Pro-convulsant alkenyl amides from Piper nigrum fruits identified with a zebrafish larvae locomotor activity model

A petroleum ether extract of Piper nigrum (Piperaceae) fruits was previously found to potentiate GABA induced chloride currents in a two-microelectrode voltage clamp assay using Xenopus laevis oocytes transiently expressing human GABA_A receptors, and piperine was identified as a positive GABA_A receptor modulator interacting at a benzodiazepine-independent allosteric binding-site [1]. When testing the same extract in a zebrafish-larvae locomotor activity assay [2], it significantly increased larval locomotion provoked by the GABA_A receptor antagonist pentylenetetrazol (PTZ). Pro-convulsant activity in the extract was tracked by HPLC-based activity profiling. Increased larval locomotion was observed in two distinct micro-fractions, while the fraction containing piperine (1) decreased PTZ provoked locomotion. The constituents responsible for the pro-convulsant effect of the extract were purified, and their structures established by 1D and 2D NMR and MS. Geometry of double bonds was resolved by 2D JRES spectra. The pro-convulsant amides were identified as 2E,4E-alkenyl amides with a common structure feature as shown in 2. In the zebrafish larval locomotor assay, the alkenyl amides increased PTZ provoked larval convulsions in a concentration-dependent manner (120 to 130%, < 6 mM) when compared to the PTZ-only group.

[1]. Zaugg J, Barburin I, Strommer B, Kim H-J, Hering S, Hambueger M. J. Nat. Prod 2010; 73: 185 – 191.

[2].Moradi-Afrapoli F, Ebrahimi SN, Smiesko M, Hamburger M. J Nat Prod 2017, advance online publication 9 May 2017; doi:10.1021/acs.jnatprod.7b00081.