*4.1. General Experimental Procedures*

GC-MS-FID analyses of essential oils and their fractions were performed on a Trace GC Ultra Gas Chromatograph coupled with DSQII mass spectrometer (Thermo Electron, Waltham, MA, USA). Simultaneous GC-FID and GC-MS analysis were performed using a MS-FID splitter (SGE Analytical Science, Ringwood, VIC, Australia). Mass range was 33–550 amu, ion source-heating: 200 ◦C; ionization energy: 70 eV. One microliter of essential oil solution (80% *v*/*v*) diluted in pentane:diethyl ether was injected in split mode at split ratios (50:1). Operating conditions: capillary column Rtx-1 MS (60 m × 0.25 mm i.d., film thickness 0.25 μm), and temperature program: 50 ◦C (3 min)—300 ◦C (30 min) at 4 ◦C/min. Injector and detector temperatures were 280 ◦C and 300 ◦C, respectively. Carrier gas was helium (constant pressure: 300 kPa). The relative composition of each essential oil sample was calculated from GC peak areas according to total peak normalization—the most popular method used in the essential oil analysis. 1H-NMR (250 MHz) and 13C-NMR (62.90 MHz) spectra for components of essential oils were recorded with a Bruker DPX 250 Avance spectrometer in CDCl3, with TMS as an internal standard.

#### *4.2. Plant Material*

Seeds of *Carpesium divaricatum* Sieb. and Zucc, provided by the Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, Tsukuba (Japan), were sown in the end of March 2015, into multipots with garden soil. In the stage of 4–5 mature leaves, the plants were transferred to plastic pots with a substrate composed of garden soil, peat and sand (2:1:1, *v*/*v*). Plants were grown in a glasshouse of the Garden of Medicinal Plants, Maj Institute of Pharmacology PAS in Krakow, under controlled conditions (temperatures by day 18–38 ◦C; by night 12–18 ◦C), without any chemical treatment. In the third week of May, the plants were divided into two groups. First group was left in the glasshouse for further growth and the second one was transplanted into the open field. Data on cultivation conditions (type of soil, average annual temperature, annual rainfall and agrotechnical procedures applied) are available elsewhere [34]. Aerial parts and roots of the plants were collected in the beginning of flowering period (August/September) and dried under shade at room temperature. Voucher specimen (3/15) was deposited in the collection kept at the Garden of Medicinal Plants, Institute of Pharmacology, Kraków, Poland. The dry plant material was stored no longer than five months.

#### *4.3. Isolation of Essential Oil*

Essential oils from aerial (dried leaf, branches, flowers) and underground parts (dried roots) of *C. divaricatum* were obtained by hydrodistillation using a Clevenger-type apparatus. Each hydrodistillation was conducted for 4 h using 100–300 g of plant material. The yellowish essential oils were dried over anhydrous magnesium sulphate, and stored at 4 ◦C in the dark, until tested and analyzed.

### *4.4. Isolation and NMR Analysis of Volatile Components*

To isolate the volatiles of interest, the essential oils from aerial parts (i.e., dried leaves with petioles, stems and flowers, 504 mg) and from roots (dried plant material, 973 mg) of the plants grown in the open field were separately flash-chromatographed (FC) on a glass column (500 × 30 mm) filled with silica gel 60 (0.040–0.063 mm, Merck, EM Science, NJ USA), starting the elution with *n*-hexane and gradually increasing the polarity by addition of diethyl ether. The elution was accelerated by means of pressurized nitrogen (flow rate 100 mL/min). The separation was monitored by TLC and GC-MS. Twenty fractions (1a–20a) of essential oil distilled from the aerial parts of the plant and twenty fractions (1b–20b) of root essential oil were obtained and analyzed by GC-MS-FID. Structures of 11 volatiles from the following fractions were confirmed using NMR spectroscopy (1H and/or 13C; see Supplementary Material): 1a: (42 mg) neryl-isobutyrate (26%); 13a: (22mg) (*E*)-nerolidol (25%); 15a: (22 mg) τ-cadinol (21%); 17a: (58 mg) nerol (25%); 18a: (48 mg) α-cadinol (23%); 3b: (17 mg) thymol-methyl-ether (33%); 7b: (32 mg) thymyl isobutyrate (57%); 8b: (51 mg) 6-methoxythymyl-isobutyrate (62%); 13b: (13 mg) caryophyllene-oxide (52%); 14b: (36%) 10-isobutyryloxy-8,9-didehydrothymyl-isobutyrate (46%); 15b: (65 mg) 9-isobutyryloxythymyl-isobutyrate (55%); 17b: (53 mg) 10-isobutyryloxy-8,9-epoxythymyl-isobutyrate (63%); 18b: (25 mg) nerol (43%).

#### *4.5. Identification of Essential Oil Constituents*

Constituents of the essential oils were identified based on their MS spectra and their comparison with those from mass spectra libraries: NIST 2012, Wiley Registry of Mass Spectral Data 8th edition and MassFinder 4.1, along with the relative retention indices (RI) on DB-1 column (available from MassFinder 4.1) and on Rtx-1MS column found in the literature [35]. Isolated compounds were also identified by the comparison of their 1H-NMR and 13C-NMR spectral data with those of the compounds isolated previously in our laboratory or those from the literature.

#### **5. Conclusions**

This was the first study on composition of essential oils from *C. divaricatum*. Aerial parts of *C. divaricatum* occurred to be a poor source of volatiles. Essential oil from roots of the plant was rich in thymyl ester derivatives of various structures. As some of the compounds, according to the literature [36], demonstrated moderate antibacterial, antifungal and anti-inflammatory activities, the essential oil from roots of *C. divaricatum* as well as its components are worth further studies.

**Supplementary Materials:** The following are available online: Figure S1: Mass spectra and retention indices (RI) together with chemical structures of thymol derivatives detected in *C. divaricatum* essential oils, Figure S2: Mass spectra and experimental retention indices (RI) of unidentified compounds from *C. divaricatum* essential oils, Figure S3: Results of NMR analyses of crude fractions (obtained by flash chromatography) from *C. divaricatum* essential oils.

**Author Contributions:** Conceptualization, A.S.; methodology, A.W.-B. and A.S.; investigation, A.W.-B., J.M., A.S.; resources, A.W.-B., J.M., A.S.; data curation, A.W.-B. and A.S.; writing—original draft preparation, A.W.-B., J.M., A.S.; writing—review and editing, A.S.; project administration, A.S.

**Funding:** This research received no external funding.

**Acknowledgments:** We are greatly indebted to the workers of the Garden of Medicinal Plants, Maj Institute of Pharmacology PAS in Kraków, for cultivation of plants.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

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