**1. Introduction**

Plant genera of the subtribe Inuleae-Inulinae (family Asteraceae), e.g., *Inula*, *Pulicaria*, *Telekia*, *Dittrichia*, *Blumea* and *Chiliadenus*, are known to produce essential oils containing biologically active mono- and sesquiterpenoids [1–3]. Essential oils from *Carpesium* spp. are less studied. To our knowledge, only two communications on essential oil from the herb of *C. abrotanoides* L., the species included in the Chinese pharmacopoeia, have been published to date [4,5]. *C. divaricatum* Sieb. and Zucc. is a medicinal and food plant rich in terpenoid metabolites [6–9]. Recently, hydroxycinnamates and biologically active oxylipin from aerial parts of the plant have been also described [10]. According to the recent taxonomic studies [11,12], *Telekia speciosa* (Schreb.) Baumg. as well as some species of the genus *Inula*, known as essential oil bearing plants, are closely related to *C. divaricatum*. However, the content and composition of essential oils from *C. divaricatum* has remained unknown until now. The aim of the present study was to investigate the volatile compounds from roots and aerial parts

of *C. divaricatum* and to compare the newly generated data with those reported previously for the related species.

#### **2. Results**

Unlike in its natural habitat, in our climate *C. divaricatum* is an annual plant. Moreover, due to late flowering, the plants grown in the open field failed to produce seeds. Fertile seeds were obtained only from the plants cultivated in a glasshouse. Yields of essential oils produced by the aerial parts of the plants were low (<0.02%, see Table 1) and except for the variations in percentages of individual compounds, oils distilled from shoots of the field grown plants and from the aerial parts of plants cultivated in a glasshouse demonstrated some minor qualitative differences in their composition (112 versus 89 identified constituents). The major compounds found in the essential oil from shoots of *C. divaricatum* were: α-pinene (c. 40% of oil), nerol (2.1%–3.7%) and neryl isobutyrate (3.2%–3.9%). Identified thymol derivatives (compounds: **54**, **73**, **84**, **85**, **111**, **130**, **142**, **148** and **149**, see Figure 1) constituted c. 6% of the oil. Roots of the plant turned out to be much better source of volatile terpenoids (yield of essential oil—0.15%). In contrast to the aerial parts (Figure 2), they contained only low amount of α-pinene (up to 1.8% of the essential oil). Thymol derivatives (17 identified structures, see Figure 1) accounted for over 60% and 44% of the essential oil from roots of the garden grown plants and plants cultivated in the glasshouse, respectively. 10-Isobutyryloxy-8,9-epoxythymyl isobutyrate was the major constituent of the analyzed root oils (18.1%–29.2%).


**Table 1.** Chemical composition of essential oils from aerial parts and roots of *Carpesium divaricatum*.



**Table 1.** *Cont.*



**Table 1.** *Cont.*

<sup>a</sup> Essential oils isolated from aerial parts and roots of *Carpesium divaricatum* cultivated in the open field. <sup>b</sup> Essential oils isolated from aerial parts and roots of *Carpesium divaricatum* cultivated in a greenhouse. <sup>c</sup> Experimental retention index measured on non-polar column. <sup>d</sup> Literature retention index from non-polar column. <sup>e</sup> Identification based on retention index. <sup>f</sup> Identification based on mass spectrum. Tr.—<0.05%.

**85 111 130 131** 

**132 142 143 146** 

**Figure 1.** *Cont.*

**Figure 1.** Structures of thymol derivatives identified in essential oils from roots of *Carpesium divaricatum*. **50**: thymol-methyl-ether; **54**: thymol; **73**: 6-methoxythymol-methyl-ether; **84**: 8,9-didehydrothymyl-isobutyrate; **85**: thymyl-isobutyrate; **111**: thymyl-2-methylbutyrate; : 6-methoxythymyl-isobutyrate; **131**: 6-methoxy-8,9-didehydrothymyl-isobutyrate; : 10-isobutyryloxy-8,9-didehydrothymol-methyl-ether; **142**: 9-isobutyryloxythymyl-isobutyrate; **143**: 10-isobutyryloxy-8,9-didehydrothymyl-isobutyrate; : 7-Isobutyryloxythymyl-isobutyrate; **147**: 9-(2-methylbutyryloxy)-thymyl-isobutyrate; : 10-(2-methylbutyryloxy)-8,9-didehydrothymyl-isobutyrate; **149**: 10-isobutyryloxy-8,9-epoxythymyl-isobutyrate; **151**: 10-(2-methylbutyryloxy)-8,9 epoxythymyl-isobutyrate; **152**: 10-isovaleryloxy-8,9-epoxythymyl-isobutyrate.

The essential oils from *C. divaricatum* contained some volatiles, which were difficult to identify based on GC-MS only. Flash chromatography (FC), monitored by thin-layer chromatography (TLC), was used to obtain fractions of oils rich in components of interest (purity 19%–63%, by GC-FID). The fractions were subsequently subjected to NMR analysis and the experimental chemical shifts of the chosen volatiles were compared to the literature data (see Supplementary Material).

Structures of 11 components remained unresolved, due to the small available amounts of the compounds, insufficient to perform full spectral analysis. MS spectra and retention indices of the compounds are shown in Supplementary Material.

**Figure 2.** Gas chromatograms of essential oils from aerial parts and roots of *Carpesium divaricatum*. The numbering of the compounds corresponds to that in Table 1.

#### **3. Discussion**

Though the essential oil content in aerial parts of *C. divaricatum* was very low, the occurrence of α-pinene (40% of the oil) is worth to note. The compound demonstrated anxiolytic and moderate anti-inflammatory effect in mice [13,14]. Essential oils obtained from plants of different provenience can markedly vary in their composition. Aerial parts of *Pulicaria gnaphalodes* (Vent.) Boiss., collected in four different locations, contained extremely different quantities of α-pinene (0.0–34.1% of the essential oil) [15]. Thus, some data on the composition of essential oils from *C. divaricatum* plants grown in their natural habitat would be of interest, to establish whether or not the high α-pinene content is typical of *C. divaricatum* aerial parts. Not much is known from the literature on essential oils from plants of the genus *Carpesium*. To date, only two studies on volatiles from the whole herb of *C. abrotanoides* have been published [4,5]. However, the authors managed to identify 14–44 components of the oils and neither α-pinene nor thymol derivatives have been detected. The major constituents were β-bisabolene (7.3–24.7%), caryophyllene-oxide (c. 13%) and eudesma-5,11(13)-dien-8,12-olide (c. 22%). Volatile constituents from other species of the Inuleae-Inulinae subtribe are better investigated. Thymol and its derivatives seem to be widespread within the plants of the subtribe, except for *Blumea* spp. [16–18]. The genus *Pulicaria* comprises species with essential oils rich in thymol and its methyl ether, like *Pulicaria vulgaris* Gaertn. [19] and *Pulicaria sicula* (L.) Moris [15] together with some species

devoid of thymol derivatives [20]. The content of thymol derivatives in essential oil from aerial parts of *C. divaricatum* (6.4%) is similar to those detected in oils from aerial parts of other species of the subtribe, e.g., *Schizogyne sericea* (L.F.) DC. [21,22], *Telekia speciosa* (Schreb.) Baumg. [23,24] and *Limbarda crithmoides* (L.) Dumort. (formerly *Inula crithmoides* L.) [25]. Structural diversity of the compounds was also similar, with numerous thymol esters.

Essential oils from roots of the Inuleae-Inulinae plants have rarely been studied. Literature data on a few species are available, including *Dittrichia viscosa* (L.) Greuter (formerly *Inula viscosa* (L.) Aiton) [26], *Inula racemosa* Hook. f. [27,28], *Inula helenium* L. [1], *Pulicaria mauritanica* Coss. [29] and *T. speciosa* [24,30]. The common feature of essential oils from *I. helenium*, *I. racemosa* and *T. speciosa* is a very high content of eudesmane-type sesquiterpene lactones (up to 82%). Such composition of essential oils seems to be correlated with a presence of resin canals in roots of the plants. Thymol derivatives were not described as constituents of essential oil from roots of *I. racemosa*. The compounds, however, were found in the oils from the remaining species. Juvenile roots of *I. helenium* and *I. viscosa* contained higher amounts of the monoterpenoids than the old ones [26,31]. Two derivatives of thymol methyl ether constituted nearly 80% of the volatile fraction from *I. viscosa* roots [26]. Thymol, thymol methyl ether and eight thymyl ester derivatives accounted for c. 5.5% of the essential oil from roots of *T. speciosa*. *P. mauritanica* root oil contained c. 16% of the structurally related compounds. Though there are no any data on essential oils from roots of *Carpesium* spp., some thymol derivatives were described as constituents of methanol extract from aerial parts of *C. divaricatum* [32]. All of the compounds were found in essential oils from the plants analyzed in this study. Volatile fraction from roots of *C. divaricatum* is exceptional, in respect of both thymol derivatives content (over 60%) and their structural diversity (17 compounds; for MS spectra see Supplementary Material). 10-Isobutyryloxy-8,9-epoxy-thymyl isobutyrate, major constituent of the analyzed essential oil, demonstrated moderate activity against *Staphylococcus aureus* and *Candida albicans* [33].

#### **4. Materials and Methods**
