New neryl esters from Helichrysum italicum (Roth) G. Don (Asteraceae) essential oil

Article abstract of DOI:10.1080/14786419.2020.1839462

Helichrysum italicum (immortelle) is a dwarf aromatic shrub native to the Mediterranean region. The typical subspecies (italicum) produces an essential oil rich in neryl acetate and characteristic β-diketones, italidiones, highly valued in the perfume industry. As esters are an important group of aroma-active volatiles, herein the composition of the ester fraction of this immortelle chemotype essential oil was studied in detail. Chromatographic separation of Corsican immortelle essential oil enabled the discovery of numerous potentially olfactory-interesting esters of nerol and/or angelic acid, undetectable by direct GC-MS analyses of the unfractioned oil. Four esters of nerol and medium-chain branched fatty acids represent new natural products, while several other esters have a rather restricted occurrence in the Plant Kingdom.

Full text of DOI:10.1080/14786419.2020.1839462

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
New neryl esters from Helichrysum italicum (Roth)
G. Don (Asteraceae) essential oil
Marija S. Genčić , Jelena M. Aksić , Milena Z. Živković Stošić , Miljana R.
Đorđević , Marko Z. Mladenović & Niko S. Radulović
To cite this article: Marija S. Genčić , Jelena M. Aksić , Milena Z. Živković Stošić , Miljana
R. Đorđević , Marko Z. Mladenović & Niko S. Radulović (2020): New neryl esters from
Helichrysumꢀitalicum (Roth) G. Don (Asteraceae) essential oil, Natural Product Research, DOI:
10.1080/14786419.2020.1839462
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1839462
New neryl esters from Helichrysum italicum (Roth)
G. Don (Asteraceae) essential oil
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Marija S. Gencic, Jelena M. Aksic, Milena Z. Zivkovic Stosic, Miljana R. Dordevic,
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Marko Z. Mladenovic and Niko S. Radulovic
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Department of Chemistry, Faculty of Sciences and Mathematics, University of Nis, Nis, Serbia
ABSTRACT
ARTICLE HISTORY
Received 18 June 2020
Accepted 15 October 2020
Helichrysum italicum (immortelle) is a dwarf aromatic shrub native
to the Mediterranean region. The typical subspecies (italicum)
produces an essential oil rich in neryl acetate and characteristic
b-diketones, italidiones, highly valued in the perfume industry.
As esters are an important group of aroma-active volatiles, herein
the composition of the ester fraction of this immortelle
chemotype essential oil was studied in detail. Chromatographic
separation of Corsican immortelle essential oil enabled the discov-
ery of numerous potentially olfactory-interesting esters of nerol
and/or angelic acid, undetectable by direct GC-MS analyses of the
unfractioned oil. Four esters of nerol and medium-chain branched
fatty acids represent new natural products, while several other
esters have a rather restricted occurrence in the Plant Kingdom.
KEYWORDS
Helichrysum italicum;
essential oil; neryl esters;
iso- and anteiso-fatty acid
esters; medium-chain fatty
acids; Corsica
1. Introduction
Helichrysum italicum (Roth) G. Don (Asteraceae) is a small Mediterranean aromatic
shrub with golden yellow flowers that do not wither, and their lively color and signa-
ture curry-like scent prevail even when the flowers are collected and dried. This is why
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it is also called immortelle or curry plant (Sarkic and Stappen 2018; Andreani et al.
2019; Oliva et al. 2020). This taxon shows remarkable adaptability in terms of growing
conditions, thriving both on the coast and in the mountains, that translates into
a heterogeneous morphology and phytochemical polymorphism. There are several
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CONTACT Niko S. Radulovic
nikoradulovic@yahoo.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1839462.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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M. S. GENČIĆ ET AL.
Figure 1. Neryl (series A) and angeloyl esters (series B) standards synthesis by the Steglich
method (a), the synthetic route to 4-methylhexanoic acid (b), and the structures of the
newly found neryl esters (c). The designations of ester synthetics samples correspond to
the designations given in Table S1.
chemotypes of H. italicum characterized by distinct essential-oil composition, and
besides the natural variability, the change in the oil composition may occur due to
geographical origin, plant growth stage and various environmental factors (soil type,
altitude, sun exposure, etc.; Appendino et al. 2015; Ornano et al. 2015; Tundis et al.
2005; Venditti et al. 2016). The typical subspecies (italicum) from Corsica produces an
essential oil rich in the acetate (ꢀ30%) and propionate (ꢀ5%) of nerol, and italidiones
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characteristic b-diketones of this species (ꢀ10%; Cavar Zeljkovic et al. 2015; Andreani
et al. 2019). This Corsican oil is the most appreciated immortelle chemotype by per-
fumers because of its spicy saffron character, well complemented with curry, nutty,
and celery facets (Hellivan 2009). A recent GC-O study of the Corsican oil revealed the
identity of several key odorants (i.e. 4,6-dimethyloctane-3,5-dione, 1,8-cineole, and
nerol). However, it was found that many other, still unidentified constituents present
in low amounts also participate in the global odor of this essential oil (Andreani
et al. 2019).
Esters of short- and medium-chain fatty acids and acyclic monoterpene alcohols are
extremely important both as fragrances and flavor substances. For example, neryl acet-
ate has a very sweet, refreshing, fruity-floral scent and it is used in perfumery for blos-
som compositions (e.g. orange blossom or jasmine; Bauer et al. 2008). Also, it has
been established that with the increase of the number of C-atoms in the acid part of
the ester the fruity note starts to dominate over the flower one, so neryl hexanoate
has a refreshing fruity-herbaceous odor (Arctander 1969). Although angelates do not
represent standard items on a perfumer’s shelf, mostly due to the cost of their produc-
tion, some of them have quite complex fragrances – e.g. butyl angelate has a warm-

NATURAL PRODUCT RESEARCH
3
herbaceous, winey odor with a distinct caramel undertone, while hexyl angelate has
an additional tea-like note instead of the caramel undertone (Arctander 1969).
As esters are an important group of aroma-active volatiles, in this work, we aimed
to study in detail the composition of the rather neglected ester fraction of the men-
tioned immortelle chemotype essential oil.
2. Results and discussion
The chromatographic separation on SiO2 of a Corsican oil sample, using n-hexane/
Et2O mixtures with a very shallow gradient at low Et2O content, yielded fractions that
were dominated either by the acetate or propionate of nerol. In this work, we focused
our attention on the first eluting ester-containing fraction (VIII) as it was proved to
contain numerous minor neryl and angeloyl esters initially undetectable in the direct
GC-MS analyses of the unfractioned oil (Figure S1; Table S1).
Although, esters of the most common isomeric acyclic monoterpene alcohols, i.e.
nerol, geraniol, linalool, and lavandulol, exhibit very similar mass spectra characterized
by the base ion at m/z 93 and a very intense ion at m/z 69 (Paolini et al. 2005; Figure
S1), we anticipated that these esters from fraction VIII were those of nerol due to the
well-known preference of H. italicum to biosynthesize neryl esters (NEs). Good match-
ing of RI values for some of the detected NEs with the RI values available in the litera-
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ture (Radulovic et al. 2017) confirmed this. The fraction total ion chromatogram
contained three groups of NEs showing regularities in their GC-retention behavior (a
constant RI difference of ca. 100 units; Table S1). The most plentiful group was the
one containing NEs of short- and medium-chain n-fatty acids (n-FAs; C1-C4, C6, C8, C10,
and C12). The identity of these NEs was unambiguously corroborated by GC co-injec-
tion runs of synthetic standards prepared from nerol and the corresponding FAs by
the general Steglich procedure with fraction VIII (Figure 1(a)). Interestingly, neryl dec-
anoate [34] has been only previously reported as a major volatile compound present
in the extracts of pheromone glands of the click beetle Betarmon bisbimaculatus
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(Konig et al. 2016), and as a minor volatile of the stingless bee Geotrigona mombuca
mandibular gland extract (Stangler et al. 2009). Moreover, neryl dodecanoate [36] has
been thus far only found as a minor constituent of the Bulgarian rose concrete
(Watanabe et al. 1977).
Compared to the mentioned n-homologs, two other groups of NEs eluted slightly
faster from the GC column implying that these were their branched-chain isomers.
Neryl 2-methylpropanoate [19], neryl 2-methylbutanoate [23], and neryl 3-methylbuta-
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noate [24], common constituents of essential oils, were readily identified (Radulovic
et al. 2017). Five additional branched NEs, eluting at RI 1693, 1789, 1799, 1996, and
2195, were detected, as well, but their identity was not inferred from MS and RI com-
parison. Based on the established biosynthetic pathways, by which higher plants pro-
duce branched medium-chain FAs (Kroumova et al. 1994), it was assumed that these
were also NEs of methyl branched, iso- or anteiso- (C6, C7, C9, and C11) FAs. Thus far,
branched NEs, with iso-C4-C8 FAs, have been exclusively found in the Australian gera-
nium essential oil (Doimo et al. 1999). In this paper, it was reported that neryl 4-meth-
ylpentanoate had a RI value of 1692 on a BPX-5 column, which fits one of the RI

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M. S. GENČIĆ ET AL.
values of our detected branched NEs (1693). However, there appears to exist an exces-
sive disparity between the reported RI value for neryl 5-methylhexanoate (1825) by
Doimo and coworkers (1999), and the RI values of the herein detected NEs with
branched C7 FAs (1789 and 1799).
To rectify the situation, synthetic samples of esters of nerol with both iso- and ante-
iso-C6 and C7 FAs were prepared and spectrally characterized (NMR, IR, MS; Figures
S2–S21; Table S2). Commercial standards of 3-methylpentanoic, 4-methylpentanoic,
and 5-methylhexanoic acids were in our stock, while we needed to prepare a synthetic
sample of 4-methylhexanoic acid using the malonic ester synthesis (Figure 1(b)). After
GC-coinjection experiments of the obtained NEs standards and fraction VIII, it was
unequivocally confirmed that the ester eluting at RI 1693 was neryl 4-methylpenta-
noate [27], the ester eluting at RI 1789 – neryl 5-methylhexanoate [29], while the ester
eluting at RI 1799 – neryl 4-methylhexanoate [30]. It seems that the alleged neryl 5-
methylhexanoate [29], reported by Doimo et al. (1999), was most probably misidenti-
fied for neryl heptanoate for which we also prepared a synthetic standard and found
the RI value of 1827. In addition, other inconsistencies between our RI data and the RI
data reported by Doimo et al. (1999) for several other NEs were noted. It appears that
they have most probably misidentified neryl heptanoate (RI ¼ 1882) for neryl 6-meth-
ylheptanoate, neryl 6-methylheptanoate for neryl octanoate (RI ¼ 1919; we found the
RI of 1926 units for synthetic neryl octanoate) and neryl heptanoate (RI ¼ 1762) for an
ester of some other isomeric monoterpenol. As the identification of the mentioned
NEs in this study was solely based on MS-library matching and comparison with litera-
ture RI values, and a dozen of geranyl esters with both normal- and branched-chains
were detected as well, it would be wise to re-examine the chemical composition of
the essential oil of this Australian Pelargonium hybrid and to unambiguously confirm
the identification of these esters by coinjections.
Interestingly, NEs of anteiso-C5 and anteiso-C6 FAs eluted from the herein used DB-
5MS column faster than the corresponding iso-isomers, while the opposite elution order
was observed for the esters with iso- and anteiso-C7 FAs (Figure S22). Thus, a careful
analyst is warned of this elution-order change in the branched C5-C8 acid range, and it
appears necessary to confirm identifications by coinjections, while a relatively constant
difference in retention index values (DRIs) of 36 and 28 units, between iso- (C9–C20) and
anteiso-isomers (C11–C19) of medium and long-chain FA esters, respectively, and the
related n-chain isomer is well established (Ilic-Tomic et al. 2015). Herein, similar DRI val-
ues were found for NEs with iso- and anteiso-C7 FAs (37 and 27, respectively). As the
remaining two branched NEs, eluting at RI 1996 and 2195, had DRI values of 28 units,
we are quite certain that these two NEs also belonged to the anteiso-series, i.e. that
they were esters of nerol and 6-methyloctanoic acid [33], and nerol and 8-methyldeca-
noic acid [35], respectively (Figure 1(c)). To the best of our knowledge, these three ante-
iso-NEs ([30], [33], and [35]) represent new natural products.
Several angeloyl esters (AEs) were detected in fraction VIII as well. The tentative
identification of the compounds in this ester series was based on the presence of a
characteristic base ion at m/z 100 (free acid) and intense ions at m/z 55 and 83 that
are derived from a-cleavage next to the C ¼ O bond in their mass spectra (Thomas
and Willhalm 1976; Figure S1). As mass spectrometry with electron-impact ionization

NATURAL PRODUCT RESEARCH
5
sometimes fails to provide distinguishing features of angelates and their geometric
isomers, tiglates, and that there are scarce RI data in the literature, we had to
unequivocally confirm these tentative identifications by co-injections of AE synthetic
samples with fraction VIII. Again, synthetic samples were prepared by the standard
Steglich esterification DCC/DMAP method (Figure 1(a)). The most abundant esters
were AEs with lower weight branched-chain saturated alcohols (i.e. iso-C4 [3], iso-C5
[6], and anteiso-C5 [7]). Octyl and neryl angelate ([18] and [25]) were also present in
relatively higher percentage, while AEs with C4-C7 n-chain saturated alcohols ([6], [9],
and [12]), (Z)-3-hexen-1-ol [10] and 2-phenyl-1-ethanol [21] were found in trace
amounts. We find it worth mentioning that octyl and heptyl angelates, [18] and [12],
have been previously found only in Anthemis nobilis L. essential oil (Akio and Takashi
1989), while phenethyl angelate [21] has been previously reported only as a minor
constituent of Pinus halepensis Miller essential oil (Fekih et al. 2014), and as a volatile
of Malay rose apple (Pino et al. 2004).
In addition to the already mentioned neryl and angeloyl esters, we have identified
four esters of low weight branched alcohols with low weight branched FAs ([1], [2],
[4], and [5]), three esters of benzoic acid ([13], [16], and [22]), one tiglate [26], as well
as one ester of geraniol [31] in fraction VIII (Table S1). To verify the presence of the
newly detected constituents in a non-commercial oil sample, we analogously analyzed
a sample of H. italicum oil hydrodistilled in our laboratory. A targeted screening of
the composition for compounds [1]–[36], combined with dry-flash chromatography,
revealed the presence of all esters.
3. Conclusions
In conclusion, in total 36 esters were identified in the studied ester-containing chroma-
tographic fraction of a H. italicum subsp. italicum essential oil sample from Corsica.
Among them, 29 represented minor constituents that were not detected during the
GC-MS runs of the unfractionated oil. The identity of the majority of the detected
esters was unequivocally confirmed by GC co-injection experiments with synthetic
standards and the chromatographic fraction. The most numerous were neryl esters (18
in total), followed by angeloyl esters (10 in total). Among them, neryl 5-methylhexa-
noate [29], neryl 4-methylhexanoate [30], neryl 6-methyloctanoate [33], and neryl
8-methyldecanoate [35] (Figure 1(c)) were found for the first time in the samples
of natural origin, while several other esters (i.e. neryl 4-methylpentanoate, decanoate
and dodecanoate, as well as phenethyl, heptyl, and octyl angelates) have a rather
restricted occurrence in the Plant Kingdom.
Disclosure statement
The authors declare that there are no conflicts of interest.
Funding
The work was supported by the Ministry of Education, Science and Technological Development
of Serbia under Grant No. 172061 (contract number: 451-03-68/2020-14/200124).

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M. S. GENČIĆ ET AL.
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