Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect

Abstract

Urginea maritima L. (squill) species is widely spread at the Mediterranean region as two main varieties, i.e., white squill (WS) and red squill (RS), that are recognized for several health potentials. The major secondary metabolite classes of the squill are cardiac glycosides, mainly, bufadienolides, flavonoids, and anthocyanins. Herein, a multiplex MS and NMR metabolomics approach targeting secondary and aroma compounds in WS and RS was employed for varieties classification. Solid-phase micro extraction-gas chromatography/mass spectroscopy (SPME-GC/MS), ultra-high-performance liquid chromatography/mass spectrometry (UPLC/MS), as well as nuclear magnetic resonance (NMR) provided fingerprinting and structural confirmation of the major metabolites for both types of the squill. For comparison of the different platforms' classification potential, multivariate data analysis was employed. While Bufadienolides, viz. "hydroxy-scilliglaucosidin-O-rhamnoside, desacetylscillirosidin-O-rhamnoside and bufotalidin-O-hexoside" as well as oxylipids, were enriched in WS, flavonoids, i.e., dihydro-kaempferol-O-hexoside and its aglycon, taxifolin derivative, were predominant in RS. A cytotoxicity screening against three cancer cell lines, including breast adenocarcinoma (MCF-7), lung (A-549), and ovarian (SKOV-3) cell lines was conducted. Results revealed that WS was more effective on A-549 and SKOV-3 cell lines (WS IC₅₀ 0.11 and 0.4 µg/mL, respectively) owing to its abundance of bufadienolides, while RS recorded IC₅₀ (MCF7 cell line) 0.17 µg/mL since is is rich inflavonoids.

Keywords: NMR; SPME-GC/MS; UPLC/MS; Urginea martima; bufadienolides; metabolomics; squill.

Figure 1

SPME-GC/MS chromatogram of WS and RS headspace volatiles. The corresponding volatile names for each peak followed that listed in Table 1.

Figure 2

UPLC/MS base peak chromatogram of both RS and WS squill varieties. Detections in “positive ionization mode” showing major secondary metabolites.

Figure 3

Chemical structure of bufadienolides in both RS and WS detected using UPLC/MS.

Figure 4

PCA scoring plot (A), loading plot, (B) OPLS-DA scoring (C), and S-plot (D) models in negative ionization mode of RS and WS squill varieties.

Figure 5

PCA scoring plot (A), loading plot (B), OPLS-DA Scoring (C), and S-plot (D) models in positive ionization mode of RS and WS squill varieties.

Figure 6

Structure of the major primary and secondary metabolites detected in squill. Metabolite numbers follow those listed in Table 3 for metabolite identification using 1D and 2DNMR.

Figure 7

¹H-NMR of WS extract. (A) WS at δ 0–5.6 ppm (B) at δ 5.6–8.00 ppm. (C) RS at δ 5.7–7.0 ppm, prescribing characteristic signals for primary and secondary metabolites. Peaks annotated at the spectra labeled as follows: ω-9 fatty acid (M1), ω-6 fatty acid (M2), ω-3 fatty acid (M3), rhamnose (M4), β-glucose (M5), α-glucose (M6), sucrose (M7), alanine (M8), aspartic acid (M9), glycine (M10), tyrosine (M11), tryptophan (M12), bufalin (M13), scillaridin (M14), dihydro kaempferol (M15), and hydroxy coumarin (M16). The compounds spectral data were listed in Table 3.

Figure 8

Cytotoxicity screening viability percentile versus RS and WS concentration (µg/mL) on different cancer cell lines. (A) MCF7 breast adenocarcinoma, (B) A-549 lung, and (C) SKOV-3 ovarian cancer cell lines.