ABSTRACT
The ability to rapidly identify active compounds in a complex mixture (e.g., natural products extract) is still one of the major problems in natural products screening programs. An elegant way to overcome this problem is to separate the complex mixture by gradient liquid chromatography followed by online biochemical detection parallel with chemical characterization, referred to as high-resolution screening (HRS). To find and identify phosphodiesterase (PDE) inhibitors in natural products extracts using the HRS technology, the authors developed a continuous-flow PDE enzymatic assay. The suitability of the continuous-flow PDE enzymatic assay for natural products screening was demonstrated. After optimization of the continuous-flow PDE assay, the limit of detection for 3-isobutyl-1-methyl-xanthine (IBMX) was 1 muM, with a dynamic range from 1 to 100 muM IBMX. The applicability of the HRS technology for the detection of PDE inhibitors in natural products extracts was demonstrated by the analysis of a plant extract spiked with 2 naturally occurring PDE inhibitors. The plant extract was analyzed with 2 assay lines in parallel, enabling background fluorescence correction of the sample. The simultaneous quantification of the active compounds using evaporative light-scattering detection allowed the estimation of the IC(50) value of the active compounds directly in the crude extract.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Chromatography, Liquid/methods , Drug Evaluation, Preclinical/methods , Phosphodiesterase Inhibitors/analysis , Plant Extracts/chemistry , Plant Extracts/pharmacology , 1-Methyl-3-isobutylxanthine/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Adenosine Monophosphate/metabolism , Combinatorial Chemistry Techniques , Guanosine Monophosphate/metabolism , Light , Mass Spectrometry/methods , Phosphodiesterase Inhibitors/pharmacology , Scattering, RadiationABSTRACT
Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (gamma-Glu-Cys)(n)()-Gly, with n = 2-11, induced in plants upon exposure to excessive amounts of heavy metals and some metalloids, such as arsenic. Two types of PC analyses are currently used, i.e., acid extraction and separation on HPLC with either precolumn derivatization (pH 8.2) with monobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's reagent [5, 5'-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods were satisfactory for analysis of Cd-induced PCs, formation of (RS)(3)-As complexes during extraction of As-induced PCs rendered the DTNB method useless. This paper shows that precolumn derivatization with mBBr, during which the (RS)(3)-As complexes are disrupted, provides a qualitative and quantitative analysis of both Cd- and As-induced PCs. In addition, derivatization efficiencies of both methods for the oligomers with n = 2-4 (PC(2)(-)(4)) are compared. Derivatization efficiency decreased from 71.8% and 81.4% for mBBr and DTNB derivatization, respectively, for PC(2) to 27.4% and 50.2% for PC(4). This decrease is most likely due to steric hindrance. Correction of measured thiol concentration is therefore advised for better quantification of PC concentrations in plant material.
Subject(s)
Arsenates/pharmacology , Bridged Bicyclo Compounds/chemistry , Cadmium/pharmacology , Dithionitrobenzoic Acid/chemistry , Metalloproteins/chemistry , Plant Proteins/chemistry , Plants/drug effects , Glutathione , Metalloproteins/biosynthesis , Phytochelatins , Plant Proteins/biosynthesis , Plants/metabolismABSTRACT
A 20-year-old man presented with signs of thornapple intoxication: restlessness, disorientation, hallucinations, euphoria, and furthermore dry and red skin and symmetrical dilation of the pupils. Thornapple intoxication mimics atropine intoxication. Thorn apples (Datura stramonium) are seemingly becoming popular as a hallucinogenic drug. When the symptoms are mild gastric lavage leaving active charcoal in the stomach afterwards will suffice. In case of severe symptoms treatment with physostigmine is indicated.
