Iphiona mucronata (Forssk.) Asch. & Schweinf. A Comprehensive Phytochemical Study via UPLC-Q-TOF-MS in the Context of the Embryo- and Cytotoxicity Profiles

Iphiona mucronata (Family Asteraceae) is widely distributed in the Eastern desert of Egypt. It is a promising plant material for phytochemical analysis and pharmacologic studies, and so far, its specific metabolites and biological activity have not yet been thoroughly investigated. Herein, we report on the detailed phytochemical study using UPLC-Q-TOF-MS approach. This analysis allowed the putative annotation of 48 metabolites belonging to various phytochemical classes, including mostly sesquiterpenes, flavonoids, and phenolic acids. Further, zebrafish embryotoxicity has been carried out, where 100 µg/mL extract incubated for 72 h resulted in a slow touch response of the 10 examined larvae, which might be taken as a sign of a disturbed peripheral nervous system. Results of in vitro testing indicate moderate cytotoxicity towards VERO, FaDu, and HeLa cells with CC50 values between 91.6 and 101.7 µg/mL. However, selective antineoplastic activity in RKO cells with CC50 of 54.5 µg/mL was observed. To the best of our knowledge, this is the first comprehensive profile of I. mucronata secondary metabolites that provides chemical-based evidence for its biological effects. A further investigation should be carried out to precisely define the underlying mechanisms of toxicity.


Introduction
Tribe Inuleae, from the family Asteraceae, comprises a wide variety of plants with diverse ethnomedicinal uses. One good example is the genus Inula L., where several members of this genus have been used for various diseases and health conditions. Some of Inula spp. distributed in China are used as expectorants, antitussives, or bactericides. Other Inula spp. were described in Ayurvedic medicine and some ancient cultures for treating diseases such as inflammation, neoplasm, diabetes, and hypertension. This makes this genus a rich material for investigators [1]. Iphiona Cass. is another genus from the tribe Inuleae which holds some chemotaxonomical relation to Inula. Inula salsoloides Ostenf., also known as Iphiona radiata, has been reported to be used in treating fever and diuresis [1]. This evidence suggests that Iphiona Cass. might also have potential medicinal importance.

Phytochemical Profiling
Metabolite profiling of plant extracts offers more insights into their complex phytochemical nature [7][8][9]. Chemical constituents of I. mucronata aerial parts were analyzed via UHPLC-PDA-CAD-ESI-Q-TOF-MS, which allowed for comprehensive chemical characterization of plant analytes (Table 1 and Supplementary Figures S1-S25). Extracts were analyzed in both positive and negative ion modes as genus Iphiona was reported to contain sesquiterpenes and flavonoids, which preferentially ionize under positive and negative ionization, respectively [10][11][12]. A total of 48 chromatographic peaks belonging to various metabolite classes were detected, including mostly sesquiterpenes, flavonoids, and phenolic acids (Table 1), of which only 3 flavonoids were previously identified in the genus Iphiona. A representative UHPLC-CAD chromatogram of I. mucronata extract is displayed in Figure 1. Structures of selected metabolites identified in the ethanol extract of Iphiona mucronata aerial parts are shown in Figure 2. To the best of our knowledge, this is the first comprehensive profile of I. mucronata secondary metabolites that provides chemical-based evidence for its biological effects. The following paragraphs describe the tentative identification of I. mucronata-specific metabolites.    Table 1.  Figure S2). Nevertheless, the appearance of a product ion at m/z 503 [M-H-60] − characteristic for C-pentoside and the higher abundance of C-hexose fragment relative to C-pentose indicate hexose attachment at the C-6 position. All these findings confirmed the identification of compound 15 as apigenin 6-C-pentoside-8-Chexoside [16].
The product ion spectrum of peak  Figure S5). Similarly, the MS/MS spectrum of compound 30 showed product ions at m/z 476 and 461, due to successive losses of two methyl groups suggesting a methoxylated flavone derivative (dimethoxy-trihydroxyflavone), and another ion at m/z 329 [Ag-H] − corresponding to the elimination of a hexose moiety confirmed the annotation of 30 as dimethoxy-trihydroxyflavone-O-hexoside (tricin-O-hexoside) [20].
Peak 24 [m/z 609.1814 (C 28 H 33 O 15 )] + was annotated as trihydroxy-methoxy-flavone-O-deoxyhexosyl-hexoside (Table 1), showing a product ion at m/z 463 due to the loss of a sugar moiety or deoxyhexose, and a base peak at m/z 301 corresponding to loss of two sugar units [21].

Chlorogenic Acids
The fragmentation behavior of five detected chlorogenic acids has been investigated using LC-MS/MS analysis. Namely, two caffeoylquinic acid (CQA) isomers and three feruloylquinic acid (FQA) isomers could be discriminated against and annotated. These assignments were consistent and in agreement with the reported literature.
It is easy to distinguish 5-CQA (neochlorogenic) and 3-CQA (chlorogenic) acids by their base peaks at m/z 191 after the loss of a caffeoyl moiety. Moreover, it is possible to discriminate between the two isomers by a comparatively higher intense deprotonated caffeic acid ion at m/z 179 in neochlorogenic acid [24]. Accordingly, peaks 6 and 9 [m/z 353.0878 (C 16 Figures S10-S12), a base peak at m/z 193 corresponding to deprotonated ferulic acid is characteristic of 3-FQAs, while a base peak at m/z 191 due to loss of feruloyl moiety differentiates 5-FQA isomers [24]. This led to the identifications of compound 10 as 3-FQA and peaks 14 and 16 as 5-FQAs. It was previously reported that cis-5-acyl chlorogenic acids, being comparatively more hydrophobic, elute appreciably later from reversed-phase column packings than their trans counterparts [25]. Thus, compounds 14 and 16 were identified as trans-5-FQA and cis-5-FQA, respectively.
A discussion is given of the fragmentation processes of 14 guaianolides with voluminous substituents at C-8, thus causing instability of the molecular ions. The compositions of the fragment ions have been determined, and it has been shown that the breakdown of the lactone skeleton takes place only after the elimination of these voluminous substituents at C8, i.e., (M -R 2 OH) + [26].
In the present paper, we consider compounds of the GUAI series ( Figure 2) with a voluminous substituent at C- 8 ) + as ammonium adduct] revealed a fragment ion at m/z 245 and 247, respectively, characteristic of a guaianolide substituted with an exocyclic methylene and a methyl group at C-4, respectively. Such ions were formed after splitting out of 2-hydroxymethyl-acrylic acid [M + H-102] + in both compounds (Supplementary Figures S20 and S21) and led to the identification of compounds 34 and 35 as 8-(2-hydroxymethyl)acryloyloxy-4-methylene-GUAI (cynaropicrin) and 8-(2-hydroxymethyl)acryloyloxy-4-methyl-GUAI, respectively [30,31]. Notably, other sesquiterpene acetates as represented by eudesmol xylopyranosides acetates were previously reported from I. mucronata polar fraction and thus the acetate derivatives could be of chemotaxomomic importance for the Genus Iphiona [32].   Figures S22-S24). All chlorinated guaianolides showed a fragment ion at m/z 297, corresponding to a guaianolide substituted with hydroxyl and chloromethyl groups at C-4, after the loss of

Zebrafish Toxicity
Zebrafish embryo toxicity model is a new approach method that might overcome celland protein-based assays and become a substitute for mammalian testing. The development of zebrafish embryos might be perturbed by tested chemicals and could be manifested in morphological malformations, behavioral abnormalities, or the death of the embryos. The model gives many advantages, fast development and transparency of embryos allow to monitor any sign of toxicity already on the cell level using microscope techniques [39,40].
It was shown that I. mucronata extract has no toxic effects at low concentrations tested (5-40 and 50, 75 µg/mL). The first sign of toxicity was noticed after 72 h incubation at the concentration of 100 µg/mL when all 10 larvae exhibited a slow touch response after a poke at the end of the tail, which might be a symptom of a disturbed peripheral nervous system. Certain sesquiterpenes characterized by Asteraceae plants were previously evaluated for zebrafish embryotoxicity. For instance, an artemisinin metabolite; dihydroartemisinin (DHA) (1-10 mg/L) caused abnormality in embryonic phenotypes, while 10 mg/L of DHA also affected the developmental zebrafish embryo by increasing angiogenesis [41]. On the other hand, the effect of flavonoids on zebrafish embryos is widely varied. In a comparative study by Bugel et al., [42], 15 out of 24 investigated flavonoids evoked negative effects on the tested developmental or behavioral endpoints of zebrafish embryos at concentrations of 1-50 µM.

Cytotoxicity and Antineoplastic Selectivity
The results of cytotoxicity testing presented in Table 2 show that I. mucronata ethanolic extract exerts similar toxicity towards normal VERO cells and two of the cancer cell lineshypopharyngeal squamous cell carcinoma and cervical adenocarcinoma, with CC 50 values between 91.6 and 101.7 µg/mL. Moreover, dose-response curves shown in Figure 3 exhibit similar patterns for VERO, FaDu, and HeLa cells. However, statistically significant (p < 0.05) antineoplastic selectivity (SI = 1.84) was observed in human colon carcinoma (RKO) cells with CC 50 of 54.5 µg/mL. To the best of our knowledge, this is the first report on the in vitro cytotoxicity of I. mucronata. Interestingly, the review of available literature showed the absence of any information concerning cytotoxicity studies of other plants belonging to the genus Iphiona. The observed cytotoxic effect could be owed to the enriched profile with guaianolides, particularly chlorinated ones. Previous studies report on the potential of these sesquiterpenes to induce cytotoxic activity. Sary et al. [43] isolated two chlorinated guaianolides; cenegyptin A and cenegyptin B, from the aerial parts of Centaurea aegyptiaca, where the first compound showed a potent cytotoxic effect against HEPG2 and HEP2 cell lines (IC 50 = 7.2 ± 0.04 and 7.5 ± 0.02 µM). Notably, other chlorinated guaianolides; including chlorojanerin (also identified in the current study) and 19-deoxychlorojanerin, showed high cytotoxic activity against MDA-MB-231 cell lines (IC 50 ; 2.21 and 2.88 µM, respectively) [30]. Centaurea aegyptiaca, where the first compound showed a potent cytotoxic effect against HEPG2 and HEP2 cell lines (IC50 = 7.2 ± 0.04 and 7.5 ± 0.02 µM). Notably, other chlorinated guaianolides; including chlorojanerin (also identified in the current study) and 19-deoxychlorojanerin, showed high cytotoxic activity against MDA-MB-231 cell lines (IC50; 2.21 and 2.88 µM, respectively) [30].

Plant Material
The aerial parts of Iphiona mucronata (Forssk.) Asch. & Schweinf., as a composite sample, were collected during the flowering stage (March 2020) in plastic bags from Wadi Arabah, Northeastern Desert (29 • 1 23.20" N 32 • 10 9.42" E), Egypt. The collected plant was identified according to Boulos (2002) and Tackholm (1974) [2]. Plant material was cleaned of any impurities and air-dried at room temperature (25 ± 3 • C) in shade for 7 days. A voucher specimen (Mans. 0010913004) was prepared and deposited in the Herbarium of Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt.

Preparation of Plant Extract
A total of 100 g of the powdered plant material was extracted by maceration for 10 days using 70% ethanol till complete exhaustion. The filtered extract was then evaporated to dryness at 50 • C using a rotary evaporator. The dried extract was kept at −80 • C for further chemical and biological analysis.

LC-MS and Qualitative Analysis
The extract was analyzed using a high-resolution LC-MS Thermo Scientific Ultimate 3000RS chromatographic system. The separation was carried out on a Waters Acquity HSS T3 column (150 × 2.1 mm i.d.; 1.8 µm, Milford, CT, USA) at 45 • C using a linear gradient from 5% to 70% phase B (acetonitrile with 0.1% formic acid) in phase A (0.1% formic acid in Milli-Q water) for 30 min, with a flow rate of 0.4 mL/min.
The photodiode array detector recorded absorbances in the 190-600 nm wavelength range with 5 nm bandwidth and 10 Hz acquisition frequency. A flow splitter was used to divert the column effluent in a proportion of 1:3 between qTOF MS (Bruker Impact II HD, Bruker, Billerica, MA, USA) and charged aerosol detector (CAD, Thermo Corona Veo RS) linked in parallel. The acquisition frequency for CAD was 10 Hz.
The MS analyses were operated in both positive and negative ion modes, using electrospray ionization. Linear spectra were obtained in the m/z 80 to m/z 1800 mass range, with 5 Hz acquisition frequency and the following parameters of the mass spectrometer: negative ion capillary voltage 3.0 kV; positive ion capillary voltage 4.0 kV; dry gas flow 6 L/min; dry gas temperature 200 • C; collision cell transfer time 90 µs; nebulizer pressure 0.7 bar. The obtained data were calibrated internally with sodium formate introduced into the ion source via a 20 µL loop at the start of each separation. The chromatographic data were acquired and processed using Bruker DataAnalysis 4.4 software, and metabolite structure elucidation and identification were achieved mostly using SIRIUS 4.8.2 software integrating CSI: FingerID for searching in molecular structure databases [44,45].

Zebrafish Embryo Toxicity (ZET) Assay
Zebrafish (Danio rerio) stocks of the AB strain were maintained at 28.5 • C on a 14/10 h light/dark cycle under standard aquaculture conditions, and fertilized eggs were collected via natural spawning. Embryos were reared under 14/10 h light/dark conditions in embryo medium: 1.5 mM HEPES, pH 7.1-7.3, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO 4 , and 0.18 mM Ca(NO 3 ) 2 at 28.5 • C. The 4-hpf embryos were placed in 48-well plates, 5 embryos per well, and then incubated in 4 different concentrations of tested extract-10 embryos per concentration (n = 10). After 24, 48, and 72 h, the embryos were checked under the microscope for any signs of cytotoxicity such as coagulation of the embryo, a lack of somite formation, non-detachment of the tail, and/or a lack of heartbeat. Each day extract solution was changed. Two ranges of concentrations were tested, first 20-50 µg/mL and then 40-100 µg/mL.
The cytotoxicity testing was carried out on VERO (ECACC, 84113001, kidney of a normal adult African Green monkey), FaDu (ATCC, HTB-43, human hypopharyngeal squamous cell carcinoma), HeLa (ECACC 93021013, human cervical adenocarcinoma), and RKO (ATCC CRL-2577, human colon carcinoma) cell lines using MTT assay as previously described [46]. In short, selected cell lines seeded in 96-well plates were incubated with serial dilution (500-1 µg/mL) of Iphiona mucronata extract stock solution in cell media for 72 h. Cells supplemented with complete media were used as a non-treated control. Subsequently, extract containing cell media was discarded, plates were washed with PBS, cell media with MTT was added, and plates were further incubated for 4 h. Finally, to dissolve the violet formazan product, the SDS/DMF/DMSO-based solvent was added, and the plates were incubated overnight. The absorbance (540 and 620 nm) was measured using Synergy H1 Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). The GraphPad Prism (version 7.04) software was used for data analysis, and the CC 50 values (concentrations decreasing the cellular viability by 50%) were calculated from doseresponse curves. The CC 50 values were expressed as mean ± SD (n ≥ 3). The antineoplastic activity was evaluated based on selectivity indexes (SI; SI = CC 50 VERO/CC 50 Cancer), where SI > 1 indicates selectivity.