Carpesium divaricatum Sieb. & Zucc. Revisited: Newly Identified Constituents from Aerial Parts of the Plant and Their Possible Contribution to the Biological Activity of the Plant

Carpesium divaricatum Sieb. & Zucc. has a long history of use as both a medicinal and a food plant. However, except for terpenoids, its chemical constituents have remained poorly investigated. The composition of hydroalcoholic extract from aerial parts of C. divaricatum was analyzed by HPLC-DAD-MSn, revealing the presence of numerous caffeic acid derivatives that were formerly unknown constituents of the plant. In all, 17 compounds, including commonly found chlorogenic acids and rarely occurring butyryl and methylbutyryl tricaffeoylhexaric acids, were tentatively identified. Fractionation of lipophilic extract from cultivated shoots led to the isolation of 12-oxo-phytodienoic acid (12-OPDA), which is a newly identified constituent of the plant. The compound, at concentrations of 0.5, 1.0, and 2.5 μM, significantly reduced IL-8, IL-1β, TNFα, and CCL2 excretion by lipopolysaccharide (LPS)-stimulated human neutrophils. Reactive oxygen species (ROS) production induced by f-MLP was also significantly diminished in the neutrophils pretreated by 12-OPDA. The newly identified constituents of the plant seem to be partly responsible for its pharmacological activity and elevate the value of C. divaricatum as a potential functional food.


Introduction
The genus Carpesium (Asteraceae, tribe Inuleae), following recent taxonomic studies, comprises 25 species distributed in Asia and southeastern Europe [1]. Plants of the genus, due to their anti-inflammatory, antipyretic, detoxifying, anti-oedematous haemostatic, and antimalarial properties, have long been used as traditional medicines in China, Japan, and Korea to treat various ailments [2,3]. Whole herbs or aerial parts of the plants are also eaten, by local communities, as a seasonal vegetable, functional food, or vitamin source [4,5]. Aerial parts of Carpesium plants are also used to prepare brewed beverages or serve as forage [2,5].
The whole plant of Carpesium divaricatum Sieb. & Zucc. has been used in traditional medicine of the Far East as a remedy in colds, fevers, diarrhea, infections of the urinary tract, sore throat, toothache, and ascaridiasis [3]. Phytochemical studies on specialized metabolites of the plant are almost entirely devoted to terpenoids, especially sesquiterpene lactones, which reportedly possess anti-inflammatory and cytostatic properties [3,6,7]. almost entirely devoted to terpenoids, especially sesquiterpene lactones, which reportedly possess anti-inflammatory and cytostatic properties [3,[6][7].

Identification of Trans-12-Oxo-phytodienoic Acid (trans-12-OPDA)
Chromatographic separation of the chloroform extract from aerial parts of C. divaricatum, except for the mixtures of sesquiterpene lactones, which are known constituents of the plant [7], gave several fractions containing substantial amounts of the compound that showed a UV absorption maximum at 221 nm (HPLC-DAD). After multiple fractionation by means of chromatographic methods, the compound was isolated and identified, on the basis of its spectral data (see Section 4.5.), as trans-12-oxo-phytodienoic acid (12-OPDA, Figure 2).

Identification of Trans-12-Oxo-phytodienoic Acid (trans-12-OPDA)
Chromatographic separation of the chloroform extract from aerial parts of C. divaricatum, except for the mixtures of sesquiterpene lactones, which are known constituents of the plant [7], gave several fractions containing substantial amounts of the compound that showed a UV absorption maximum at 221 nm (HPLC-DAD). After multiple fractionation by means of chromatographic methods, the compound was isolated and identified, on the basis of its spectral data (see Section 4.5.), as trans-12-oxo-phytodienoic acid (12-OPDA, Figure 2).   Cytotoxicity of trans-12-OPDA to human polymorphonuclear leukocytes (PMNs) was assessed using propidium iodide (PI) staining and flow cytometry (FACS) analysis. The compound was essentially nontoxic in PMNs at 2.5 μM or lower concentrations ( Figure 3). Solvent DMSO did not induce any cytotoxicity to the cells (data not shown). On the basis of these data, all further experiments were performed using trans-12-OPDA at concentrations up to 2.5 μM.

Reactive Oxygen Species (ROS) Production
Activation of PMNs at a site of inflammation induces an oxidative burst in these cells. The phenomenon is characterized by intense ROS production and liberation of proteolytic enzymes from azurophilic granules. An effect of trans-12-OPDA on ROS generation in PMNs was examined in response to cytochalasin A/N-formyl-Met-Leu-Phe (f-MLP) stimulation. The examined oxylipin efficiently reduced ROS release at 1-2.5 μM (Figure 4). (IL-8, TNFα, IL-1β, CCL2) Neutrophils, in response to stimulation with proinflammatory agonists, e.g., LPS or f-MLP, secrete an array of cytokines and chemokines, including TNFα, IL-1β, IL-8, and CCL2 [18,19]. Human neutrophils were pretreated with trans-12-OPDA before their priming with LPS. Using ELISA, we determined levels of IL-8, TNFα, IL-1β, and CCL2 in the culture medium 24 h after LPS stimulation. Preincubation of human neutrophils with trans-12-OPDA resulted in significant and dose-dependent inhibition of IL-8 production upon LPS stimulation ( Figure 5A). The oxylipin was also active as an inhibitor of TNFα production. A statistically significant reduction in the release of this cytokine was achieved with all tested doses of the compound (see Figure 5B). Both IL-1β and CCL2 production by LPS-treated neutrophils were moderately and dose-dependently lowered by pretreatment with trans-12-OPDA at a dose of 0.5-2.5 μM ( Figure 6A,B).

Reactive Oxygen Species (ROS) Production
Activation of PMNs at a site of inflammation induces an oxidative burst in these cells. The phenomenon is characterized by intense ROS production and liberation of proteolytic enzymes from azurophilic granules. An effect of trans-12-OPDA on ROS generation in PMNs was examined in response to cytochalasin A/N-formyl-Met-Leu-Phe (f-MLP) stimulation. The examined oxylipin efficiently reduced ROS release at 1-2.5 µM (Figure 4).   Neutrophils, in response to stimulation with proinflammatory agonists, e.g., LPS or f-MLP, secrete an array of cytokines and chemokines, including TNFα, IL-1β, IL-8, and CCL2 [18,19]. Human neutrophils were pretreated with trans-12-OPDA before their priming with LPS. Using ELISA, we determined levels of IL-8, TNFα, IL-1β, and CCL2 in the culture medium 24 h after LPS stimulation. Preincubation of human neutrophils with trans-12-OPDA resulted in significant and dose-dependent inhibition of IL-8 production upon LPS stimulation ( Figure 5A). The oxylipin was also active as an inhibitor of TNFα production. A statistically significant reduction in the release of this cytokine was achieved with all tested doses of the compound (see Figure 5B). Both IL-1β and CCL2 production by LPS-treated neutrophils were moderately and dose-dependently lowered by pretreatment with trans-12-OPDA at a dose of 0.5-2.5 µM (Figure 6A,B).

Discussion
Hydroxycinnamates, including chlorogenic acids, as common constituents of fruit, vegetables, and widely consumed beverages (coffee) are intrinsic components of the human diet with potential health-enhancing properties [20,21]. Some recent reports suggest their implication in prevention of so-called lifestyle diseases, such as diabetes [22,23], cardiovascular and neoplastic diseases [24,25],

Discussion
Hydroxycinnamates, including chlorogenic acids, as common constituents of fruit, vegetables, and widely consumed beverages (coffee) are intrinsic components of the human diet with potential health-enhancing properties [20,21]. Some recent reports suggest their implication in prevention of so-called lifestyle diseases, such as diabetes [22,23], cardiovascular and neoplastic diseases [24,25],

Discussion
Hydroxycinnamates, including chlorogenic acids, as common constituents of fruit, vegetables, and widely consumed beverages (coffee) are intrinsic components of the human diet with potential health-enhancing properties [20,21]. Some recent reports suggest their implication in prevention of so-called lifestyle diseases, such as diabetes [22,23], cardiovascular and neoplastic diseases [24,25], oxidative-stress-induced cognitive impairment [26][27][28][29], and obesity [30]. The Asteraceae plants are rich in hydroxycinnamates representing different structural types [21,31]. Plants of the Inuleae tribe are also good producers of acylquinic acids [31][32][33]. Despite their proven value as active plant metabolites, hydroxycinnamates have remained unexplored constituents of Carpesium sp. [3]. In the hydroalcoholic extract from aerial parts of C. divaricatum, we managed to detect 17 caffeic acid derivatives, using the HPLC-DAD-MS n method. Seven of them, including two monocaffeoylquinic acids, four dicaffeoylquinic acids (DCQAs), and 3,4,5-tri-O-caffeoylquinic acid, are quite common within the Asteraceae. Their chromatographic behavior and mass fragmentation patterns have been thoroughly studied before. Thus, their identification was not challenging. Conjugates of caffeic acid with hexaric acid (or acids) have been less investigated. Referring to our previous study, on caffeic acid derivatives from tissue culture of Inula helenium [14], we were able to identify three further compounds as dicaffeoylhexaric acid ([M − H] − 533) and two tricaffeoylhexaric acids ([M − H] − 695). The compounds were formerly reported as constituents of the Asteraceae plants belonging to the tribes Eupatorieae [11], Gnaphalieae [10,12], and Millerieae of the Heliantheae alliance [13,34]. While investigating phenolic compounds from flowerheads of Buphthalmum salicifolium L. (Asteraceae, Inuleae), Heilmann and coworkers [15] isolated 1-isobutyryl-3,5-DCQA. Its molecular ion-586 [M] + and fragmentation pattern in negative ion mode nicely corresponded to those of the compound eluted at 19.8 min. The compound of the same molecular mass, eluted at 19.0 min, is very likely another isobutyryl ester of DCQA. Taking into consideration that esterification of both terpenoid and phenolic metabolites with isobutyric acid and/or 2-methylbutyric/isovaleric acid is quite common within Inuleae [7,35,36], we postulated that the compounds eluted at 22.9 min and 23.8 min were 2-methylbutyryl/isovaleryl-DCQAs. Finally, on the basis of spectral data of leontopodic acid, given by Schwaiger et al. [17], we tentatively identified the three remaining compounds as isobutyryl-tricaffeoylhexaric acid (m/z at 765 [M − H] − ) and 2-methylbutyryl/isovaleryl-tricaffeoylhexaric acids (m/z at 779 [M − H] − ). The estimated contents of 5-CQA (1.23% dry weight) and 3,5-DCQA (1.46% dry weight), together with the relation of peak areas of the two compounds to the areas of remaining hydroxycinnamate signals visible on the chromatogram, allow us to conclude that aerial parts of C. divaricatum could be a rich dietary source of these compounds [37].
12-OPDA is an intermediate in jasmonic acid biosynthesis in plants. The compound itself demonstrates physiological activity as a hormone regulating seed dormancy, embryo development, redox homeostasis, and response to stress [38]. As an optically active molecule, 12-OPDA may exist in four possible absolute configurations (9S,13S; 9S,13R; 9R,13R; and 9R,13S). On the basis of 1 H-NMR spectral data, we were able to exclude cis orientation of side chains in our compound (i.e., 9S,13S and 9R,13R configurations) [39]. Cis or trans orientation of the side chains in 12-OPDA molecule can be easily determined by comparison of the chemical shift values for protons at C-8, C-9, and C-13 of the compound with those reported by Baertschi et al. [39]. Two protons at C-8 of the cis isomer give rise to two multiplets at δ H 1.15 and 1.73 (1H each), whereas both protons at C-8 of the trans-12-OPDA give one signal at δ H 1.49 (2H). Signals of protons at C-9 and C-13 of trans-12-OPDA are shifted upfield of those in the cis-OPDA by c. 0.4 ppm. Discrimination between the 9S,13R trans isomer and the 9R,13S trans isomer was possible by optical activity measurement. The dextrorotary activity of 12-OPDA isolated by us (see Section 4.5.) proved that the compound was (9S,13R)-12-OPDA, i.e., (+)-trans-12-OPDA [40]. Except for the activity as a plant hormone, 12-OPDA exerted cytoprotective effects in human neuroblastoma SH-SY5Y cells subjected to oxidative stress [41], mitigated LPS-induced inflammation in microglia [42], inhibited proliferation of human breast cancer cells [43], and demonstrated antileishmanial activity [44]. Thus, we decided to check whether or not the compound may contribute to the overall activity of C. divaricatum.
PMNs are an essential part of human immune system. They are, among other things, engaged in the inflammation process, which could be a consequence of bacterial infection, environmental stress, or some kinds of neoplastic disease. The neutrophils, except for performing phagocytosis, generating toxic molecules, releasing enzymes, and forming extracellular traps, participate in fine regulation of the immune response and inflammatory process via a capability to respond to and to produce a variety of cytokins [19]. Cytokins produced by human neutrophils include the proinflammatory cytokine IL-1β, TNFα, and the chemokines CCL2 and IL-8 (CXCL8). In order to establish whether or not trans-12-OPDA may influence neutrophil function, we decided to monitor secretion of the mentioned cytokines by LPS-stimulated human neutrophils in the absence or presence of the compound. LPS stimulation of neutrophils is mediated via toll-like receptors (TLRs), and this process includes activation of both the NF-κB and mitogen-activated protein (MAP) kinase pathways. At a site of inflammation, leucocytes are activated and, subsequently, an intense production of ROS by the cells takes place. We observed that incubation of human neutrophils with 12-OPDA significantly and in a concentration-dependent manner diminished f-MLP-induced ROS production by the cells (Figure 4). The examined oxylipin significantly reduced secretion of proinflammatory cytokines (IL-1β, TNFα) and chemokines CCL2 and IL-8 by LPS-stimulated human neutrophils (Figures 5 and 6). The inhibitory effect exerted by the lowest concentration tested (0.5 µM) was slightly weaker towards IL-1β and CCL2 secretion (p < 0.05, Figure 6). The results of our tests suggested that 12-OPDA may be implicated in the anti-inflammatory activity of C. divaricatum preparations. Effects observed in vitro nicely corresponded to the traditional use of the plant.
In conclusion, we would like to emphasize that the aerial parts of C. divaricatum are a rich and still not completely explored source of bioactive molecules. The variety of their chemical structures and bioavailability after ingestion are far from being completely investigated. Though sesquiterpene lactones isolated from the plant have proven activity as cytotoxic and anti-inflammatory agents, they are not the sole compounds responsible for the pharmacological activity of the plant. The set of specialized metabolites produced by C. divaricatum suggests considerable potential for the future application of the plant either as a functional food or as an herbal medicinal product.

Extract Preparation and Characterization by the HPLC-DAD-MS n Method
Dried and pulverized aerial parts of C. divaricatum (0.1 g) were 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. The obtained residue (10 mg) was dissolved in a mixture of MeOH and 0.1% HCOOH (8:2, v/v) and then filtered through a 0.45 µm Chromafil membrane (Machery-Nagel, Duren, Germany). HPLC-DAD-MS n analysis was performed on a UHPLC-3000 RS system (Dionex, Germany) with a photodiode array (DAD) detection and an AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonics GmbH, Germany). Separation was performed on a Kinetex XBC18 column (150 × 2.1 mm, 1.7 µm) Phenomenex (Torrance, CA, USA). The column temperature was 25 ºC. The mobile phase (A) was water/formic acid (100:0.1, v/v) and the mobile phase (B) was acetonitrile/formic acid (100:0.1, v/v). A gradient system was used: 0-10 min 10-25% B; 10-40 min 25-35% B. The flow rate was 0.3 mL/min. The column was equilibrated for 7 min between injections. UV spectra were recorded over a range of 200-450 nm, and chromatograms were acquired at 325 nm. The LC eluate was introduced directly into the ESI interface without splitting. The nebuliser pressure was 40 psi; dry gas flow 9 L/min; dry temperature 300 • C; and capillary voltage 4.5 kV. Analysis was carried out using a scan from m/z 90 to 2200. Compounds were analyzed in a negative ion mode. The MS 2 fragmentation was obtained for the most abundant ion at the time.

Isolation of Human Neutrophils
Peripheral venous blood was taken 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 routine laboratory tests showed all values to be within the normal ranges. The study conformed to the principles of the Declaration of Helsinki. 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.

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 trans-12-OPDA (at concentrations of 0.5, 1.0, 2.5, or 5.0 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 with PI at room temperature, cells were analyzed by a 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 a 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. 4.7.3. 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 trans-12-OPDA (final concentration in a range of 0.5-5 µM), at 37 • C with 5% CO 2 . After 24 h, the neutrophils were harvested and centrifuged (2000 r.p.m.; 10 min; 4 • C). The amount of released cytokines was measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions (BD Biosciences, USA). The effects on IL-8, IL-1β, CCL2, and TNFα 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 ± standard error of 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.
Author Contributions: A.S.: concept of the work, analysis of spectroscopic data, and review of the manuscript; N.K. and J.M.: phytochemical work, isolation of compounds, and involvement in manuscript preparation; B.M., N.K., and A.K.K.: in vitro activity assays; A.K.K.: performance of HPLC-DAD-MSn analyses and involvement in manuscript preparation.
Funding: This research was funded by the Ministry of Science and Higher Education, Poland (statutory activity funding).