@article{3020734,
    title = "Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles",
    author = "Brillatz, T. and Jacmin, M. and Vougogiannopoulou, K. and Petrakis, E.A. and Kalpoutzakis, E. and Houriet, J. and Pellissier, L. and Rutz, A. and Marcourt, L. and Queiroz, E.F. and Crawford, A.D. and Skaltsounis, A.-L. and Wolfender, J.-L.",
    journal = "Journal of Ethnopharmacology",
    year = "2020",
    volume = "259",
    publisher = "Elsevier Ireland Ltd",
    issn = "0378-8741",
    doi = "10.1016/j.jep.2020.112954",
    keywords = "21 hydroxymethyl 24 [(o beta dextro glucopyranosyl(1-4)beta dextro fucopyranosyl)oxy] 3beta,23 dihydroxyspirosta 5,25 (27)diene 1beta yl o (alpha levo rhamnopy ranosyl)(1-2) o [beta dextro xylopyranosyl(1-3)]alpha levo arabinopyranoside;  4 hydroxy 5 [(beta dextro glucopyranosyloxy)methyl]dihydrofuran 2(3h) one;  aglycone;  alkaloid;  anticonvulsive agent;  bufadienolide;  deglucohellebrin;  ecdysterone;  furostanol;  hellebrigenin;  hellebrin;  helleodoraside;  plant extract;  plant glycoside;  saponin derivative;  topiramate;  unclassified drug;  anticonvulsive agent;  methanol;  pentetrazole;  phytochemical;  plant extract;  solvent, animal experiment;  animal model;  anticonvulsant activity;  Article;  biological activity;  chemical composition;  controlled study;  drug identification;  drug isolation;  drug screening;  drug synthesis;  embryo;  epilepsy;  fish model;  fractionation;  Helleborus;  Helleborus odorus subsp. cyclophyllus;  locomotion;  medicinal plant;  metabolic fingerprinting;  nonhuman;  nuclear magnetic resonance spectroscopy;  pentylenetetrazole-induced seizure;  plant root;  solvent extraction;  tandem mass spectrometry;  ultra performance liquid chromatography;  zebra fish;  animal;  chemistry;  comparative study;  disease model;  dose response;  drug effect;  high performance liquid chromatography;  isolation and purification;  metabolism;  metabolome;  metabolomics;  pathophysiology;  seizure, Animals;  Anticonvulsants;  Chromatography, High Pressure Liquid;  Disease Models, Animal;  Dose-Response Relationship, Drug;  Helleborus;  Locomotion;  Metabolome;  Metabolomics;  Methanol;  Pentylenetetrazole;  Phytochemicals;  Plant Extracts;  Plant Roots;  Seizures;  Solvents;  Tandem Mass Spectrometry;  Zebrafish",
    abstract = "Ethnopharmacological relevance: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. Aim of the study: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. Materials and methods: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. Results: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 μg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 μM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 μM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35–37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. Conclusions: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction. © 2020 The Authors"
}