ABSTRACT
To identify the structures of flavonoid glycosides in bee pollen collected from rapeseed plants (Brassica napus L.), we utilised an approach that combined liquid chromatography-diode array detector-electrospray ionization-mass spectrometry (LC-DAD-ESI-MS) and nuclear magnetic resonance (NMR) technology with a step-wise separation strategy. We identified four constituents of high purity in rape bee pollen samples: (1) quercetin-3-O-ß-D-glucosyl-(2âl)-ß-glucoside, (2) kaempferol-3, 4'-di-O-ß-D-glucoside, (3) 5, 7, 4'-trihydroxy-3'-methoxyflavone-3-O-ß-D-sophoroside and (4) kaempferol-3-O-ß-D-glucosyl-(2âl)-ß-D-glucoside. This study will also provide useful reference standards for qualification and quantification of four flavonoid glycosides in natural products.
Subject(s)
Brassica napus/chemistry , Glycosides/chemistry , Pollen/chemistry , Animals , Bees , Cell Line/drug effects , Chemical Fractionation , Chromatography, Liquid , Drug Evaluation, Preclinical/methods , Flavonoids/chemistry , Glycosides/isolation & purification , Glycosides/pharmacology , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy/methods , Protective Agents/chemistry , Protective Agents/pharmacology , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
Sudden unexplained death in epilepsy (SUDEP) is the most common cause of premature mortality in epilepsy and was linked to mutations in ion channels; however, genes within the channel protein interactome might also represent pathogenic candidates. Here we show that mice with partial deficiency of Sentrin/SUMO-specific protease 2 (SENP2) develop spontaneous seizures and sudden death. SENP2 is highly enriched in the hippocampus, often the focus of epileptic seizures. SENP2 deficiency results in hyper-SUMOylation of multiple potassium channels known to regulate neuronal excitability. We demonstrate that the depolarizing M-current conducted by Kv7 channel is significantly diminished in SENP2-deficient hippocampal CA3 neurons, primarily responsible for neuronal hyperexcitability. Following seizures, SENP2-deficient mice develop atrioventricular conduction blocks and cardiac asystole. Both seizures and cardiac conduction blocks can be prevented by retigabine, a Kv7 channel opener. Thus, we uncover a disease-causing role for hyper-SUMOylation in the nervous system and establish an animal model for SUDEP.
Subject(s)
Cysteine Endopeptidases/metabolism , Death, Sudden , KCNQ Potassium Channels/metabolism , Seizures/genetics , Seizures/physiopathology , Acoustic Stimulation , Action Potentials/drug effects , Action Potentials/genetics , Analysis of Variance , Animals , Animals, Newborn , Cells, Cultured , Cysteine Endopeptidases/genetics , Disease Models, Animal , Electric Stimulation , Electrocardiography , Electroencephalography , Hippocampus/cytology , Immunoprecipitation , In Vitro Techniques , Mice , Mice, Transgenic , Neurons/drug effects , Neurons/physiology , Potassium Channel Blockers/pharmacology , Seizures/pathologyABSTRACT
A high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method was developed for simultaneous determination of melittin and apamin in crude bee venom lyophilized powder (CBVLP) as the traditional Chinese medicine possessing specific biological activity. Melittin and apamin were extracted with pure water from CBVLP samples followed by HPLC-DAD-MS/MS analysis. The method was validated to demonstrate its selectivity, linearity, limit of quantification (LOQ), intraday precision, interday precision, accuracy, recovery, matrix effect, and stability. The assay was linear over the concentration ranges of 1-100 and 0.2-25 microg/ml with limit of quantifications (LOQs) of 1.0 and 0.3 microg/ml for melittin and apamin, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 2.2% to 11.4% for intraday repeatability and from 3.2% to 13.1% for interday intermediary precision. The concentrations of endogenous melittin and apamin in CBVLP samples ranged from 46% to 53% and from 2.2% to 3.7% of dry weight, respectively. This rapid, simple, precise, and sensitive method allowed the simultaneous determination of melittin and apamin to evaluate authenticity and quality of CBVLP samples.