1. Introduction
Vitamin E is a collective term for a group of naturally occurring lipophilic antioxidants (Kamal-Eldin et al., 1996). In addition, a variety of beneficial effects on human health have been attributed to this substance class, e.g. prevention of arteriosclerosis (Saremi and Arora, 2010), reduction of blood cholesterol levels (Prasad, 2011), and inhibition of tumour promotion (Goh et al., 1994). Likewise, anti-inflammatory and anti-angiogenetic effects were referred to vitamin E (Birringer et al., 2018; Miyazawa et al., 2004). The common structural feature of vitamin E compounds is a 6-chromanol backbone, and hence individual variants (vitamers) are commonly termed tocochromanols (Fig. 1 ) (Kamal-Eldin et al., 1996). One or two methyl groups in addition to the compulsory one on C-8 of the aromatic ring of the 6-chromanol backbone lead to a theoretical variety of four homologue groups, i.e. α-tocochromanols (5,7,8-trimethyl substituted), β-tocochromanols (5,8-dimethyl substituted), γ-tocochromanols (7,8-dimethyl substituted) and δ-tocochromanols (8-methyl substituted) (Kamal-Eldin et al., 1996; Sen and Khanna, 2006; IUPAC-IUB, 1982). Both a methyl and a branched alkyl substituent (side chain) are attached at 2S - and 2R -position, respectively, to the heterocyclic ring moiety. The four tocopherols (α-T, β-T, γ-T and δ-T) contain no double bond (db) in the side chain (Fig. 1a ), whereas the four tocotrienols (α-T3, β-T3, γ-T3 and δ-T3) contain three db (Fig. 1b ) [1,7,8]. The resulting four tocopherols and four tocotrienols are regarded as the eight original vitamin E forms (Kamal-Eldin et al., 1996). The recommended adequate daily intake was suggested to be ~8 mg α-T or higher amounts of other tocochromanols which are ~2-4 times less bioactive (Deutsche Gesellschaft für Ernährung (DGE), 2018).
Recently, tocomonoenols (one db in the side chain, T1, Fig. 1c ) and tocodienols (two db in the side chain, T2) have been discovered in selected plants. The most prominent representative of these rare tocochromanols is 11´-α-T1 which was detected especially in palm oil (Matsumoto et al., 1995; Ng et al, 2004) and at traces in sunflower oil (Hammann et al., 2015). Yamamoto et al. (1999) discovered 12´-α-T1 in eggs of the pacific salmon Oncorhynchus keta being named marine-derived tocopher. A recent study enabled the differentiation of both isomers, i.e. 11´-α-T1 and 12´-α-T1, by gas chromatography (GC) with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) (Müller et al, 2018). Moreover, two α-T2 isomers (i.e. 3´,11´-α-T2 and 7´,11´-α-T2) were successively detected in palm oil (Gee et al., 2016; Müller et al., 2020).
Next to one report on traces of δ-T1 in kiwi fruits (Fiorentino et al, 2009), γ-T1 was found at trace levels in sesame and corn oil (Mariani and Bellan, 1996), green leaves of alligator plant (Kalanchoe daigremontiana ) (Kruk et al., 2011), as well as with most relevant shares in pumpkin seed oil (PSO) (Butinar et al., 2011). Specifically, Butinar et al. (2011) detected γ-T1 with a content of ~120 µg/g in roasted seeds of the Slovenian pumpkin variety Slovenska golica . The position of the db was provisionally allocated to C-11´-position based on data collected for α-T1 (Ng et al, 2004; Puah et al., 2007) and the diagnostic allylic ion at m/z 69 (Müller et al., 2020; Fiorentino et al, 2009). Moreover, they also tentatively indicated the presence of a γ-T2 isomer in the same samples (Butinar et al., 2011). However, none of the minor γ-tocochromanols were isolated and available in milligram-amounts, which would be important in order to study the bioactivity relative to α-T.
The goal of this study was the isolation as well as the structural characterisation and verification of γ-T1 in high purity from PSO. Given previous experience with the isolation of tocochromanols (Müller et al, 2018; Müller et al., 2020; Vetter et al., (2019), countercurrent chromatography (CCC) appeared to be well-suited for this purpose. Furthermore, CCC fractionation followed by gas chromatography with mass spectrometry (GC/MS) analysis of the individual fractions enabled the detection of more minor lipid components than without this step (Schröder and Vetter, 2012). Hence, this CCC fractionation and GC/MS screening strategy was adopted in order to describe additional minor tocochromanols in PSO including the verification of γ-T2.