Abstract
Ethnopharmacological relevanceEthnopharmacological 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 studyA 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 methodsThe 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. ResultsThe 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. ConclusionsThis 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.
Highlights
Epilepsy is one of the oldest and most critical central nervous system (CNS) disorders that affects more than 50 million people worldwide (Megiddo et al, 2016; WHO, 2019)
The root methanolic extract (RME) demonstrated a significant antiseizure activity with a reduction of 80% of PTZinduced locomotor activity (p < 0.01 at 300 μg/ml; Fig. 1A, C), while the ethyl acetate root extract was toxic at this concentration but exhibited a 45% reduction in seizures at 100 μg/ml (p < 0.05) (Fig. 1A and B)
The root decoction extract (RDE) was less potent than the RME and reduced seizures up to 45% (300 μg/ml, p < 0.05) (Fig. 1A, D), which is probably due to the presence of inactive components such as sugars
Summary
Epilepsy is one of the oldest and most critical central nervous system (CNS) disorders that affects more than 50 million people worldwide (Megiddo et al, 2016; WHO, 2019). The intense manifestations of epilepsy in the form of seizures have been described in numerous ancient texts dating to 2000 BC. Vivid descriptions of epileptic seizures have been found in inscriptions from Mesopotamia, ancient Egypt, the Babylonian and Roman empires, and ancient Greece (Magiorkinis et al, 2010). Epilepsy was considered to have a divine emanation. Several treatments for the prevention of seizures were proposed, such as shaving the head, walking, exercising, vomiting, use of diuretics, phlebotomy, and even sneezing (Magiorkinis et al, 2010)
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