Countercurrent chromatography isolation of 11′-γ-tocomonoenol from pumpkin seed oil with detection of novel minor tocochromanols

Abstract

A non-refined, organic pumpkin seed oil (PSO) was chosen for the isolation and structure verification of the rare vitamin E compound γ-tocomonoenol (γ-T1). Initial measurements indicated the presence of ~0.4 mg γ-T1 per 100 g PSO. Saponification of ~2 L of PSO, followed by repeated countercurrent chromatography (CCC) with the solvent system n-hexane/benzotrifluoride/acetonitrile (v/v/v) and silica gel column chromatography enabled the isolation of 6.8 mg γ-T1 with a purity of 96.0% according to gas chromatography with mass spectrometry (GC/MS) analysis. Structural analysis by 1H nuclear magnetic resonance (NMR) spectroscopy and GC/MS of the γ-T1 isolate confirmed the presence of a double bond in C-11′-position (11′-γ-tocomonoenol). In addition, CCC fractionation enabled the detection of 18 different tocochromanols, many of which were reported for the first time in PSO. This unmatched variety included among others α-/γ-tocopherol, α-/γ-tocomonoenol, two α- and two γ-tocodienol isomers, α-/γ-tocotrienol as well as the rare 11′-β-tocomonoenol (β-T1) and δ-T1. Three uncommon tocochromanols were also detected whose origins and structure remained unclear.