High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae)

Phytomedicine. 2019 Feb 15;54:259-264. doi: 10.1016/j.phymed.2018.10.030. Epub 2018 Oct 26.

Abstract

Background: Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity.

Purpose: To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesterase ELISAs and zebrafish epilepsy models.

Research methods and procedure: In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABAA antagonist pentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior.

Results: The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62-1.75 µg/ml) and bergamottin (2-10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10-16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A.

Conclusion: Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.

Keywords: AChE; ASD; BChE; CNS; Coumarins; Counter-current chromatography; DAD; DMSO; ESI-TOF-MS; Epilepsy; HPCCC; HPLC; MAO; MTC; NMR; Natural products; Neurodegenerative disorders; PTZ; RP; VHC; Zebrafish; acetylcholinesterase; anti-seizure drug; butyrylcholinesterase; central nervous system; dimethyl sulfoxide; diode array detection; electrospray ionization time-of-flight mass spectrometry; high-performance counter-current chromatography; high-performance liquid chromatography; maximum-tolerated concentration; monoamine oxidase; nuclear magnetic resonance; pentylenetetrazol; reverse phase; vehicle.

MeSH terms

  • Animals
  • Anticonvulsants / therapeutic use
  • Apiaceae / chemistry*
  • Cholinesterase Inhibitors / therapeutic use
  • Coumarins / chemistry
  • Coumarins / isolation & purification*
  • Countercurrent Distribution / methods*
  • Disease Models, Animal
  • Enzyme-Linked Immunosorbent Assay
  • Epilepsy / drug therapy
  • Furocoumarins / chemistry
  • Furocoumarins / isolation & purification*
  • Plant Extracts / pharmacology
  • Plant Extracts / therapeutic use
  • Plants, Medicinal / chemistry
  • Zebrafish

Substances

  • Anticonvulsants
  • Cholinesterase Inhibitors
  • Coumarins
  • Furocoumarins
  • Plant Extracts