Hypaconitine Research Articles

An online coupled cell membrane chromatography with LC/MS method for screening compounds from Aconitum carmichaeli Debx. acting on VEGFR-2

An online analytical method coupling high expression vascular endothelial growth factor receptor (VEGFR) cell membrane chromatography (VEGFR-CMC) with high performance liquid chromatography mass spectrometry (LC/MS) for screening and identification of active component from traditional Chinese herb Aconitum carmichaeli Debx. acting on VEGFR-2 was established. Through a 10-port column switcher, factions separated by VEGFR-CMC column (first dimension) were transferred and were adsorbed on an enrichment column. Then, these fractions were sent into LC/MS system (second dimension) immediately and directly for separation and preliminary identification, respectively. Sunitinib malate (SN) was used as positive control, while nifedipine (NF), dexamethasone acetate (DX), methoxyamine hydrochloride (MT) and atenolol (AT) as negative controls. The specification of this VEGFR-CMC-online-LC/MS method was validated by competitive displacement test. As a result, mesaconitine (MSC), aconitine (AC), and hypaconitine (HPC) were identified as the active constituents acting on VEGFR-2. The in vitro inhibition activity of starting extract of Aconitum carmichaeli Debx., MSC, AC, and HPC on HEK293/VEGFR cell viability by MTT test, separately. The in vitro inhibition activity of MSC, AC, and HPC on vascular endothelial growth factor (VEGF) secretion of HEK293/VEGFR cell was tested by VEGF-ELISA assay. The screening results given by the system offered additional exemplification supporting this online coupling method and gave new evidence to the development of anti-tumor drug from natural products.

Development and Validation of a High-Performance Liquid Chromatography—Tandem Mass Spectrometry Method for the Rapid Simultaneous Quantification of Aconitine, Mesaconitine, and Hypaconitine in Rat Plasma after Oral Administration of Sini Decoction

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

Simultaneous quantitation of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine in human plasma by liquid chromatography–tandem mass spectrometry and pharmacokinetics evaluation of “SHEN-FU” injectable powder

A rapid, specific and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids, i.e. aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in human plasma collected from 18 healthy volunteers after intravenous drop infusion of “SHEN-FU” injectable powder in three different dosages. Lappaconitine was selected as the internal standard (IS). LC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. The transitions of the Aconitum alkaloids executed as following: m/ z 646.3 → 586.0 for AC; m/ z 632.4 → 573.1 for MA; m/ z 616.2 → 556.1 for HA; m/ z 604.2 → 104.8 for BAC; m/ z 590.1 → 104.8 for BMA; m/ z 574.1 → 104.8 for BHA; m/ z 585.2 → 161.8 for IS. Sample preparation was performed with solid-phase extraction (SPE) on a 1 mL HLB cartridge prior to analysis. The separation was applied on a Waters C 18 column (1.7 μm, 2.1 mm × 100 mm) and a gradient elution of methanol and 0.1% formic acid–water was used as mobile phase. The retention time was less than 4.5 min. The concentrations ranged from 0.1 to 1000 ng/mL for all six Aconitum alkaloids and showed a good linearity with the correlation coefficient ( r 2) >0.995. The validated method was employed to simultaneous quantitation and successfully used for the first time for the pharmacokinetic evaluation of the six Aconitum alkaloids after intravenous drop administration of “SHEN-FU” injectable powder in phase I clinical trial.

Facile separation and determination of Aconitine alkaloids in traditional Chinese medicines by CE with tris(2,2'‐bipyridyl) ruthenium(II)‐based electrochemiluminescence detection

A facile CE method coupled with tris(2,2'-bipyridyl) ruthenium(II)-based electrochemiluminescence [Ru(bpy)(3) (2+)] detection was developed for simultaneous determination of Aconitum alkaloids, i.e., hypaconitine (HA), aconitine (AC), and mesaconitine (MA) in baseline separation. The optimal separation of these Aconitum alkaloids was achieved in a fused-silica capillary column (50 cm x 25 microm id) with 30 mM phosphate solution (pH 8.40) as running buffer at 12 kV applied voltage. The three alkaloids can be determined within 10 min by a single run. The calibration curves showed a linear range from 2.0 x 10(-7) to 2.0 x 10(-5) M for HA, 3.4 x 10(-7) to 1.7 x 10(-5) M for AC, and 3.8 x 10(-7) to 1.9 x 10(-5) M for MA. The RSDs for all analytes were below 3.01%. Good linear relationships were found with correlation coefficients for all analytes exceeding 0.993. The detection limits were 2.0 x 10(-8) M for HA, 1.7 x 10(-7) M for AC, and 1.9 x 10(-7) M for MA under optimal conditions. This method was successfully applied to determine the three alkaloids in Aconitum plants.

Separation and determination of aconitine alkaloids in traditional Chinese herbs by nonaqueous capillary electrophoresis.

An easy, rapid, and simple nonaqueous capillary electrophoresis (NACE) method was developed for the identification and determination of three aconitine alkaloids, hypaconitine (HN), aconitine (AN), and mesaconitine (MN) within 6 min. The most suitable running buffer was composed of 60 mM ammonium acetate, 0.5% acetic acid, and 15% acetonitrile (ACN) in methanol with a fused-silica capillary column (50 cm x 75 microm ID). In the concentration range 12.5-1000 mg/L the calibration curves reveal linear relationships between the peak area for each analyte and its concentration (correlation coefficients: 0.9997 for HN, 0.9999 for AN, and 0.9995 for MN). The relative standard deviations of the migration time and peak area of the three alkaloids were 0.13, 0.57, 0.33 and 2.87, 1.06, 3.49%, respectively. The method was successfully applied to determine the three alkaloids in two commonly used traditional Chinese herbal medicines, the recoveries of the three constituents ranging between 94.7-101.9% for HN, 98.3-102.3% for AN, and 98.1-104.6% for MN.

Hypaconitine, the Dominant Constituent Responsible for the Neuromuscular Blocking Action of the Japanese-Sino Medicine “Bushi” (Aconite Root)

The neuromuscular blocking actions of several constituents extracted from Japanese-smo medicine, aconite, were compared in mouse phrenic nerve-diaphragm muscle preparations. Hypaconitine (HAT) was more potent than aconitme (ATN), mesaconitme (MAT) and deoxyaconitine. Lipohypaconitine, coryneine and lipodeoxyaconitine were less effective. Lipoaconitine, benzoyl-mesaconine, higenamine, kobusine and chasmanine were not effective. The blockades by HAT, ATN and MAT were not recovered by neostigmine. The mechanisms of blockade were similar to that of aconite crude extract. These results suggest that aconite action is dependent on HAT, a main constituent.

Pharmacological actions of aconitine alkaloids.

Aconitine (AC), mesaconitine (MA), hypaconitine (HA) (50 μg/kg i.v.), benzoylaconine (BA), benzoylmesaconine (BM) and benzoylhypaconine (BH) (5 mg/kg i.v.) produced a weak temporary hypotension in rats which was blocked to some extent by atropine. In the isolated guinea pig right atria, MA and HA (3×10-8 g/ml) mediated a positive inotropic action and a positive chronotropic action. At the higher concentration of 10-7 g/ml, AC, MA and HA exhibited the above actions followed by inhibition of contractions and disorder of the beating rate. In the isolated guinea pig ileum, AC, MA and HA (>10-6 g/ml) caused a contraction which was completely blocked by atropine. In the isolated guinea pig vas deferens, AC, MA and HA (>10-5 g/ml) elicited a contraction which was completely obliterated by phentolamine. In the isolated guinea pig hypogastric nerve-vas deferens, the isolated rabbit mesenteric nerve-jejunum and the isolated rat phrenic nerve-diaphragm, responses induced by the electrical stimulation of the corresponding nerves were inhibited by AC, MA and HA (10-6-10-5 g/ml). Some of the mechanisms of the above actions induced by these alkaloids are discussed. These alkaloids more or less potentiated the hexobarbital anesthesia, inhibited the revolution of the wheel cage, and reduced the rectal temperature in mice, indicating that they have a depressant activity on the central nervous system.