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.