Integrated Analysis by GC/MS and 13 C NMR of Moroccan Cladanthus mixtus Essential Oil; Identiﬁcation of Uncommon Epoxyfarnesanes

: Cladanthus mixtus (L.) Chevall., Asteraceae, also known as Moroccan chamomile, is a spontaneous, annual plant growing wild in North-Western Morocco. Economically, the essential oil of C. mixtus is of high interest, Morocco being the only supplier on the international market. Two essential oil samples (EO) were isolated from aerial parts of Cladanthus mixtus (L.) Chevall., and analyzed by a combination of chromatographic and spectroscopic techniques (gas chromatography (GC) in combination with retention indices (RI), gas chromatography-mass spectrometry (GC/MS), and 13 C NMR spectroscopy). Computer matching against the in-house 13 C NMR library allowed the identiﬁcation of the eight components at appreciable contents, namely 3,6,6,9-bis-epoxy-farnesa-1,7(14),10-triene, and its 3-epi, 9-epi, and 3,9-diepi epimers, and 6,9-epoxy-farnesa-1,7(14),10-trien-3-ol and its 3-epi, 6-epi, and 3,6-diepi epimers. Our results conﬁrm the tremendous chemical variability of Moroccan C. mixtus essential oil and the usefulness of 13 C NMR analysis, in combination with GC(RI), for the identiﬁcation of uncommon oxygenated sesquiterpenes that induce an original composition.


Introduction
Cladanthus mixtus (L.) Chevall. (synonyms: Anthemis mixta (L.), Chamaemelum mixtum (L.) All., Ormenis mixta (L.) Dumort, Ormenis multicaulis Braun-Blanq. & Maire), also known as Moroccan chamomile, Asteraceae, is a spontaneous, annual plant 10 to 40 cm tall with numerous erect, lying, or ascending stems terminated by fragrant flower heads with ligulate, white, and sterile external flowers decorated with yellow at their base and fertile yellow tuberous internal flowers. This species is a sialophyte that abounds in the voids of Colibacillary colitis [4]. Thanks to its pleasant smell, the essential oil of C. mixtus is sought after in perfumery, cosmetics, and medicine [4]. Concerning the chemical composition of the essential oil of Cladanthus mixtus (Moroccan chamomile), various studies have shown a very important chemical polymorphism.
The chemical composition of C. mixtus essential oil has been investigated, most of the time, by GC/MS in combination with retention indices on the non-polar or semi-polar chromatography column. Various chemical compositions have been reported describing an important chemical variability, and they have recently been reviewed [5]. The authors listed the 264 compounds that have been identified at least once in C. mixtus essential oil isolated from plants harvested all around the Mediterranean Sea (Algeria, Morocco, France, and Italy).
The aim of this paper that reports on the composition of two C. mixtus oil samples submitted to combined analysis by chromatographic and spectroscopic techniques is to demonstrate overall the importance of 13 C NMR in identifying uncommon oxygenated sesquiterpenes, the presence of which induces an unusual composition.

Plant Material and Essential Oil Isolation
Aerial parts of C. mixtus have been collected in two locations ( Figure 1, Table 1). Hydrodistillation (2 h) using a Clevenger-type apparatus of C. mixtus aerial parts (300 g) in 2 L flask yielded 0.3 mL of essential oil for both samples. To avoid any damage, the samples were stored at 5 • C in amber vials. of C. mixtus was ranked ninth among the 20 best essential oils produced in Morocco. In Morocco, C. mixtus is advised as an anxiolytic for the rebalancing of the central nervou system; it has great value in nervous breakdowns and for mild hepatic and gastric insuf ficiency and Colibacillary colitis [4]. Thanks to its pleasant smell, the essential oil of C. mix tus is sought after in perfumery, cosmetics, and medicine [4]. Concerning the chemica composition of the essential oil of Cladanthus mixtus (Moroccan chamomile), various stud ies have shown a very important chemical polymorphism.
The chemical composition of C. mixtus essential oil has been investigated, most of th time, by GC/MS in combination with retention indices on the non-polar or semi-pola chromatography column. Various chemical compositions have been reported describing an important chemical variability, and they have recently been reviewed [5]. The author listed the 264 compounds that have been identified at least once in C. mixtus essential oi isolated from plants harvested all around the Mediterranean Sea (Algeria, Morocco France, and Italy).

Plant Material and Essential Oil Isolation
Aerial parts of C. mixtus have been collected in two locations ( Figure 1, Table 1). Hy drodistillation (2 h) using a Clevenger-type apparatus of C. mixtus aerial parts (300 g) in L flask yielded 0.3 mL of essential oil for both samples. To avoid any damage, the sample were stored at 5 °C in amber vials.

GC-FID Analysis
GC-FID analyses were carried out using a Clarus 500 Perkin Elmer (Perkin Elmer, Courtaboeuf, France) chromatograph equipped with two FID and two fused-silica capillary columns (50 m length × 0.22 mm internal diameter, film thickness 0.25 µm), BP-1 (polydimethylsiloxane), and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 • C to 220 • C at 2 • C/min and then held isothermal at 220 • C for 20 min; injector temperature, 250 • C; detector temperature, 250 • C; carrier gas, H 2 (0.8 mL/min); split, 1/60; and injected volume, 0.5 µL. The relative proportions of the essential oil constituents were expressed as percentages obtained by peak-area normalization without using correcting factors. Retention indices (RI) were determined relative to the retention times of a series of n-alkanes (C8-C28) with linear interpolation (Target Compounds software from Perkin Elmer, V1.2019, Courtaboeuf, France).

Nuclear Magnetic Resonance
13 C NMR spectra were recorded on a Bruker AVANCE 400 Fourier Transform spectrometer equipped with a 5 mm probe operating at 100.63 MHz for 13 C, in CDCl 3 , with all shifts referred to internal Tetramethylsilane (TMS) at room temperature (298 • C). The following parameters were used: pulse width = 4 µs (flip angle 45 • ); acquisition time = 2.7 s for 128 K data table with a spectral width of 25,000 Hz (250 ppm); CPD mode decoupling; and digital resolution = 0.183 Hz/pt. For each sample (40 mg of essential oil in 0.5 mL of CDCl 3 ), 3000 scans were recorded.

Identification of Individual Components
Identification of the individual components was carried out (i) by comparison of their GC retention indices (RI) on non-polar and polar columns with those of reference compounds compiled in the in-house library and with literature data [19][20][21]; (ii) on computer matching against commercial mass spectral libraries [21][22][23]; and (iii) on comparison of the signals in the 13 C NMR spectra of the samples with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software [24,25]

Methods for Identification of Individual Components of Essential Oils
Identification of individual components of essential oils is routinely performed by using a fast-scanning mass spectrometer associated with a gas chromatograph. The mass spectrum of every component is compared with those of reference compounds compiled in commercial or in-house libraries. Two-dimensional GC coupled with MS may be useful for the analysis of complex essential oils in order to individualize components that are coeluted when a unique column is employed and then to record more reliable mass spectra. Commercial libraries contain mass spectra of thousands of compounds (covering all fields of research); among these, a few thousand belong to volatile components of essential oils. In-house MS libraries are constructed with pure compounds or compounds whose identity is ascertained in one essential oil sample by spectroscopic techniques. Therefore, the reference MS spectra of various new compounds identified in essential oils and reported in the literature are not directly available to be introduced in a given MS library. In most analyses, identification of the compounds suggested by MS is confirmed by comparison of its retention indices (RI) on non-polar and polar chromatography columns with those of reference compounds compiled in the literature and/or homemade RI libraries.
In parallel, it has been shown that 13 C NMR can be used for the non-destructive, nonseparative identification of individual components of essential oils. In this computerized procedure developed at the University of Corsica, an individual component is identified by comparison of the signals of the mixture spectrum with those of reference spectra compiled in a library [24,25] (Figure 2). It should be pointed out that structural elucidation of every new compound proceeds, inter alia, via 13 C NMR spectroscopy, and therefore, the 13 C NMR spectrum is fully reported in the publication. It could be added that nowadays, a high-field spectrometer allows us to record the 13 C NMR spectrum of isolated compounds at the mg level. The diluted solutions avoid intermolecular influence, and therefore, chemical shifts are perfectly reproducible. In practice, two spectral data libraries were constructed; the first one contains spectra recorded in the lab, and the second contains spectra reported in the literature for every new compound isolated from plants or obtained by synthesis. Both libraries are continuously implemented. Each component is identified considering three parameters directly available from the in-house computer program: (i) the number of observed carbons with respect to the number of expected signals, (ii) the number of overlapped signals of carbons that possess the same chemical shift, and (iii) the difference of the chemical shift of each signal in the mixture spectrum and in the reference (Figure 2). A compound is considered as identified when at least 50% of its signals belonging solely to that molecule are observed [24,25].
In-house library of reference compounds

Methods for Identification of Individual Components of Essential Oils
Identification of individual components of essential oils is routinely performed by using a fast-scanning mass spectrometer associated with a gas chromatograph. The mass spectrum of every component is compared with those of reference compounds compiled in commercial or in-house libraries. Two-dimensional GC coupled with MS may be useful for the analysis of complex essential oils in order to individualize components that are co-eluted when a unique column is employed and then to record more reliable mass spectra. Commercial libraries contain mass spectra of thousands of compounds (covering all fields of research); among these, a few thousand belong to volatile components of essential oils. In-house MS libraries are constructed with pure compounds or compounds whose identity is ascertained in one essential oil sample by spectroscopic techniques. Therefore, the reference MS spectra of various new compounds identified in essential oils and reported in the literature are not directly available to be introduced in a given MS library. In most analyses, identification of the compounds suggested by MS is confirmed by comparison of its retention indices (RI) on non-polar and polar chromatography columns with those of reference compounds compiled in the literature and/or homemade RI libraries.
In parallel, it has been shown that 13 C NMR can be used for the non-destructive, nonseparative identification of individual components of essential oils. In this computerized procedure developed at the University of Corsica, an individual component is identified by comparison of the signals of the mixture spectrum with those of reference spectra compiled in a library [24,25] (Figure 2). It should be pointed out that structural elucidation of every new compound proceeds, inter alia, via 13 C NMR spectroscopy, and therefore, the 13 C NMR spectrum is fully reported in the publication. It could be added that nowadays, a high-field spectrometer allows us to record the 13 C NMR spectrum of isolated compounds at the mg level. The diluted solutions avoid intermolecular influence, and therefore, chemical shifts are perfectly reproducible. In practice, two spectral data libraries were constructed; the first one contains spectra recorded in the lab, and the second contains spectra reported in the literature for every new compound isolated from plants or obtained by synthesis. Both libraries are continuously implemented. Each component is identified considering three parameters directly available from the in-house computer program: (i) the number of observed carbons with respect to the number of expected signals, (ii) the number of overlapped signals of carbons that possess the same chemical shift, and (iii) the difference of the chemical shift of each signal in the mixture spectrum and in the reference (Figure 2). A compound is considered as identified when at least 50% of its signals belonging solely to that molecule are observed [24,25].
The benefit of using various chromatographic and spectroscopic techniques for the analysis of essential oil has been demonstrated and exemplified. For instance, the key role of 13 C NMR analysis in the identification of individual components of Ivoirian Polyalthia longifolia leaf oil and of Xanthocyparis vietnamensis wood oil has been highlighted [26,27]. 13 C NMR analysis also appeared suited for the identification of stereoisomers [28,29].

Chemical Composition of the Two Oil Samples
EOs were isolated from aerial parts of C. mixtus harvested in North-Western Morocco, at Souk Had (sample SH) and at Ain Chkef forest, near Sidi Slimane (Sample SS). Yields were measured as 0.10% (v/w) for both oil samples, which were submitted to GC(RI) on two column (non-polar and polar phases), GC/MS, and 13 C NMR analyses.
In total, 81 compounds were identified. They accounted for 90.1 and 87.6% of the whole composition, respectively ( Table 2). The chromatogram of the SS sample (non-polar column) is reported in Figure 3.
The eight components were identified by comparison of their 13 C NMR chemical shifts, measured in the recorded 13 C NMR spectra of both oil samples with those of reference spectra compiled in our library ( Figure 5, Table S1).

Discussion
Despite the similarity of the structures and the low percentage of some components, the chemical shifts of all carbons were observed, except those of the quaternary carbons of the minor isomers. The difference in chemical shifts between the experimental spectra

Discussion
Despite the similarity of the structures and the low percentage of some components, the chemical shifts of all carbons were observed, except those of the quaternary carbons of the minor isomers. The difference in chemical shifts between the experimental spectra and the reference data was always acceptable, as well as the number of overlapped signals. It could be noticed that the eight components were first isolated from the essential oil of Tanacetum fruticulosum and spectroscopically characterized by Weyerstahl et al., who reported, inter alia, their 13 C NMR data [46]. To the best of our knowledge, since that time, the eight compounds have only been identified, by using 13 C NMR spectroscopy, in essential oil isolated from aerial parts of Corsican Dittrichia viscosa [38].

Conclusions
Although oil samples whose chemical composition was dominated by (i) α-pinene and/or 1,8-cineole, (ii) (E)-nerolidol, or (iii) (E)-β-farnesene have been reported, the occurrence of oxygenated farnesane derivatives at appreciable contents (up to 25%) brings originality to the investigated C. mixtus oil samples. Our results confirm the tremendous chemical variability of Moroccan C. mixtus essential oil and the potential of 13 C NMR analysis, in combination with GC(RI), for the identification of uncommon oxygenated sesquiterpenes.