Constituents of Xerolekia speciosissima (L.) Anderb. (Inuleae), and Anti-Inflammatory Activity of 7,10-Diisobutyryloxy-8,9-epoxythymyl Isobutyrate

Xerolekia speciosissima (L.) Anderb., a rare plant from the north of Italy, is a member of the Inuleae-Inulinae subtribe of the Asteraceae. Despite its close taxonomic relationship with many species possessing medicinal properties, the chemical composition of the plant has remained unknown until now. A hydroalcoholic extract from the aerial parts of X. speciosissima was analyzed by HPLC-DAD-MSn, revealing the presence of caffeic acid derivatives and flavonoids. In all, 19 compounds, including commonly found chlorogenic acids and less frequently occurring butyryl and methylbutyryl conjugates of dicaffeoylquinic and tricaffeoylhexaric acids, plus two flavonoids, were tentatively identified. Chromatographic separation of a hydroalcoholic extract from the capitula of the plant led to the isolation of (+)-dehydrodiconiferyl alcohol 4-O-β-glucopyranoside, quercimeritrin, astragalin, isoquercitrin, 6-hydroxykaempferol-7-O-β-glucoside, quercetagitrin, methyl caffeate, caffeic acid, protocatechuic acid, chlorogenic acid and 1,5-dicaffeoylquinic acid. Composition of a nonpolar extract from the aerial parts of the plant was analyzed by chromatographic methods supported with 1H-NMR spectroscopy. The analysis revealed the presence of loliolide, reynosin, samtamarine, 2,3-dihydroaromaticin, 2-deoxy-4-epi-pulchellin and thymol derivatives as terpenoid constituents of the plant. One of the latter compounds—7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate—at concentrations 0.5, 1.0 and 2.5 μM, significantly reduced IL-8, IL-1β and CCL2 excretion by LPS-stimulated human neutrophils.


Introduction
Xerolekia speciosissima L. Anderb. (synonyms: Buphthalmum speciosissimum Ard., Telekia speciosissima (L.) Less.), a species which belongs to the newly created Xerolekia genus [1] is considered a member of the tribe Inuleae, subtribe Inulinae, of the family Compositae (Asteraceae) [2]. X. speciosissima is a perennial herb, 20-60 cm tall, with a stem having resin canals. Leaves of the plant are up to 25 cm long and are broadly lanceolate; inflorescences are solitary, terminal, heterogamous and have yellow flowers. The species, growing in nature, is a pre-Alpine endemite, listed in Italian and regional (Lombardy) red lists, that naturally inhabits crevices on limestone or dolomite rocks, between 1000 and 1900 m.a.s.l. Its distribution is limited to the area from Lake Como to Lake Garda and to the Ledro Valley [1,3].

Caffeic Acid Derivatives in Leaves of X. speciosissima, Buphthalmum salicifolium and Telekia speciosa
Results of HPLC-DAD analysis of caffeic acid derivatives in young leaves of X. speciosissima and the two closely related species are summarized in Table 2. The plant material was collected in May, at the start of vegetation. Moreover, five unidentified caffeic acid derivatives that were exclusively present in T. speciosa leaves and one specific for B. salicifolium were detected.

Constituents of a Hydroalcoholic Extract from Capitula of X. speciosissima
A hydroalcoholic extract from the dried inflorescences of X. speciosissima yielded caffeic acid and its derivatives, flavonols-derivatives of quercetin and kaempferol (see Figure 2), one lignan and one hydroxybenzoic acid. The isolated compounds were identified on the basis of their chromatographic behavior and their spectral data (UV, 1 H-NMR 400.17 MHz), with reference to those of the standard compounds and to those from the literature. The only isolated lignan, of neolignan type, (+)-dehydrodiconiferyl alcohol 4-O-β-glucopyranoside (1) [16] and protocatechuic acid (3,4-dihydroxybenzoic acid, 2) [17], were minor constituents of the examined extract. Caffeic acid (3) [18] and its methyl ester (4) [19] were present in majority of fractions eluted from the polyamide column. The most abundant conjugates of caffeic and quinic acids were 5-CQA (7) [20], and 1,5-DCQA (11) [21], which was the main component of the complex mixture of hydroxycinnamates from fractions P87-P91.
Results of HPLC-DAD analysis of caffeic acid derivatives in young leaves of X. speciosissima and the two closely related species are summarized in Table 2. The plant material was collected in May, at the start of vegetation. Moreover, five unidentified caffeic acid derivatives that were exclusively present in T. speciosa leaves and one specific for B. salicifolium were detected.

Constituents of a Chloroform Extract from Aerial Parts of X. speciosissima
Four sesquiterpene lactones: reynosin (12), santamarine (13) [25], 2,3-dihydroaromaticin (14) [26] and 2-deoxy-4-epi-pulchellin (16) [27] (see Figure 3) together with one apocarotenoide-loliolide (15) [28] were isolated from a chloroform extract of X. speciosissima aerial parts. The compounds were identified based on their experimental 1 H-NMR spectroscopic data and their chromatographic parameters. The data were directly compared either with those of the standard compounds or with those from the literature. A pair of isomeric eudesmanolides, reynosin and santamarine, have been frequently found in different, taxonomically distant species, also outside the Asteraceae. It has been known for a long time that the compounds might be artifacts formed from costunolide-1,10-epoxide during chromatographic separation on silica gel [29]. This could explain their occurrence in fractions of different polarity. A mixture of monoterpenoid thymol derivatives was also separated from the extract. The mixture was not further purified, as it contained multiple compounds in relatively low amounts. Thymol derivatives were major terpenoid constituents in roots of X. speciosissima [6].

Constituents of a Chloroform Extract from Aerial Parts of X. speciosissima
Four sesquiterpene lactones: reynosin (12), santamarine (13) [25], 2,3-dihydroaromaticin (14) [26] and 2-deoxy-4-epi-pulchellin (16) [27] (see Figure 3) together with one apocarotenoide-loliolide (15) [28] were isolated from a chloroform extract of X. speciosissima aerial parts. The compounds were identified based on their experimental 1 H-NMR spectroscopic data and their chromatographic parameters. The data were directly compared either with those of the standard compounds or with those from the literature. A pair of isomeric eudesmanolides, reynosin and santamarine, have been frequently found in different, taxonomically distant species, also outside the Asteraceae. It has been known for a long time that the compounds might be artifacts formed from costunolide-1,10-epoxide during chromatographic separation on silica gel [29]. This could explain their occurrence in fractions of different polarity. A mixture of monoterpenoid thymol derivatives was also separated from the extract. The mixture was not further purified, as it contained multiple compounds in relatively low amounts. Thymol derivatives were major terpenoid constituents in roots of X. speciosissima [6].

Cytotoxicity
The cytotoxicity of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate to human polymorphonuclear leukocytes (PMNs) was investigated using propidium iodide (PI) staining and flow cytometry (FACS) analysis. The compound did not cause toxicity in PMNs at 2.5 μM or lower concentrations ( Figure 4). Solvent (DMSO) was not toxic to the cells either (data not shown). On the basis of these data, all further experiments were performed using the tested compound at concentrations up to 2.5 μM.  The cytotoxicity of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate to human polymorphonuclear leukocytes (PMNs) was investigated using propidium iodide (PI) staining and flow cytometry (FACS) analysis. The compound did not cause toxicity in PMNs at 2.5 µM or lower concentrations ( Figure 4). Solvent (DMSO) was not toxic to the cells either (data not shown). On the basis of these data, all further experiments were performed using the tested compound at concentrations up to 2.5 µM.

Reactive Oxygen Species (ROS) Generation
Activation of PMNs at a site of inflammation induces an oxidative burst in these cells. The phenomenon is characterized by intense ROS generation and liberation of proteolytic enzymes from azurophilic granules. An effect of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate on ROS production in PMNs was assessed in response to N-formyl-Met-Leu-Phe (f-MLP) stimulation. The examined monoterpenoid efficiently reduced ROS release at 1-2.5 µM ( Figure 5).

Reactive Oxygen Species (ROS) Generation
Activation of PMNs at a site of inflammation induces an oxidative burst in these cells. The phenomenon is characterized by intense ROS generation and liberation of proteolytic enzymes from azurophilic granules. An effect of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate on ROS production in PMNs was assessed in response to N-formyl-Met-Leu-Phe (f-MLP) stimulation. The examined monoterpenoid efficiently reduced ROS release at 1-2.5 μM ( Figure 5). In response to stimulation with pro-inflammatory agonists, e.g., LPS or f-MLP, human neutrophils secrete several cytokines and chemokines, including TNF-α, IL-1β, IL-8 and CCL2 [30,31]. In the present study, neutrophils were pretreated with the examined thymol derivative before their priming with LPS. Using ELISA, levels of IL-8, TNF-α, IL-1β and CCL2 were determined in the culture medium, 24 h after LPS stimulation. Preincubation of human neutrophils with 7,10diisobutyryloxy-8,9-epoxythymyl isobutyrate resulted in significant and dose-dependent inhibition In response to stimulation with pro-inflammatory agonists, e.g., LPS or f-MLP, human neutrophils secrete several cytokines and chemokines, including TNF-α, IL-1β, IL-8 and CCL2 [30,31]. In the present study, neutrophils were pretreated with the examined thymol derivative before their priming with LPS. Using ELISA, levels of IL-8, TNF-α, IL-1β and CCL2 were determined in the culture medium, 24 h after LPS stimulation. Preincubation of human neutrophils with 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate resulted in significant and dose-dependent inhibition of IL-8 production upon LPS stimulation ( Figure 6A). The compound was less active as an inhibitor of TNF-α production. Statistically significant reduction in release of this cytokine was achieved only with the highest of the tested concentrations (2.5 µM, see Figure 6B). Both IL-1β and CCL2 production by LPS treated neutrophils were significantly and dose-dependently lowered by pretreatment with the examined monoterpenoid at a dose of 0.5-2.5 µM (Figure 7A,B). of IL-8 production upon LPS stimulation ( Figure 6A). The compound was less active as an inhibitor of TNF-α production. Statistically significant reduction in release of this cytokine was achieved only with the highest of the tested concentrations (2.5 μM, see Figure 6B). Both IL-1β and CCL2 production by LPS treated neutrophils were significantly and dose-dependently lowered by pretreatment with the examined monoterpenoid at a dose of 0.5-2.5 μM (Figure 7A,B).

Discussion
Total phenolic contents in shoots and roots of X. speciosissima were higher than those estimated for Cichorium intybus, a food plant rich in polyphenols (approximate total phenolic content (TPC) value: 7.5-42.0 mg g −1 GAE) [32] and the other Asteraceae species studied, in which TPC values ranged from 3.7 to 15.2 GAE (mg g −1 ) [33,34]. In contrast to chicory plants, roots of X. speciosissima demonstrated higher reducing capacity (80.08 ± 1.10 mg g −1 GAE) than the aerial parts of the plant (61.66 ± 2.14 mg g −1 GAE).
As can be seen in the Table 2, caffeic acid derivatives constitute major part of the polyphenolic fraction from the plant shoots. By looking at the hydroxycinnamate profiles of X. speciosissima, T. speciosa and B. salicifolium, one can observe that X. speciosissima shares more similarity with B.

Discussion
Total phenolic contents in shoots and roots of X. speciosissima were higher than those estimated for Cichorium intybus, a food plant rich in polyphenols (approximate total phenolic content (TPC) value: 7.5-42.0 mg g −1 GAE) [32] and the other Asteraceae species studied, in which TPC values ranged from 3.7 to 15.2 GAE (mg g −1 ) [33,34]. In contrast to chicory plants, roots of X. speciosissima demonstrated higher reducing capacity (80.08 ± 1.10 mg g −1 GAE) than the aerial parts of the plant (61.66 ± 2.14 mg g −1 GAE).
As can be seen in the Table 2, caffeic acid derivatives constitute major part of the polyphenolic fraction from the plant shoots. By looking at the hydroxycinnamate profiles of X. speciosissima, T. speciosa and B. salicifolium, one can observe that X. speciosissima shares more similarity with B. salicifolium. Isobutyryl-and 2-methylbutyryl/isovaleryl-dicaffeoylquinic acids especially seem to be distinctive metabolites of the two species. The compounds were also found in Carpesium divaricatum Sieb. and Zucc. [13], another closely related species of the Inuleae-Inulinae. On the other hand, acylated glycosides of caffeic and ferulic acids [35] appear to be characteristic metabolites of T. speciosa.
Except for hydroxycinnamates, HPLC-DAD-MS n analysis revealed the presence of two flavonol glycosides, in aerial parts of X. speciosissima. Flavonols, namely, 3-O-β-glucopyranosides of quercetin and kaempferol and 7-O-β-glucopyranosides of quercetin, quercetagetin (6-hydroxyquercetin) and 6-hydroxykaempferol, were also found in capitula of the plant. In contrast to B. salicifolium, flowerheads of X. speciosissima did not accumulate acylated flavonol glucosides [15]. Moreover, methylated flavonols (patuletin, isorhamnetin) were not found in the investigated plant material. Chemical diversity of the flavonoids produced by X. speciosissima is limited also in comparison with that of T. speciosa [36].
The majority of phenolic constituents found in X. speciosissima possess well documented biological activity, especially as antioxidative, free radical scavenging and anti-inflammatory agents [24,[37][38][39][40]. Astragalin, isoquercitrin, protocatechuic acid and caffeoylquinic acids are among the most extensively studied plant metabolites, with respect to the potential health implications of their dietary intake.
Sesquiterpene lactones are a group of terpenoid metabolites considered to be useful taxonomic markers within the Asteraceae. In X. speciosissima the compounds are represented by two eudesmanolides and two pseudoguaianolides. The lactones were isolated exclusively from aerial parts of the plant. In the subaerial organs, monoterpenoid thymol derivatives were the only lower terpenoids that we managed to isolate [6]. Reynosin and santamarine, the two eudesmanolides found in stems and leaves of X. speciosissima, might have been partly of artifactual origin. Their occurrence in apolar fractions of the plant extract may suggest the presence of their germacranolide precursor costunolide-1,10-epoxide in the analyzed material. Both reynosin and santamarine, as was mentioned before, are of limited usefulness as taxonomic markers due to their occurrence in a number of taxonomically distant species. The compounds demonstrated anti-inflammatory activity in in vitro assays [25,41]. Reynosin inhibited platelet aggregation induced by arachidonic acid, ADP and platelet activating factor (PAF) [42], and protected neurons against dopamine-induced toxicity [43]. The two remaining lactones, pseudoguaianolides, 2,3-dihydroaromaticin and 2-deoxy-4-epi-pulchellin, are frequently found in plants of Inulae-Inulinae, e.g., in Carpesium spp., Inula spp., Ondetia linearis Benth and T. speciosa [44][45][46][47][48][49][50]. The compounds were active in in vitro assays as antiproliferative and anti-inflammatory agents [45][46][47][48].
Phytochemical analysis of aerial parts and flowerheads of B. salicifolium did not reveal the presence of sesquiterpene lactones [15,51]. Four bithiophenes were major non-polar constituents of the plant shoots. In contrast to B. salicifolium, T. speciosa is rich in sesquiterpene lactones of eudesmanolide, pseudoguaianolide and xanthanolide types [39,52]. The compounds could be found in both aerial and subaerial parts of the plant. The roots of T. speciosa contain a large amount of isoalantolactone and may be considered a substitute for Inula helenium roots. X. speciosissima is rather a poor source of sesquiterpene lactones when compared to T. speciosa and Carpesium spp.
Monoterpenoid thymol derivatives are synthesized in B. salicifolium [15], T. speciosa [53], Carpesium spp., [5] Inula spp. [4], X. speciosissima [6] and many other species. Outside the Inuleae, the compounds are frequently found in the Eupatorieae, Helenieae and other members of the Heliantheae alliance. Despite the frequent occurrence in many species of known medicinal use, only few studies have been devoted to the biological activities of the compounds [7]. Recently, a thymol derivative, 8-hydroxy-9,10-diisobutyryloxythymol (constituent of X. speciosissima), was found to inhibit the interaction between p53 (tumor suppressor protein) and its inhibitor, MDM2 protein [54]. Our investigation on anti-inflammatory activity of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate was meant to support the role of monoterpenoid thymol derivatives as active ingredients of plant preparations.
Polymorphonuclear leukocytes (PMNs) play a pivotal role in human immune system. They, among others, participate in fine regulation of the immune response and inflammatory process via the capability to respond to and to produce a variety of cytokins [31]. Cytokines produced by human neutrophils include pro-inflammatory cytokines IL-1β and TNF-α, and chemokines CCL2 and IL-8 (CXCL8) that are implicated in the pathogenesis of inflammatory diseases in humans. To assess an effect of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate on neutrophile function, secretion of the mentioned cytokines by LPS-stimulated human neutrophils in the absence or presence of the tested compound was monitored. Preincubation of human neutrophils with the examined thymol derivative significantly, and in a concentration-dependent manner, diminished f-MLP-induced ROS production by the cells (Figure 5). The tested compound significantly reduced secretion of pro-inflammatory cytokine IL-1β and chemokines CCL2 and IL-8 ( Figures 6A and 7). Only its highest concentration (2.5 µM) significantly affected secretion of TNF-α by LPS-stimulated neutrophils ( Figure 6B). This might be due to very small amounts of TNF-α produced by human neutrophils. The results of our tests suggested that monoterpenoid thymol derivatives, together with sesquiterpene lactones and phenolic compounds, are involved in the anti-inflammatory activity of the Inuleae.

Plant Material
Seeds of X. speciosissima were supplied by the Alpine Botanical Garden "Rezia" (Bormio, Italy). That garden specializes in preservation of the plant species occurring in the Stelvio National Park (Lombardy, Italy). X. speciosissima seeds were germinated following the protocol by Brusa et al. [3]. The seedlings were initially grown in a glasshouse, and later on were planted into the garden. Plants for the phytochemical analysis were collected in the second season of vegetation, in June 2016, from the Garden of Medicinal Plants, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, where the voucher specimen (1/16) was deposited. Roots; stems with leaves and buds; and capitula in bloom were separated and dried under shade, at room temperature.
For the HPLC-DAD analysis of caffeic acid derivatives in leaves of X. speciosissima, and in leaves of Telekia speciosa (Schreb.) Baumg. and Buphthalmum salicifolium L., two species closely related to the examined taxon [2], plant material was collected in May 2017 from perennial plants cultivated in the Garden of Medicinal Plants, Institute of Pharmacology, Polish Academy of Sciences, Kraków. Samples of the leaves were harvested from five individual plants of each of the investigated species at the rosette stage. Each sample (4 or 5 tiny leaves collected from one plant) was processed separately. Results are means of five measurements (± SD).

Estimation of Total Phenolic Content (TPC)
The reducing capacity of the plant material, referred as TPC, was estimated by using a Folin-Ciocalteu colorimetric method. The dry plant material (0.01 g) was extracted twice, for 2 h, with 2 mL of 80% MeOH containing 1% HCl, at room temperature, on a reciprocal shaker. The combined extracts were further analyzed as described by Velioglu et al. [55]. Results are expressed as mg of gallic acid equivalents (GAE) per 1 g of the plant material dry weight and are means of three measurements (± SD).

Preparation of Samples for HPLC-DAD and UHPLC-DAD-MS n Analysis
The dry and pulverized plant material (0.1 g) was extracted twice with 10 mL of 70% MeOH, at room temperature, for 3 h, on a rotary shaker (100 r.p.m.). The extracts were combined and evaporated to dryness under reduced pressure, to give a residue which was either redissolved in 1 mL of 70% MeOH and centrifuged (11,340× g, 5 min) prior to analytical HPLC/DAD separation, or had an aliquot (0.01 g) dissolved in a mixture of MeOH and 0.1% HCOOH (8:2), filtered through 0.45 µm Chromafil membrane (Machery-Nagel, Duren, Germany) and subjected to UHPLC-DAD-MS n analysis.  [56] were applied. The compounds were identified based on their retention time values, online UV spectra, co-chromatography with standard samples and comparison with the results of HPLC-DAD-MS n analysis. Quantification was done using an external standard method as it was described previously [12].

Isolation of Chemical Constituents from a Chloroform Extract of X. speciosissima Aerial Parts
Dried and pulverized aerial parts (325 g) of X. speciosissima (without capitula in bloom) were extracted five times with 1.7 L of CHCl 3 at room temperature with shaking. The combined extracts were concentrated in vacuo, at 40 • C, providing c. 35 g of an oily residue. The residue was subjected to CC on silica using gradients of EtOAc in n-hexane (up to 100% EtOAc) and subsequently MeOH in EtOAc (up to 10% of MeOH). The separated fractions (50 mL each) were combined, as shown by TLC, and further fractionated either by preparative TLC or by semipreparative RP-HPLC. Fractions 74-79 and 86-87, eluted with n-hexane-EtOAc 9:1 (v/v) and n-hexane-EtOAc 4:1 (v/v), respectively, were subjected to prep TLC (n-hexane-EtOAc, 4:1) to give subfractions containing monoterpene thymol derivatives that were previously isolated from roots of the plant [6]. The subfractions were not further separated.  Peripheral venous blood was obtained from healthy human donors (18-35 years old) in the Warsaw Blood Donation Centre. Donors did not smoke or take any medications. They were clinically recognized to be healthy and a routine laboratory tests showed all values to be within the normal ranges. Neutrophils were isolated by dextran sedimentation and centrifugation in a Ficoll Hypaque gradient and then resuspended in (Ca 2+ )-free HBSS buffer or RPMI 1640 medium. Blood samples from three donors were used in each experiment.

Cytotoxicity Measurement
Cytotoxicity was assessed by a standard flow cytometric probe using propidium iodide (PI) staining. After 24 h of incubation in the absence or presence of the tested compound (at concentrations of 0.5, 1.0 and 2.5 µM), the neutrophils (3.5 × 10 5 ) were harvested and centrifuged (1500 r.p.m.; 10 min; 4 • C), washed once with cold PBS and resuspended in 400 µL of PBS. A 5 µL aliquot of PI solution (50 µg/mL) was added to the cell suspension. After 15 min of incubation, in the dark, with PI at room temperature, cells were analyzed by BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and 10,000 events were recorded per sample. The number of cells that displayed high permeability to PI, expressed as a percentage of PI(+) cells, was determined.

ROS Production by Neutrophils
ROS production was measured using luminol-dependent chemiluminescence test. A 70 µL aliquot of neutrophil suspension (3.5 × 10 5 ) in (Ca 2+ )-free HBSS buffer, 50 µL of the tested compound solution and 50 µL of luminol (100 µM) were added to a well in a 96 well plate. ROS production was initiated by the addition of f-MLP (30 µL of 0.1 µg/mL solution) to obtain a total volume of 200 µL per well.
Chemiluminescence changes were measured for 40 min, at 2 min intervals, in a microplate reader (37 • C). The background chemiluminescence produced by non-stimulated cells was also determined. The percentage of inhibition was calculated by comparison to the stimulated control without the tested compound, at the maximum luminescence. 4.8.4. IL-8, IL-1β, CCL-2 and TNFα Production by Neutrophils Neutrophils (2 × 10 6 ) were cultured in 24-well plates in RPMI 1640 medium with 10% FBS, 10 mM HEPES, and 2 mM L-glutamine, in the presence or absence of LPS (100 ng/mL) and in the absence or presence of 7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate (final concentration in a range of 0.5-2.5 µM), at 37 • C with 5% CO 2 . After 24 h, the supernatants were harvested and centrifuged (2000 r.p.m.; 10 min; 4 • C). The amounts of released cytokines were measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions (BD Biosciences, USA). The effects on IL-8, IL-1β, CCL2 and TNF-α production were calculated by comparing the percentages of the released agents to the stimulated control, which lacked the test compound.

Statistical Analysis
The results were expressed as the mean ± SEM of three independent experiments performed at least in duplicate. All analyses were performed using Statistica 13 software. The statistical significance of the differences between means was established by ANOVA with Dunnett's post hoc test p values.

Conclusions
Our research supported a close relationship of X. speciosissima with Carpesium spp. and some species of the Inula genus. The species clearly differs from B. salicifolium with respect to sesquiterpene lactone and tiophene content. In contrast to T. speciosa (and resiniferous species of Inula), X. speciosissima does not accumulate substantial amounts of essential oil, rich in eudesmanolides, in its roots. The composition of polyphenolic fraction of X. speciosissima, despite some differences, places the taxon close to Carpesium spp. and B. salicifolium. The results of the phytochemical investigation are in agreement with the current taxonomic position of X. speciosissima as a separate monotypic genus.
X. speciosissima, a rare plant of the pre-Alpine area, produces specialized metabolites typical of the Inuleae. The majority of them, obtained from other sources, have been extensively studied in respect of their pharmacological activity in vitro. The most distinctive chemical feature of the plant is the occurrence of thymol derivatives of uncommon structures in its roots.