Constituents of Pulicaria inuloides and Cytotoxic Activities of Two Methoxylated Flavonols

Plants of the genus Pulicaria are known for providing traditional medicines, spices, herbal teas, and insect deterrents. Pulicaria inuloides (Poir.). DC. is one of the less chemically studied species within the genus. Hydroalcoholic extracts from roots and aerial parts of P. inuloides were analyzed using the UHPLC-PAD-MSn technique and revealed the presence of six caffeoylquinic and eleven caffeoylhexaric conjugates together with hydroxykaempferol dimethyl ether and quercetagetin trimethyl ether. Moreover, constituents of chloroform extract from the whole P. inuloides plants were isolated and identified by spectroscopic methods. One new and four known caryophyllene derivatives, three thymol derivatives, and four polymethoxylated flavonols were found in the analyzed extract. The structure of the new compound was established by spectroscopic methods (HRESIMS, 1H NMR, 13C NMR, COSY, HSQC, HMBC, NOESY). The cytotoxicity of 6-Hydroxykaempferol 3,7-dimethyl ether and quercetagetin 3,7,3’-trimethyl ether (chrysosplenol C), which are major flavonols isolated from the plant, were tested on prostate epithelial cells (PNT2), prostate cancer cells (DU145 and PC3), human keratinocytes (HaCaT), and melanoma cells (HTB140 and A375). Both flavonols demonstrated moderate cytotoxic activity against PC3 cells (IC50 = 59.5 µM and 46.6 µM, respectively). The remaining cell lines were less affected (IC50 > 150 µM).


Structure Elucidation
Compound 2 was isolated as a colorless amorphous solid. An adduct ion peak at m/z 299.1620 [M + Na] + , which was observed in the HRESIMS spectrum of 2, corresponded to the molecular formula of C 17 H 24 O 3 Na (calculated mass 299.1623). The molecular formula of the compound, established as C 17 H 24 O 3 (Figure 2), indicated six degrees of unsaturation that might be accounted for the two rings system, two olefinic double bonds, and two carbonyl groups.  The NOESY spectrum verified the proximity of H-1β to H-4β and H-12β; H-12β to H-1β and H-8β; H-4β to H-1β, H-3β, and H-5β; H-3β to H-4β and H-5β, as well as the proximity of H-9α to H-8α and H-13α ( Figure 3).

Cytotoxic Activities of 6-Hydroxykaempferol 3,7-Dimethyl Ether and Quercetagetin 3,7,3 -Trimethyl Ether
Compounds 10 and 11, as major flavonoid constituents of the plant, were assayed for their cytotoxic effects against normal and cancer cells (Table 3). Normal prostate epithelial cells (PNT-2), two prostate cancer cell lines (DU145 and PC3), human keratinicytes (HaCaT), and two melanoma cell lines were used in the experiments. The tested compounds were not active toward the keratinocytes and melanoma cells at a dose range of 5-100 µg/mL. Both 10 and 11 demonstrated moderate cytotoxic activity against PC3 cells (IC 50 = 19.64 ± 0.83 and 16.79 ± 0.77 µg/mL, respectively). However, normal prostate epithelial cells and DU145 cells were less susceptible.

Cytotoxic Activities of 6-Hydroxykaempferol 3,7-Dimethyl ether and Quercetagetin 3,7,3'-Trimethyl Ether
Compounds 10 and 11, as major flavonoid constituents of the plant, were assayed for their cytotoxic effects against normal and cancer cells (Table 3). Normal prostate epithelial cells (PNT-2), two prostate cancer cell lines (DU145 and PC3), human keratinicytes (Ha-CaT), and two melanoma cell lines were used in the experiments. The tested compounds were not active toward the keratinocytes and melanoma cells at a dose range of 5-100 µg/mL. Both 10 and 11 demonstrated moderate cytotoxic activity against PC3 cells (IC50 = 19.64 ± 0.83 and 16.79 ± 0.77 µg/mL, respectively). However, normal prostate epithelial cells and DU145 cells were less susceptible.

Discussion
Recent phytochemical investigations of P. inuloides have led to the identification of components in the essential oils from aerial parts of the plant [25] and to the isolation of several polyphenolic and terpenoid constituents, including ent-kaurane diterpenoids, β-sitosterol, daucosterol, and methoxylated flavonols (10,11,13) [22,24]. The bioguided fractionation of the P. inuloides root extract yielded 8,9-epoxy-10-isobutyryloxythymyl isobutyrate (1) as a constituent responsible for the antileishmanial activity of the plant [23]. Despite the interest in the biological activity of P. inuloides extracts, data on secondary metabolites produced by the plant are sparse.
Plants of the Inuleae tribe are rich in hydroxycinnamates that are largely responsible for the antioxidant activity of the plant extracts, and possess potential health-enhancing properties [28,29,[41][42][43]. To our knowledge, hydroxycinnamates of P. inuloides have not been studied before. Extracts from P. dysenterica, analyzed using high-performance liquid chromatography coupled with electrospray ionization and time-of-flight mass spectrometry (HPLC-ESI-TOF-MS), revealed the presence of three isomers of chlorogenic acid (CQA) and four dicaffeoylquinic acids (DCQAs) [11]. However, structural details (such as positions of substitution) were not given. Another metabolomic study, devoted to the composition of extracts from P. crispa and P. incisa, enabled the tentative identification of two isomeric CQAs, caffeic acid, caffeoyl-O-shikimic acid, O-coumaroylquinic acid, dehydro-O-dicaffeoyl-hydroxyferulic acid, tri-O-caffeoyl-hydroxyferulic acid, five DCQA isomers, three isomers of p-coumaroyl-O-caffeoylquinic acid, CQA methyl ether, and two tri-O-caffeoylquinic acids [9]. Again, more detailed structural data are missing.
Except for hydroxycinnamates, HPLC-DAD-MS n analysis of the extract from aerial parts of P. inuloides showed the presesnce of two flavonoids: hydroxykaempferol dimethyl ether and quercetagetin trimethyl ether that correspond to 6-hydroxykaempferol 3,7dimethyl ether (10) and quercetagetin 3,7,3'-trimethyl eter (11) isolated from the CHCl 3 extract of the plant. These two compounds were also isolated by Galala et al. [24] from aerial parts of P. inuloides, and there seemed to be major flavonoid constituents in the plant. According to a study on lipophilic and vacuolar flavonoids from the European Pulicaria species [46], 6-hydroxykaempferol 3,7-dimethyl ether is a constituent of P. dysenterica, and chrysosplenol C (11) is the most common flavonoid in European Pulicaria plants. The latter compound was the subject of several studies that revealed its antiviral [47], cytotoxic [48,49], and inotropic [50] activity. Quercetagetin-3,5,7,3'-tetramethyl ether (13) was isolated from aerial parts of P. inuloides as a compound responsible for the antileishmanial activity of the examined extract [22]. Its isomer, quercetagetin-3,7,3',4'-tetramethyl ether (12), was isolated from P. inuloides for the first time, although the compound was detected earlier in some other species of Pulicaria [46].
Methoxylated flavonols of Artemisia annua L. have recently raised some interest as compounds active against different cancer cell lines in vitro [51,52]. The inhibition of topoisomerase IIα and ERK1/2-mediated apoptosis have been proposed as a possible explanation for their activity. This prompted us to investigate the cytotoxic activity of 10 and 11 against normal and cancer cell lines of prostate and skin origin. The results (Table 3) indicated some selectivity of the cytotoxic effect exerted by the tested compounds. The selective activity of chrysosplenol C (11) against PC3 cells is worth further studies.
In summary, P. inuloides is a plant rich in phenolic and terpenoid metabolites that demonstrates a wide range of biological activities. The most important seems to be the modulatory effect on subcellular signaling pathways that may hold promise for future therapeutic use.

General Methods
NMR spectra were recorded either in CDCl 3 or in DMSO-d 6 on a Bruker AVANCE III HD 400 (resonance frequency 400.17 MHz for 1 H). Optical rotation was determined using a PolAAr31 polarimeter (Optical Activity Ltd., Ramsey, UK). RP-HPLC separations were performed using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector (PAD). Analytical chromatographic separations were carried out either on a Kinetex XB-C18 column (4.6 × 250 mm, 5 µm total particle size; Phenomenex, Torrance, CA, USA; nonpolar compounds) or on a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm; Agilent Technologies, USA; phenolic compounds). Semipreparative RP-HPLC was conducted on a Vertex Plus column (Eurospher II 100-5 C18, 8 × 250 mm; Knauer GmbH, Berlin, Germany), with an isocratic elution, using MeOH-H 2 O mixtures of different polarities, at a flow rate of 1.0-2.0 mL min −1 . The column was coupled to a Knauer P4.1S pump and a dual wavelength UV/VIS detector operating at 210 and 260 nm. Conventional column chromatography was carried out on Silica gel 60 (0.063-0.2 mm, Merck, Germany) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). TLC separations were performed using precoated plates (Silica gel 60 without fluorescence indicator, Art. No 5553, Merck, Germany).

Plant Material
The seeds of Pulicaria inuloides DC. were delivered by the Jerusalem Botanical Gardens of the Hebrew University of Jerusalem (Jerusalem, Israel). Plants obtained from the seeds were cultivated in the greenhouse of the Garden of Medicinal Plants, Maj Institute of Pharmacology, Polish Academy of Sciences in Kraków (Cracow, Poland), and the voucher specimens (3/2019, 10/2020) were deposited there. Plants (roots and aerial parts) were collected at blooming in July 2021 and dried under shade at room temperature.

Estimation of Total Phenolic Content (TPC)
The total phenolic content (the reducing capacity of the plant material) was estimated 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. [59]. In brief, a 0.1 mL aliquot of the extract was mixed with 0.75 mL of the Folin-Ciocalteu reagent diluted with water (10-fold). After 5 min, 0.75 mL of the sodium bicarbonate solution (60 g/L) was added to the mixture. After the following two hours at room temperature, absorbance was measured at 725 nm. The results are expressed as the mg of gallic acid equivalents (GAeq) per 1 g of the plant material dry weight and are the means of three measurements (±SD).

Preparation of Samples for HPLC-PAD and UHPLC-PAD-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 a reduced pressure to give a residue that was either redissolved in 1 mL of 70% MeOH and centrifuged (11.340× g, 5 min) prior to analytical HPLC-PAD separation or its aliquote (0.01 g) was dissolved in a mixture of MeOH and 0.1% HCOOH (8:2), before being filtered through a 0.45 µm Chromafil membrane (Machery-Nagel, Duren, Germany) and subjected to UHPLC-PAD-MS n analysis.

Characterization of P. inuloides Shoot and Root Extracts by HPLC-DAD-MS n Method
UHPLC-PAD-MS n analysis was performed on a UHPLC-3000 RS system (Dionex, Germany) with PAD detection and an AmaZon SL ion trap mass spectrometer with ESI interface (Bruker Daltonik GmbH, Bremen, Germany). Separation was performed on a Zorbax SB-C18 column (150 × 2.1 mm, 1.9 µm) Agilent (USA). The column temperature was 25 • C. 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: 0-60 min, 5-40% B. The flow rate was 0.2 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; the dry gas flow was 9 L/min; the dry temperature was 300 • C; and the capillary voltage was 4.5 kV. Analysis was carried out using a scan from m/z 90 to 2200. Compounds were analyzed in the negative ion mode. The MS 2 fragmentation was obtained for the most abundant ion at the time.

Isolation of Chemical Constituents from a Chloroform Extract of P. inuloides
Dried and pulverized whole plants of P. inuloides (118.4 g) were extracted five times with 0.7 L of CHCl 3 at room temperature with shaking. The combined extracts were concentrated in vacuo at 40 • C, providing c. 4.6 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 if required. Fractions 46-49, eluted with n-hexane-EtOAc 97:3 (v/v), were subjected to a semipreparative RP-HPLC (solvent system: MeOH-H 2 O 7:3; v/v; isocratic elution, flow rate: 2 mL/min) to give subfractions containing monoterpene thymol derivatives 1 (t R = 47.5 min; 6.8 mg) and 4 (t R = 24.5 min; 6.1 mg) together with a new caryophyllene derivative 2 (t R = 62.5 min; 1.5 mg). Fractions 54-55, eluted from the silica gel column with n-hexane-EtOAc 19:1 (v/v), after HPLC separation, conducted as described above, yielded a mixture of compounds containing 3 as a major constituent (t R = 12.0 min; 4.6 mg). The mixture was not further separated as the signals of the compound were clearly visible in the corresponding 1 H NMR spectrum. Combined fractions 56-59 (n-hexane-EtOAc 19:1; v/v) were partly dissolved in MeOH and the MeOH insoluble residue was identified as stigmasterol (6,

Cell Culture and Cytotoxicity Assessment
Cytotoxic activity was tested on human cancer and normal cells, namely: prostate cancer cell lines DU145 (ATCC HTB-81) and PC3 (ATCC CRL-1435), prostate epithelial cells PNT-2 (ECACC 95012613), melanoma cell lines A375 (ATCC CRL-1619) and HTB140 (ATCC Hs 294T) and skin keratinocytes HaCaT (obtained as a kind gift from prof. Marta Michalik, Department of Cell Biology, Jagiellonian University). DU145 cells were grown in a modified Eagle's medium with a low (1.0 g/L) glucose concentration, as well as PC3 and PNT-2 cells in Dulbecco's modified Eagle's media: F12 HAM nutrient mixture. At the same time, melanoma cells and keratinocytes were maintained in a modified Eagle's medium with a high (4.5 g/L) glucose concentration. The culture media (all supplied by Sigma-Aldrich Co.; St. Louis, MO, USA) contained antibiotics and 10% fetal bovine serum (FBS). All cultures were maintained at 37 • C in a humidified 5% CO 2 -containing atmosphere.
The examined flavonols were diluted in the culture media from freshly made stock solutions in MeOH (10 mg/mL) to the working concentrations (from 5 to 100 µg/mL).
Cells were seeded in 96-well plates (1.5 × 10 4 cells/well) and preincubated for 24 h (37 • C, 5% CO 2 ). Then, the culture medium was replaced with a fresh medium containing different concentrations of the tested compounds (5-100 µg/mL). The incubation lasted 48 h. Cell viability was measured by an MTT assay, as previously described [60]. The absorbance at 490 nm was measured using a Synergy II Biotek (BioTek Instruments, Winooski, VT, USA) microplate reader. Cytotoxic activity was assessed based on the cell viability expressed as the percentage of living cells. The results were the means of three independent measurements (±SD). Doxorubicin (Ebewe Pharma GmbH., Unterach, Austria) was used as a reference cytostatic drug. The IC 50 values were determined by plotting the percentage viability of the cells versus the concentration and adequate calculations made using either Excel or the AAT Bioquest website program (https://www.aatbio.com/tools/ic50-calculator, accessed on 4 September 2022).