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.

Free and Esterified Tocopherols, Tocotrienols and Other Extractable and Non-Extractable Tocochromanol-Related Molecules: Compendium of Knowledge, Future Perspectives and Recommendations for Chromatographic Techniques, Tools, and Approaches Used for Tocochromanol Determination

“…T1s and T2s are less common in nature and can be found in lower concentration than Ts and T3s [ 14 , 16 ]. 11′-α-T1 was first isolated and purified from palm and rice bran oils in 1995 [ 69 ].…”

“…Apart from this, in studies on T1s in plants with emphasis on thorough structure identification, only 11′-T1s have been structurally confirmed, and in cases where precise identification was not possible, detected T1s were assumed to be 11′-T1s ( Figure 8 ). T1s were identified in palm oil (11′-α-T1) [ 72 ], T3-rich fraction of palm oil (11′-α-T1) [ 52 ], countercurrent chromatography (CCC) fraction of palm oil (11′-α-, 11′-β-, and 11′-γ-T1) [ 73 ], in pumpkin seed oil and its CCC fraction (11′-α-, 11′-β-, 11′-γ-, and 11′-δ-T1) [ 16 ], leaves of Kalanchoe daigremontiana and Phaseolus coccineus (11′-β-, 11′-γ-, and 11′-δ-T1) [ 74 ], kiwi ( Actinidia chinensis ) peel and pulp (11′-δ-T1) [ 75 ], in cyanobacteria and microalgae (11′-α-T1 and MDT) [ 15 ] ( Figure 9 ). By analogy with T3s, T2s theoretically can have two unsaturated carbons at positions 3′ and 7′, 3′ and 11′ or 7′ and 11′ ( Figure 6 ).…”

“…By analogy with T3s, T2s theoretically can have two unsaturated carbons at positions 3′ and 7′, 3′ and 11′ or 7′ and 11′ ( Figure 6 ). α-T2 was identified in 1996 in the palm oil [ 76 ], and later, T2s were identified in the T3-rich fraction of palm oil (7′,11′-α-T2) [ 52 ], in CCC fraction of palm oil (3′,11′-α-T2 and 7′,11′-α-T2) [ 73 ], and in pumpkin seed oil and its CCC fraction (3′,11′-γ-T2, 7′,11′-γ-T2, 3′,11′-α-T2 and 7′,11′-α-T2) [ 16 ]. Lastly, it was proposed to call 3′,11′ T2s, 7′,11′ T2s and 11′-T1s ‘tocoflexols’ due to an increased side-chain flexibility in comparison with T3s [ 77 , 78 ] ( Figure 10 ).…”

“…Isolation of a novel 11′-δ-T1 homologue from kiwi fruits has been performed by extraction with n -hexane; subsequently, column chromatography (silica—petroleum ether/acetone mixture and NH 2 -silica—petroleum ether/ethyl acetate mixture) and RPLC preparative chromatography (RP-8 column—methanol:acetonitrile:H 2 O mixture) were used to fractionate the extract [ 75 ]. Counter-current chromatography (CCC) is the latest trend in fraction enrichment with T1s and T2s [ 16 , 73 ]. Higher sample amounts can be loaded into the system due to the absence of the stationary phase; this advantage makes CCC beneficial for the fractionation or isolation of compounds with low content in the plant material, including tocochromanols.…”

“…The challenge with the determination of esterified tocochromanols is not only associated with the procedure of tocol determination but also with lack of standards. Similarly, commercial standards for some other tocochromanols are not available, e.g., the well-known plastochromanol-8 (PC-8) [ 12 , 13 ], tocodienols (T2s) and tocomonoenols (T1s) [ 14 , 15 , 16 , 17 ]. Research on the latter two has been gaining momentum in the past decade, and new commercial standards of tocochromanols or their metabolites are released to the market every year.…”