Efficient separation of N-Alkylamides from Piper longum leL. using off-line two-dimensional coupled with gradient high-speed counter-current chromatography

Piper longum L. (Piperaceae) is famous for flavoring food and treatment purposes. N-Alkylamides (NAAs) are the most common secondary metabolites found in Piper species with broad spectrum of biological properties. Because of the similar structures and lower polarities, it is difficult to separate NAAs by traditional chromatographic methods. Herein, an efficient off-line two-dimensional coupled with gradient high-speed counter-current chromatography (HSCCC) method is reported for preparative separation of N-alkylamides from P. longum. The HSCCC solvent system with n-hexane/ethyl acetate/methanol/water (5:4:5:4, v/v) was used for the first dimensional separation. Two pure compounds i.e. piperlonguminine (1) and piperine (2) were successfully separated, coupled with an enriched trace ingredient. Furthermore, the enriched ingredient was used for the second dimensional separation with a gradient HSCCC mode by adjustment of the mobile phase with more enhanced elution ability. Eight minor compounds were successfully separated including retrofractamide A (3), retrofractamide C (4), dehydropipernonaline (5), pipernonaline (6), piperundecalidine (7), guineesine (8), brachystamide B (9), and (2E,4E,12Z)-N-isobutyl-2,4,12- octadecatrienamide (10). The structures of the isolated pure compounds were characterized by ESI-MS, H-1, and C-13 NMR spectral analysis.