Alkamide Content and Localization in Heliopsis longipes Cypselae, Obtained via Fluorescence and Double-Multiphoton Microscopy

Abstract

The alkamide content and specific tissue localization in the cypselae of Heliopsis longipes were investigated using gas chromatography-electron ionization mass spectrometry (GC-EIMS) and multiphoton fluorescence microscopy (MPFM). GC-EIMS analysis identified two olefinic alkamides: affinin (spilanthol) and N-2-methylbutyl-2E,6Z,8E-decatrienamide. Microscopic analysis revealed that alkamides are localized within the cotyledons, and specifically compartmentalized in lipid bodies, highlighting their spatial organization. The linear unmixing of fluorescence emission fingerprints showed that affinin exhibits autofluorescence at 693 nm, corresponding to the red spectral region. This emission is attributed to the conjugated double bonds in its acyl chain. This study is the first to report on the presence and precise localization of alkamides in the cypselae of H. longipes.

Keywords: GC-EIMS; Heliopsis longipes seeds; affinin; alkamides; multiphoton microscopy; spilanthol.

Figure 1

Structures of H. longipes cypselae (Cy) used for alkamides analyses: pericarp (Pr), seedcoat more cotyledon (Sc+), seedcoat (Sc), and cotyledon (Cot).

Figure 2

GC-EIMS analysis of H. longipes Cot ethanolic extract. (A) Total ion chromatogram of Cot extract; the components retained at *18.785, *21.280, *23.334 min are affinin, N-2-methylbutyl-2E,6Z,8E-decatrienamide, and farnesol acetate, respectively. (B) N-isobutyl-2E,6Z,8E-decatrienamide (affinin) mass spectrum; comparison between the component retained at 18.785 (red) and the authentic compound (blue). (C) N-2-methylbutyl-2E,6Z,8E-decatrienamide mass spectrum; comparison between the component retained at 21.280 (red) and the authentic compound (blue).

Figure 3

Transversal slice of H. longipes cypsela stained with NR (NR) observed using an optical microscope (10×). Cot = cotyledon; Ms = seedcoat; Ex = pericarp.

Figure 4

Lambda stack emission spectra: (A) affinin; (B) affinin + Nile Red; (C) N-isobutyl-decanamide.

Figure 5

Autofluorescence of selected compounds present in a transversal slice of H. longipes cotyledons observed under multiphoton microscope. On the left, triglycerides stained with Nile Red. In the middle, the autofluorescence of alkamides (affinin). On the right, the overlapping of the two previous results.