Isolation of Secondary Metabolites from Achillea grandifolia Friv. (Asteraceae) and Main Compounds’ Effects on a Glioblastoma Cellular Model

This study aims at the isolation and structural determination of the secondary metabolites of the herbaceous perennial plant Achillea grandifolia Friv. (Asteraceae). The examination of the non-volatile content of the leaves and flowers of A. grandifolia afforded the isolation of sixteen secondary metabolites. On the basis of NMR spectra, the identified compounds included ten sesquiterpene lactones; three guaianolides—rupicolin A (1), rupicolin B (2), and (4S,6aS,9R,9aS,9bS)-4,6a,9-trihydroxy-9-methyl-3,6-dimethylene-3a,4,5,6,6a,9,9a,9b-octahydro-3H-azuleno [4,5-b]furan-2-one (3); two eudesmanolides—artecalin (4) and ridentin B (5); two sesquiterpene methyl esters—(1S,2S,4αR,5R,8R,8αS)-decahydro-1,5,8-trihydroxy-4α,8-dimethyl–methylene-2-naphthaleneacetic acid methylester (6) and 1β, 3β, 6α-trihydroxycostic acid methyl ester (7); three secoguaianolides—acrifolide (8), arteludovicinolide A (9), and lingustolide A (10); and an iridoid—loliolide (11). Moreover, five known flavonoids, i.e., apigenin, luteolin, eupatolitin, apigenin 7-O-glucoside, and luteolin 7-O-glucoside (12-16) were also purified from the aerial parts of the plant material. We also investigated the effect of rupicolin A (1) and B (2) (main compounds) on U87MG and T98G glioblastoma cell lines. An MTT assay was performed to define cytotoxic effects and to calculate the IC50, while flow cytometry was employed to analyze the cell cycle. The IC50 values of reduced viability during the 48 h treatment for compound (1) and (2) were 38 μM and 64 μM for the U87MG cells and 15 μM and 26 μM for the T98G cells, respectively. Both rupicolin A and B induced a G2/M cell cycle arrest.


Introduction
The genus Achillea has a great pharmacological importance and belongs to the Asteraceae family; this genus includes more than 130 wild-growing species worldwide, which are mainly distributed in the northern hemisphere, and almost 40% occur in the Balkan peninsula [1]. Due to numerous medicinal properties (anti-microbial, antioxidant, antiinflammatory, wound healing activity, anti-diabetic activity), the aerial parts of several members of this genus have been used extensively in folk medicine according to many ethnopharmacological studies [2,3]. Numerous phytochemical studies have reported that members of the genus Achillea are usually terpenoid-rich (mainly sesquiterpene lactones) and phenolic-rich plants with phenolic acids, flavonoids, and coumarins. Moreover, many investigations have proved the cytotoxic effects of natural products (extracts, essential oils, and isolated compounds) from different Achillea species such as A. millefolium L., A. clavennae L., A. talagonica Boiss., A. wilhelmsii C. Koch, A. fragrantissima (Forssk.) Sch. Bip., A. teretifolia Willd., and A. coarctata Poir. [4][5][6][7][8].
Achillea grandifolia Friv. is an herbaceous perennial plant, which is subendemic to the Balkan Peninsula with populations in adjacent Anatolia in Turkey. Previous studies have examined infusions of A. grandifolia with a focus on the antioxidant activity, total phenol, and total flavonoid contents of the extracts, as well as on its essential oil composition [9,10]. The methanol extract from A. grandifolia collected from the Balkan Peninsula is reported to have antibacterial and antioxidant properties [11]. The first research aiming to detect secondary metabolites in A. grandifolia [12] have led to the isolation of 6-hydroxyluteolin 6methyl ether (nepetin), coumarin esculetin, and a flavonol that was assumed to be quercetin dimethyl ether; all were characterized using chromatographic technics (TLC) compared to standards and were coupled with mass and UV spectroscopy. However, no NMR spectra of these compounds have ever been presented [12]. In this line, another older study examining the roots of A. grandifolia have revealed the presence of two piperidides [13].
To date, there are no reports in the literature concerning the isolation of non-volatile terpenoid compounds from A. grandifolia; to fill the existing gap, we report herein the findings of the investigation performed separately on its flowers (compound head inflorescences) and compound leaves. Moreover, the potential anti-proliferative role of rupicoline A (1) and B (2) in cancer cells such as glioblastoma (GBM) has never been studied. Therefore, we investigated the effect of these two main compounds in two GBM cell lines as well.

General Experimental Procedures
Column chromatography (CC) was carried out on silica gel 60 (Merck Art. 9385, Darmstadt, Germany), gradient elution, with the solvent mixtures indicated in each case. Vacuum liquid chromatography (VLC) was carried out on silica gel 60 H (Merck Art. 7736), gradient elution, with the solvent mixtures indicated in each case. Thin liquid chromatography (TLC) was carried out on silica gel plates (Kieselgel F254, Merck, Art. 5554); detection on TLC plates: UV light (absorbance: 254 and 366 nm). For the visualization of the chromatograms on silica gel, vanillin-H 2 SO 4 spray reagent was used. For the highperformance liquid chromatography (HPLC), a Lab Alliance Series III pump equipped with Clarity software and a Shodex RI Detector was used using a C18 ODS1 Spherisorb with a 10µm column that measured 250 mm × 10 mm (Waters).
Spectroscopic NMR data: The 1 H-NMR and 13 C-NMR spectra were recorded in CD 3 OD using AGILENT DD2 500 (500.1 MHz for 1 H-NMR and 125.5 MHz for 13 C-NMR) spectrometers. The chemical shifts are provided in δ (ppm) values relative to TMS (3.31 ppm for 1 H-NMR and 49.05 ppm for 13 C-NMR). The leaf material was detached from stems, and the inflorescences were cut-off from stems before drying. A taxonomically identified voucher specimen (No Lazari D. 7347) has been deposited at the School of Pharmacy of the Aristotle University of Thessaloniki (Greece).

Extraction and Isolation
The air-dried flowers (328.17 g) and air-dried leaves (268.47 g) of A. grandifollia were successively extracted at room temperature with a mixture of solvents (petroleum ether: ether: MeOH/1:1:1) first and with methanol right after the extracts provided with the mixture of solvents were washed with brine. Then, the aqueous layer was re-extracted with ethyl acetate. The extraction procedure was the same as described previously [16]. The organic layer was dried over Na 2 SO 4 and concentrated under reduced pressure to obtain a viscous mass (~8.23 g for the inflorescences and~4.25 g for the leaves).

Viability Assay
The glioma cell lines employed were the U87MG (glioma cell line, used as a reference in neuro-oncology) and T98G (glioblastoma multiforme cell line, also popular in neurooncology studies). The cell lines were exposed to rupicolin A and B at increased concentrations for the viability experiments (0-100 µM). Both compounds were dissolved in dimethyl sulfoxide (DMSO) before incubation. The final concentration of DMSO was below 0.1% in all cases (maximal concentration 0.05% DMSO). Viability calculation was performed as previously described [17]. Briefly, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Life Sciences, Darmstadt, Germany) assay was used to evaluate cell viability. A total of 5000 cells were placed in 96-well plates, and, after 24 h, they were treated with rupicolin A and B at increasing concentrations for additional 48 h without medium change. After incubation, MTT was added. Following the manufacturer's protocol, we performed colorimetric analysis of absorbance which is proportional to cell viability.

Cell Cycle Analysis
Cell cycle analysis was performed as described previously [18]. Cells (10 4 ) were treated with rupicolin A and B at the IC50 value for 48 h. Equal amounts of cells were treated with plain culture media as a negative control. After being treated with trypsin, the cells were harvested, centrifuged, and then rinsed with phosphate-buffered saline solution (PBS) before being exposed to propidium iodide (PI) working solution (50 g/mL PI, 20 mg/mL RNase A, and 0.1% Triton X-100) for 20 min at 37 • C in the dark. Using a flow cytometer, information from the PI fluorescence was gathered to a total count of 10,000 nuclei (FAC-Scalibur, BD Biosciences, San Jose, CA, USA). The cell cycle fractions G0/G1, S, G2/M, and G1/S were calculated using the CellQuest software from BD Biosciences.

Biological Activity
We next analyzed the possible effects of the main compounds (1) and (2) on glioblastoma. The quantification of IC50 values were based on the quantification of MTT fluorescence, which is proportional to viable cell population. The concentration leading to a decrease in 50% of the standard MTT colorimetric absorbance for each cell line corresponds to the IC50. The concentrations used and the respective results are presented in Figure 3, panel B. The IC50 value of reduced viability during the 48-h treatment for rupicolin A (1) and rupicolin B (2) was 38 µM and 64 µM for the U87MG cells and 15 µM and 26 µM for the T98G cells, respectively. Both rupicolin A and B induced a G2/M cell cycle arrest. In the U87MG cells, the G2/M fraction was increased from 20.87% to 54.40% and 37.00% and in the T98G cells from 25.73% to 50.67% and 42.93% following rupicolin A and B treatment, respectively (Table 1). An increase in the S phase was also observed, which was more prominent in the case of the U87 line. Rupicolin A (1) and rupicolin B (2) in higher concentrations induced cytotoxic phenomena, thereby slightly inducing the subG1 cell population (Table 2 and Figure 3). A flow cytometric analysis of the DNA content with a guide for cell cycle fractions quantification is presented in Figure 3.

278
The genus Achillea is well known for its richness in flavonoids. Many studies reveal 279 that apigenin, luteolin, and their derivatives are the main flavonoids in polar extracts of 280 the aerial parts of the plants, since they are detected in many species such as Achillea mille-281 folium [31], A. collina [32], A. sivasica Ç elik & Akpulat [33]. These results are in accordance 282 with the current research on the isolation of compounds 12, 13, 14, 15, and 16.

283
All compounds referred herein were isolated from A. grandifolia for the first time. 284 Moreover, according to our knowledge, this is the first report regarding the occurrence of 285 compounds 3, 6, 11, and 14 in any Achillea species. Further studies are needed to verify 286 whether these compounds are species-specific or are to be found in other Achillea species 287 as well. 288 Rupicolin A and B (1 and 2) have been isolated for the first time from the plant Arte-289 misia tripartita Rydb. subsp. rupicola Beetle (Asteraceae) [19]. It has been suggested that the 290 occurrence of rupicolins in many species of the genus Achillea can serve as a good chemo-291 taxonomical marker [34]. In many cases, there is a co-occurrence of both rupicolin A and 292

Discussion
The genus Achillea is well known for its richness in flavonoids. Many studies reveal that apigenin, luteolin, and their derivatives are the main flavonoids in polar extracts of the aerial parts of the plants, since they are detected in many species such as Achillea millefolium [31], A. collina [32], A. sivasica Çelik & Akpulat [33]. These results are in accordance with the current research on the isolation of compounds 12, 13, 14, 15, and 16.
All compounds referred herein were isolated from A. grandifolia for the first time. Moreover, according to our knowledge, this is the first report regarding the occurrence of compounds 3, 6, 11, and 14 in any Achillea species. Further studies are needed to verify whether these compounds are species-specific or are to be found in other Achillea species as well.
The name of compound 9 (arteludovicinolide-A) has been given after Artemisia luboviciana Nutt. (Asteraceae) was found to be the plant from which this component was isolated for the very first time [24]. Until now, this compound had also been isolated from Achillea chrysocoma [40], A. coarctata [41], and A. millefolium [26].
Acrifolide is a 1,2-seco-guaianoIide hemiacetal that has only been reported, to date, in Achillea species (compound 8). The first report of this compound was in 2000 from the aerial parts of A. crithmifollia [42]. According to our knowledge, acrifolide has only been isolated from plants belonging to the genus Achillea such as A. chrysocoma [40] and A. pseudopectinata Janka [43].
Previous studies [45] have firstly described NMR data about the compound 7, namely, 1β,3β,6α-Trihydroxycostic acid methyl ester, and the triacetate derivative of this natural product was isolated from Artemisia rutifolia Stephan ex Spreng. [46]. It should be noted, however, that this study provides the first report for this compound in a member of the genus Achillea i.e., A. grandifolia.
The first isolation of the eudesmanolide ridentin B (5) was performed from Artemisia tripartita subsp. rupicola [19], but it has also been reported from other plants of the same genus [47] such as A. asiatica Nakai ex Pump. [22], as well as from Achillea coarctata and A. chrysocoma [40].
Artecalin (4) is an eudesmanolide which has also been reported in other plants of the genus Achillea, such as A. biebersteinii [35], A. coarctata and A. chrysocoma [40], and A. ligustica [21]. Artecalin is a widely distributed sesquiterpene lactone in members of the Asteraceae family, since this compound has been isolated also from Artemisia californica Less., A. tripartite subsp. rupicola [48], and Tanacetum santolina C. Winkl [49], among others.
Both rupicolin A and B are guaianolides. According to the literature, these types of sesquiterpene lactones exhibit various medicinal properties such as anthelmintic, antimicrobial, high anti-tumor, antifeedant, root-growth, and germination-inhibiting actions [55]. This report acted as a motive, and, for this reason, the isolated compounds 1 and 2 were also subjected to an anti-proliferative assay on two different glioblastoma cell lines, U87MG and T98G. It is known that natural substances have anti-glioma activity [18,56]. However, this is the first research that examined the anti-proliferative activity of rupicolin B. Notably, a G2/M cell cycle arrest coupled with an induction of the S phase fraction, especially in the case of U87MG cells, is known to be associated with the anti-glioma activity of natural compounds such as deglucohellebrin [18], thereby supporting a similar mechanism of action that may be associated with an anti-inflammatory effect and the action of NF-kB transcription factor. Previous studies have revealed that rupicolin A had a protective effect on iodixanol-induced cytotoxicity in LLC-PK1 cells at concentrations of 10 µM, with a cell survival rate of 75.1 ± 1.9% [15]. The current research offered evidence that natural substances exert anti-cancer effects, thereby suggesting that rupicolin A and B are promis-ing candidates for further research, given the continuous need for novel therapies for the second cause of human mortality [57]. To our knowledge, there are only few reports related to the anti-tumour activities of sesquiterpene lactones [58][59][60], thus also suggesting that guaianolides such as rupicolin A and rupicolin B may exhibit potential antitumor activities against various tumor cell lines. Our research confirmed this potential activity by illustrating the anti-proliferative and cytotoxic action of these compounds in glioma cell lines. To date, there is only one study concerning the anti-tumor activity of rupicolin A in cultured renal tubular cells (LLC-PK1) [15], while there is no study for rupicolin B; the results herein confirmed the anti-tumor activity of rupicolin B for the first time. Furthermore, the results of the current study expand our knowledge on the action of both rupicolin A and rupicolin B on the cell cycle distribution of cancer cells.

Conclusions
The phytochemical investigation of Achillea grandifolia proved that this species is a rich source of sesquiterpene lactones and flavonoids. We extracted and characterized sixteen known compounds from the inflorescences and leaves of A grandifolia. This is the first study leading to the isolation of non-volatile terpenoid compounds from the aerial part of this species of the genus Achillea. The most abundant compounds were rupicolin A (1) and B (2). These compounds were isolated from both the inflorescences and leaves of the species under study. Furthermore, these two main compounds of A. grandifolia were also examined for their anti-proliferative effect on U87MG and T98G glioblastoma cell lines. The preliminary results of the treatment of glioblastoma cell lines with A and B indicate that they have the potential to support novel anti-cancer therapies. Our current studies focus on the validation of the findings reported herein and on the investigation of the mechanism of their biological action with the aim to facilitate their establishment as possible anti-glioma agents.

Funding:
We acknowledge support of this work by the project "Upgrading the Plant Capital (PlantUp)" (MIS 5002803), which was implemented under the action "Reinforcement of the Research and Innovation Infrastructure", which was funded by the operational program "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable. Data Availability Statement: All data referred to or generated in this study are included in tables or figures and are available upon request.