyl ether. Chromatographic separation was accomplished on a Phenomenex Luna C(18) column (50mm×2.00mm, 5μm) utilizing a isocratic mobile phase consisting of (A) 0.1% formic acid in methanol and (B) formic acid in water. Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. The optimized fragmentation transitions for MRM were m/z 644.4→m/z 584.3 for BLA and m/z 632.4→m/z 572.3 for mesaconitine (IS). The method was linear over the concentration range of 0.02-20.0ng/mL. The intra and inter-day variance are less than 6.1% and accuracy is within ±9.0%. The validated method was successfully applied to pharmacokinetic study in rat after single oral administration of 40μg/kg of BLA and the oral pharmacokinetic data of BLA have been demonstrated for the first time., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objectives: To study the effects of hypaconitine used alone and combined with liquiritin on calmodulin (CaM) expression and connexin43 (Cx43) phosphorylation on serine368 (Ser368), as well as to investigate the intervention of liquiritin on these hypaconitine-induced effects., Methods: Adult Wistar rats were orally administered hypaconitine (0.23, 0.69, 2.07 mg/kg per day), liquiritin (20 mg/kg per day), or hypaconitine (2.07 mg/kg per day) plus liquiritin (20 mg/kg per day) for seven consecutive days. The mRNA expression levels of CaM and Cx43 in rat myocardial tissue were determined by real-time quantitative PCR. The protein contents of CaM and phosphorylated Cx43 (Ser368) were determined by Western blot., Key Findings: The results indicated that the mRNA and protein expression levels of CaM were significantly decreased by hypaconitine used alone and combined with liquiritin. Although CaM mRNA expression level was inhibited by liquiritin, its protein expression level was upregulated. Meanwhile, although no obvious effect on Cx43 mRNA expression was observed after the drug administration, the phosphorylation level of Cx43 (Ser368) was significantly inhibited. Furthermore, the coadministration of hypaconitine and liquiritin significantly reduced hypaconitine-induced inhibitory action on Cx43 (Ser368) phosphorylation., Conclusions: The study indicated that hypaconitine could inhibit CaM expression and Cx43 (Ser368) phosphorylation, and liquiritin could interfere with this kind of effect by synergistically inhibiting CaM expression and by antagonizing Cx43 (Ser368) dephosphorylation induced by hypaconitine., (© 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.)

Nicotine administration alters various forms of hippocampus-dependent learning and memory. Increasing work has found that the dorsal and ventral hippocampus differentially contribute to multiple behaviors. Thus, the present study examined whether the effects of nicotine in the dorsal and ventral hippocampus have distinct influences on contextual fear learning in male C57BL/6J mice. Direct infusion of nicotine into the dorsal hippocampus resulted in an enhancement of contextual fear learning, whereas nicotine infused into the ventral hippocampus resulted in deficits. Nicotine infusions into the ventral hippocampus did not alter hippocampus-independent cued fear conditioning or time spent in the open arm of the elevated plus maze, a measure of anxiety, suggesting that the effects are due to alterations in contextual learning and not other general processes. Finally, results from using direct infusions of MLA, a low-affinity α7 nicotinic acetylcholine receptor (nAChR) antagonist, in conjunction with systemic nicotine, provide evidence that α7-nAChRs in the ventral hippocampus mediate the detrimental effect of ventral hippocampal nicotine on contextual fear learning. These results suggest that with systemic nicotine administration, competition exists between the dorsal and ventral hippocampus for behavioral control over contextual learning., (Copyright © 2012 Wiley Periodicals, Inc.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

A rapid, specific and sensitive method was developed for the simultaneous determination of eight Aconitum alkaloids: aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine and mesaconine in rat blood by ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS system coupled with an electrospray ionization (ESI) source was operated in a positive mode via multiple-reaction monitoring (MRM). Samples were treated with methanol to remove protein prior to analysis by UPLC-MS/MS. The analytes were separated with a Waters C18 column (1.7 µm, 50 × 2.1 mm) and a gradient elution using acetonitrile and 0.1% formic acid-water as the mobile phases. The linear response range was from 0.125 to 1000 nmol/L for these eight alkaloids and the correlation coefficients (r(2) values) were all higher than 0.997. The method was validated with respect to precision, accuracy, recovery, matrix effect, carryover effect and sample stability, and found to be within the acceptable limits. The developed and validated method was successfully applied to simultaneously determine the eight Aconitum alkaloids in rats blood after intravenous administration of a mixture of AC, MA and HA.