Two new eudesman-4  -ol epoxides from the stem essential oil of Laggera pterodonta from Côte d’Ivoire

The investigation of the stem essential oil of Laggera pterodonta (DC.) Sch. Bip. ex Oliv. (Asteraceae) from Côte d’Ivoire was carried out, using a combination of chromatographic (GC-RI, CC, pc-GC) and spectroscopic (GC-MS, 13 C NMR) techniques. This study led to the identification of fifty constituents of which two new natural compounds namely 7  ,11  -epoxy-eudesman-4  -ol and 7  ,11  -epoxy-eudesman-4  -ol. Their structures were elucidated by 1D and 2D NMR spectroscopy after pc-GC purifying. Finaly 98.9% of the whole composition of the oil was identified with a high amount of 2,5-dimethoxy- p -cymene (78.9%). The other significant components were  -humulene (6.2%), ( E )-  -caryophyllene (1.7%), thymyl methyl oxide (1.7%),  -phellandrene (1.5%), p -cymene (1.2%), (3  H,4  H,6  H,1  Me)-1,6-epoxy-3-hydroxycarvotanacetone angelic acid ester (1.1%) and 10- epi -  -eudesmol (1.0%).


Plant material and isolation procedure
The plant material was harvested in Marabadiassa, (Region of Vallée du Bandama, Department of Béoumi, Central Côte d'Ivoire) in September 2016 and authenticated by Mr Jean Assi, technician at the Herbarium of the Centre National de Floristique (Abidjan, Côte d'Ivoire) and Mr Henry Téré from the Centre Suisse de Recherche (Abidjan, Côte d'Ivoire). A voucher specimen has been deposited at the herbarium of the Centre National de Floristique (CNF), Abidjan, with the reference LAA 14631.
The essential oil was obtained in 0.040% (w/w) yield, by successive hydrodistillations of a total of 14250.2g of fresh stems. Each hydrodistillation was performed using a Clevenger-type apparatus for 3 hours.

Gas chromatography with FID associated to RI
Analyses were carried out using a Clarus 500 Perkin Elmer (Perkin Elmer, Courtaboeuf, France) system equipped with a FID and two fused-silica capillary columns (50m x 0.22 mm, film thickness 0.25 m), 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: helium (0.8 mL/min); split: 1/60; injected volume: 0.5 mL. The relative proportions of the 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 with linear interpolation (Target Compounds software from Perkin Elmer).
The injected volume is 1 μL in splitless mode. The oven temperature was ramped from 70 to 120 °C at 10 °C/min, then from 120°C to 250°C at 20°C/min. The system was operated in constant pressure mode at 35 kPa (Carrier gas H 2 ). Compound was trapped at 5-10 °C in Gerstel U-type glass tubes by programming cutting times into the operating software allowing accurate automated operation. The isolation of any unknown compound in amounts sufficient for NMR analysis required 150-400 GC runs, and to avoid all contaminations, the product was collected directly in a NMR tube.

Nuclear magnetic resonance
Essential oil and fractions nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE 400 Fourier Transform spectrometer operating at 400.132 MHz for 1 H and 100.63 MHz for 13 C, equipped with a 5 mm probe, in CDCl 3 , with all shifts referred to internal TMS. The 1 H NMR spectra were recorded with the following parameters: pulse width (PW), 4.3 μs; relaxation delay 1 s and acquisition time 2.6 s for 32 K data table with a spectral width (SW) of 6000 Hz. 13 C NMR spectra of the oil samples were recorded with the following parameters: pulse width = 4 µs (flip angle 45°); acquisition time = 2.7 s for 128K data table with a spectral width of 25 000 Hz (250 ppm); CPD mode decoupling; digital resolution = 0.183 Hz/pt. Standard pulse sequences from Bruker library were used for two-dimensional spectra. Gradient-enhanced sequences were used for the heteronuclear two dimensional experiments. NMR spectra of 7,11-epoxy-eudesman-4-ol 48 and 7,11-epoxy-eudesman-4-ol 49 were recorded in C 6 D 6 at 298 K on a Bruker Avance DRX 500 spectrometer operating at 500.13 MHz for 1 H and 125.75 MHz for 13 C. In order to increase sensitivity, 13 C NMR spectra such as broadband-13 C, DEPT 135 and DEPT 90 were run with a direct probe head (5 mm PADUL 13 C-1 H Z-GRD). 1D-and 2D NMR spectra such as 1 H, COSY, NOESY, HSQC, HMBC were run with an inverse probe head (5 mm PHTXI 1 H-13 C/ 15 N ZGRD). Spectrum calibration was performed by using the C 6 D 6 signal as internal reference (7.16 ppm for 1 H NMR, 128.06 ppm for 13 C NMR). Chemical shifts (δ) are expressed in parts per million (ppm) and coupling constants (J) in hertz. All NMR experiments were carried out using pulse sequences supplied by the spectrometer manufacturer (Bruker Topspin TM ) and processed via Mestrelab MestreNOVA software (Version 6.0.2-5475).

Identification of individual components
Identification of the individual components was carried out: (i) by comparison of their GC retention indices (RI) on polar and apolar columns, determined relative to the retention times of a series of nalkanes with linear interpolation with those of reference compounds (König et al. 2001); (ii) on computer matching against commercial mass spectral libraries (National Institute of Standards and Technology. 1999; König et al. 2001;Adams 2007); (iii) on comparison of the signals in the 13 C NMR spectra of the mixtures with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software (Tomi et al. 1995;Kambiré et al. 2018). This method allows the identification of individual components of the essential oil at content as low as 0.4-0.5%. A few compounds were identified by comparison with literature data. Order of elution and percentages were given on an apolar column (BP1), except components with an asterisk (*), which percentages were taken on polar column (BP20) b RI1, Rip = Retention indices measured on apolar and polar capillary column respectively. tr = traces level (<0.05%).
All compounds were identified by GC(RI) and GC-MS. 13 C NMR, components were identified by NMR in the essential oil and obvious in at least one fraction of chromatography, 13 C NMR, components were identified by NMR in one fraction of chromatography. Compounds 48 and 49 are new natural stereoisomers.