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

Khattab, Omar M.; El-Kersh, Dina M.; Khalifa, Shaden A. M.; Yosri, Nermeen; El-Seedi, Hesham R.; Farag, Mohamed A.

doi:10.3390/plants12112078

Open AccessArticle

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

by

Omar M. Khattab

^1,†

,

Dina M. El-Kersh

^2,†

,

Shaden A. M. Khalifa

³,

Nermeen Yosri

⁴

,

Hesham R. El-Seedi

^1,5,6,* and

Mohamed A. Farag

^7,*

¹

Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Kom 32512, Egypt

²

Department of Pharmacognosy, Faculty of Pharmacy, The British University in Egypt, Cairo 11837, Egypt

³

Psychiatry and Psychology Department, Capio Saint Göran’s Hospital, Sankt Göransplan 1, 112 19 Stockholm, Sweden

⁴

Chemistry Department of Medicinal and Aromatic Plants, Research Institute of Medicinal and Aromatic Plants (RIMAP), Beni-Suef University, Beni-Suef 62514, Egypt

⁵

International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013, China

⁶

International Joint Research Laboratory of Intelligent Agriculture and Agri-Products Processing, Jiangsu Education Department, Zhenjiang 212013, China

⁷

Pharmacognosy Department, College of Pharmacy, Cairo University, Kasr El Aini St., Cairo 11562, Egypt

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Plants 2023, 12(11), 2078; https://doi.org/10.3390/plants12112078

Submission received: 14 March 2023 / Revised: 29 April 2023 / Accepted: 16 May 2023 / Published: 23 May 2023

(This article belongs to the Section Plant Physiology and Metabolism)

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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:

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

1. Introduction

Urginea, closely related to the “Drimia” genera belonging to family Asparagaceaeis, is widely spread at the Mediterranean region, Africa and India [1]. In Arabic, it is known as Basal farion, Onsul, and Samm el-Far [2]. Other common names are termed squill and sea onion [3]. U. martima species comprises two varieties to include white and red, with both recognized for their bulb’s medicinal value and abundance of cardiac glycosides [4,5]. Stoll and coworkers isolated cardiac glycosides of U. maritima for the first time in structure similar to that of the Bufo genus termed bufadienolides, though it was less potent than digitalis cardiac glycosides [6]. U. maritima bulbs displayed a wide spectrum of pharmaceutical potentials. U. maritima bulbs enriched with “cardiac glycosides” Bufadienolides, viz. glucoscillarene A, proscillaridine A, scillarene A, scilliglaucoside and scilliphaeoside. Those bufadienolides involved in the cardiovascular system (CVS) via inhibiting Na⁺/K⁺ ATPase, which increases intracellular Ca⁺², leading to enhancement of the contractility of cardiac muscles (positive inotropic effect) [6,7]. The methanolic extract and methylene chloride fraction of Urginea bulbs were reported as being antibacterial, anthelmintic, diuretic, and expectorant. Fresh bulbs were used for wound healing [7,8]. Besides its CVS effect, it possesses potential cytotoxic activity against lymphoma and breast cancer human cell lines in addition to its antirheumatic and antimicrobial actions [1,3,7]. The red variety of U. maritima differs from white squill mostly by the presence of red pigments, i.e., anthocyanins. Anthocyanins, such as cyanidin-3-monoglucoside and pelargonidin-3-monoglucoside, either free or acylated with caffeic or p-coumaric acid, were previously reported from the red bulbs of Spanish U. maritima. Taxifolin, taxifolin-4’-glucoside, quercetin-3-glucoside, C-flavone glycosides, and caffeic acid were isolated as major phenolic components [9]. Traces of fixed oil and volatile oil were also reported in addition to lignans and fatty acids [1,2,3,7,10,11,12]. Egyptian U. maritima showed more complex types of bufadienolides from its bulbs, which are from other Urginea species collected from Turkey, Greece and Tunisia [13]. Herein, we aimed to adopt a metabolomics approach for the classification of RS and WS, targeting their metabolome, including volatiles, as well as the secondary metabolites via multiple analytical platforms, viz. SPME/GC-MS, 1D, 2D NMR, and UPLC-MS, respectively. For data visualization and untargeted classification, multivariate data analyses, unsupervised learning, i.e., principal component analysis (PCA), and supervised learning, i.e., orthogonal projection to latent structures discriminant analysis (OPLS-DA) models, were applied. Both PCA and OPLS focus on identifying variances and similarities among specimens as extensively reported in our similar previous work using the same comparative MS and NMR platforms in metabolomics analysis of rosella [14,15,16].

2. Results and Discussion

2.1. Identification of Volatile Organic Compounds (VOCs)

The volatiles profiling of RS and WS was determined using headspace SPME coupled with GC-MS, as reported in the current study, for the first time. A total of 26 volatiles were identified belonging to monoterpenes (10), aliphatic hydrocarbons (6), oxygenated compounds (4), in addition to a heterocyclic, an aromatic, and a sesquiterpene, as listed in Table 1. Due to the higher percentiles of monoterpenes and aliphatic hydrocarbons in RS compared to WS (47.82% and 37.26% vs. 25.69% and 19.76%), respectively, RS had a stronger aroma profile than WS. Other classes found exclusively in WS included heterocyclics, oxygenated hydrocarbons, aromatics, sesquiterpenes, oxygenated monoterpenes, ca. 4.57%, 12.15%, 1.75%, 8.22% and 2.29%, respectively, as shown in Figure 1.

2.2. Secondary Metabolites Profiling via UHPLC/MS

UPLC-MS profiling was employed for secondary metabolites, and profiling in WS is denoted in pink color versus RS in red color. Figure 2 highlights the major differences between the two varieties. Diversity of secondary metabolite classes were identified in both squill, including mostly cardiac glycosides “bufadienolides” (50) followed by flavonoids (40) including“flavonols, flavanones and dihydroflavonols”. Minor classes of coumarins (1), anthocyanins (2), and phenolic acids (5) were detected, with full spectral data of identified peaks presented in Table 2.

The UV spectra of the major classes of secondary metabolites were observed at λ 280–299 nm. For bufadienolides glycosides, they were observed at 292–299 nm [17], while phenolics showed UV max at 280–283 and 296–340 nm [18]. Different classes have been annotated in both white and red squill, viz. bufadienolides, flavonoids, phenolic, amino, and fatty acids, as detailed in the next subsections. All identified secondary metabolites were listed in Table 2 with Supplementary Figures showing typical fragmentation pattern supporting their identification.

2.2.1. Bufadienolides

Bufadienolides are C-24 steroids with a pyranone ring at C-17β that are naturally present in plants and mammalian animals, specifically toads and snakes [19]. Bufadienolides were found more abundant in WS than RS as revealed from the inspection of the chromatograms of both WS and RS at retention time “R_t.” from 5 to 13 min. A series of different classes of bufadienolides were identified in WS and RS and included hydroxy-oxobufadienolide with a C-19 aldehyde group, i.e., bufotalidin, and hydroxybufaenolides with a C-19 methyl group, including scillarenin and scilliphosidin. Notably, identified bufadienolides structures were illustrated in Figure 3.

Hydroxyoxobufaenolide

Hydroxyoxobufaenolides were detected in multiple peaks L53, L37, L49, while L36, L63, L67 and L68 were annotated as glycosides of hydroxyoxobufaenolides [20,21,22]. L36, L63, L67, and L68 were identified as hydroxyoxobufaenolide glycosides based on the loss of −162 or 146 amu. in L36 desacetylscillirosidin-O-thevetoside (m/z 577, (C₃₁H₄₄O₁₀)⁺) (Figure S1A), L63 Hydroxyscilliroside (m/z 637, (C₃₂H₄₄O₁₃)⁺) (Figure S1B) [23], L67 m/z 579 bufatalidin-O-hexoside, and L68 m/z 545 (C₃₀H₄₁O₉)⁺ [M+H]⁺ scilliglaucosidin-O-rhamnoside (Figure S1C).

Identified hydroxybufaenolides are subdivided by the number of hydroxyl groups to mono, di, tri and tetra hydroxyl derivatives and were identified in both WS and RS as listed in Table 2.

The L64 peak m/z 367 (C₂₄H₃₁O₃)⁺ [M+H]⁺ was detected in both WS and RS as monohydroxybufa-tetraenolide “Scillaridin A” [24,25,26] as an example of a monohydroxybufa-enolide, illustrated in Figure S1D.

Dihydroxybufaenolides were detected in several peaks including L82 m/z 387 (C₂₄H₃₅O₄)⁺ [M+H]⁺ dihydroxybufadienolide “bufalin” [27] and L70 m/z 385 (C₂₄H₃₃O₄)⁺ [M+H]⁺ dihydroxy-bufa-trienolide “Scillarenin” [28,29,30]. Glycosidic conjugates were observed in L91 m/z 531, showing a loss of rhamnose (−146 amu) (Figure S1E) and L86 m/z 693 (C₃₆H₅₃O₁₃)⁺ [M+H]⁺ with a loss of hexose (−162 amu) (Figure S1F). L91 and L86 annotated as scillarenin-O-rhamnoside “Proscillaridin A” [11,31,32,33] and proscillaridin A-O-glucoside, respectively.

Acylated bufaenolides were detected in L90 at m/z 607 (C₃₂H₄₇O₁₁)⁺ [M+H]⁺ and annotated as acetyl-scilliphaeoside. L39 at m/z 403 (C₂₄H₃₅O₅)⁺ [M+H]⁺ annotated as trihydroxybufa-dienolide “gamabufotalin” [29,34,35] (Figure S1G).

Other acetylated tetrahydroxybufaenolide were detected in peak L54 at m/z 477, (C₂₆H₃₇O₈)⁺ (M+2H)⁺ identified as hydroxyscillirosidin with a loss of acetyl group likely at C-16 [26,36,37] (Figure S1H).

2.2.2. Flavonoids

Compared to WS’s abundance of bufadienolides, flavonoids were more abundant in RS, with several being reported for the first time in this current study. Beside a positive mode, improved detection of flavonoids was observed in a negative mode, with MS2 fragments found to be characteristic of flavonoids, i.e., amu 151, 125 and 153 as well as neutral loss of amu −28 “CO”, −18 “H₂O” and −44 “CO₂” [38,39]. The next subsection shall summarize identification of the different flavonoid subclasses and distribution in Urginea species.

Identification of Flavanols/Flavonols

Identified flavanols in L22 and its methylated derivative in L24 at m/z 305 (C₁₅H₁₃O₇) ⁺ [M+H] ⁺ and m/z 319 (C₁₆H₁₅O₇)⁺ were annotated as dihydroquercetin (taxifolin) and dihydroisorhmentin (methyl taxifolin), respectively [40]. Flavonols were also detected in peak L40 at m/z 303 (C₁₅H₁₁O₇)⁺ [M+H]⁺ identified as quercetin, as well as its glycosides L41 and L32 (Table 2).

Another major flavanol was detected in peak in RS L17 m/z 289 (C₁₅H₁₃O₆)⁺ [M+H]⁺ and N13 m/z 287 (C₁₅H₁₁O₆)⁻ [M-H]⁻ and were annotated as dihydrokaempferol [29,41,42] along with its glycoside in L16 (Figure S1I), N6 (Figure S1J), N7, N9 and N10. Furthermore, catechin and its hexoside belonging to flavanols, (C₂₁H₂₃O₁₁)⁻ and (C₁₅H₁₅O₆)^+, were detected in red squill as N3 and L14 (m/z 451 and 291), respectively [43].

Flavonols, such as N16 m/z 465 (C₂₁H₂₁O₁₂)⁻ and N24 m/z 447 (C₂₁H₁₉O₁₁)⁻ with loss of 180 amu (hexose + H₂O) moieties and −162 hexose moiety, were identified as kaempferol-O-glucoside [44] (Table 2).

Identification of Flavones/Flavanones

Compared to the abundance of flavonols, the flavone subclass was detected in few peaks exemplified in L20 at m/z 271 (C₁₅H₁₁O₅)⁺ [M+H]⁺ apigenin and its sugar glycoside in L28 and L33 (Table 2) [41]. L93 m/z 285 (C₁₆H₁₃O₅)⁺ was interpreted as dihydroxymethoxyisoflavone [45].

L94 m/z 317 (C₁₇H₁₇O₆)⁺, annotated as dihydroxy dimethoxy flavanone, represented an example of flavanone as well as N22 m/z 595 (C₂₇H₃₁O₁₅)⁻, with MS₂ m/z 271 attributed to the loss of two hexose units (−324 amu) and annotated as naringenin-O-dihexoside [46].

Finally, N12 m/z 479 (C₂₂H₂₃O₁₂)⁻ is annotated as noidesol A or B [47], and this is the first report of Noidesol in Urginea species (Figure S1K).

Compared to flavonols richness in red squill, anthocyanins were likewise identified exclusively in RS, accounting for its characteristic reddish color. Major anthocyanins included L3 at m/z 475 (C₂₁H₃₁O₁₂)⁺, annotated as hydroxycinnamyl-O-dihexoside [9] (Figure S1L), and L13 at m/z 307 (C₁₅H₁₅O₇)⁺ [M+H]⁺, identified as leucocyanidin [3] (Figure S1M).

Identification of Coumarins

The hydroxycoumarin class has been annotated in both squill types, represented as L29 and N15 m/z 163 (C₉H₇O₃)⁺ and 161 (C₉H₅O₃)⁻, respectively, and is in agreement with previous reports for coumarins in U. indica species [48,49].

2.2.3. Phenolic Acids

Phenolic acids are aromatic secondary plant metabolites found ubiquitously in plants and play a role in food quality and their organoleptic properties [50]. The primary detected phenolic acid was vanillic acid in L26 at m/z 169 (C₈H₉O₄)⁺, and several glycosidic conjugates in peaks L25, L27, and N5, showing MS₂ ion fragments of vanillic acid. L25 at m/z 447 (C₂₀H₂₉O₁₃)⁺ vanillic-O-rhamnosyl-O-hexoside, L27 m/z 331 (C₁₄H₁₉O₉)⁺ vanillic acid-O-hexoside [51], and N5 at m/z 491 (C₂₀H₂₇O₁₄)⁻ as vanillic acid-O-dihexoside with MS₂ m/z 167 [26] in a negative ionization mode was detected. Other detected phenolic acids included in L105 at m/z 329 (C₁₇H₂₉O₆)⁺ as spiculisporic acid and N14 at m/z 473 (C₂₁H₂₉O₁₂)⁻, a glycoside of dimethoxyhydrocinnamic acid [52].

2.2.4. Amino Acids and Fatty Acids

Few amino acids were identified in both squills, including inL12 at m/z 205 (C₁₁H₁₃N₂O₂)⁺, L8 at m/z 182 (C₉H₁₂NO₃)⁺, and L9 m/z 166 (C₉H₁₂NO₂)⁺ for tryptophan, tyrosine, and phenyl alanine, respectively. Compared to amino acids showing earlier elution, fatty acids were observed at the end of the chromatogram, considering their lypophilic nature in both varieties. An example of major fatty acids includes L114 at m/z 279 (C₁₈H₃₁O₂)⁺ [M+H]⁺ octadecatrienoic acid and L120 at m/z 281 (C₁₈H₃₃O₂)⁺ linoleic acid. No difference in amino acids was observed among squill varieties compared to flavonoids; however, they later appear as stronger marker for variety type.

2.3. Multivariate Data Analysis of UPLC-MS Dataset

To aid in identifying further markers for each squill variety in an untargeted manner, unsupervised principle component analysis (PCA) and orthogonal projection to latent structures analysis (OPLS-DA) were attempted in both negative (Figure 4) and positive ionization modes (Figure 5). Complete segregation between the two squill varieties was observed, highlighting that RS was more rich in phenolics, whereas WS was abundant in bufadienolides.

A score plot model derived from the negative ionization mode of UHPLC/MS prescribed by PC1 and PC2 accounted for 72 and 17%, respectively, of the total variance (Figure 4A), with clear segregation of RS from WS. As for revealing the metabolites mediating RS and WS segregation, a PCA-loading plot (Figure 4B) revealed an abundance of flavanols, identified as dihydrokaempferol-O-hexoside and taxifolin-O-hexoside derivatives, in RS, which are likely to serve as precursors for anthocyanins solely found in RS variety. The OPLS-DA model (Figure 4C) further confirmed the PCA results from the S-plot (Figure 4D) revealing that dihydrokaempferol-O-hexoside, its aglycon, and vanillin rhamnoglucoside, were abundant in RS compared to WS.

The results from the UHPLC/MS-derived model in the positive ionisation mode were comparable to those in the negative mode (Figure 5A). The two squill varieties, RS and WS, were clearly separated in the PCA model, which was prescribed by PC1 66% and PC2 23%. Furthermore, according to Figure 5B, in accordance with the negative ionisation mode, RS was more abundant in dihydrokaempferol, which is a biosynthetic precursor for anthocyanins in RS [53]. On the other hand, WS had higher concentrations of bufadienolides, such as hydroxy-scilliglaucosidin-O-rhamnoside, desacetylscilliglaucosidin-O-rhamnoside, and bufotalidin-O-hexoside as well as oxylipids, such as linoleic acid and linoleyl alcohol. Supervised OPLS-DA model. Figure 5C,D confirmed linoleic acid enrichment in WS and identifying dihydro-kaempferol “aromandrin” and the bufadienolide “scillipheoside-O-glucoside” as markers for RS.

2.4. NMR Metabolites Fingerprinting

To provide a broader coverage of squill metabolome, NMR was employed to provide insight on both secondary and primary metabolites, especially with the later class not detected using LCMS. NMR offers also improved structural elucidation tool aided by its extensive 2D NMR experiments and quantitative determination of the major metabolites [54] for quality control purposes. Major classes detected in squill using NMR included sugars, flavonoids, bufadienolides, phenolics, and amino and fatty acids (Figure 6 and Table 3). Nevertheless, compared to MS, NMR suffers from low sensitivity and from signal overlap, especially in aliphatic regions. To overcome the problem of signal overlap, 2D NMR experiments were employed to allow for the resolution of overlapped signal along the second dimension, i.e., carbon in the case of HMBC [55,56]. ¹H NMR spectra from WS showed the signals relative richness in both varieties (Figure 7 and Table S1).

2.4.1. Fatty Acids

A key feature of the assignment of unsaturated fatty acids (M1, M2 and M3) was based on the signals (-CH₃) δH 0.89 ppm, a long chain of methylene groups (-CH₂)_n at δH 1.2 ppm, and olefinic bond (s) at δH 5.30–5.37 ppm, and HSQC cross-peak correlation with ¹³C showed signals at δ 15.7, 31.9, 128.8–132.1 ppm, respectively. Additionally, confirmation of unsaturation in fatty acids was based on allylic methylenes resonating at δ 2.05–2.10 ppm and correlated with ¹³C at δ 29.4 ppm. Bis allylic (-CH₂) with two triplets at δ 2.76 and 2.78 with ¹³C at δ 26.2 and 27.8 ppm were correlated to ω-6 (M2) and ω-3 (M3) fatty acids, respectively. Furthermore, confirmation of these data was done using other 2D NMR experiments, as HSQC and HMBC displayed in Figures S2–S4.

2.4.2. Sugars

Rhamnose (M4), β-glucose (M5), α-glucose (M6), and sucrose (M7) were the major sugars detected in squill using NMR. Rhamnose (M4) was assigned based on its terminal CH₃ at δH 1.22 and ¹³C at δ 13.8 ppm showing COSY correlation to anomeric proton at δ 3.34 and 13.51 ppm. Moreover, M5, M6, and M7 were identified based on the anomeric protons at δ 4.48 (d, J = 7.8 Hz), 5.10 (d, J = 3.7 Hz), and 5.39 (d, J = 3.8 Hz) ppm, respectively, alongside their respective ¹³C at δ 99.5, 95.2, and 94.8 ppm detected in HSQC (Figure S5).

2.4.3. Amino Acids

A total of five amino acids were identified in squill, including alanine (M8), aspartic acid (M9), glycine (M10), tyrosine (M11), tryptophan (M12). In details, alanine (M8) showed methyl group signals at δ 1.47 (d, J = 7.2 Hz) ppm. Moreover, M9, M10, and M11 showed methylene group signals at δ 2.95, 3.87, and 2.79 ppm. In Figure S2, the previously mentioned protons signals showed HSQC cross-peak correlation with ¹³C at δ 36.8, 45.1, and 37.3 ppm for M9 and M10, respectively. There were long-range HMBC correlations with carbons at δ 51.47 for alanine (M8) (Figure S3) while 52.6, 174.9, and 174.5 for aspartic acid (M9) (Figures S3 and S4) and 53.5 for tyrosine (M11) confirmed their assignments. Furthermore, a 1,4 di-substitution benzene ring appearing at δH 6.70 and 7.12 (d, J = 8.6 Hz) showed that HSQC correlations, alongside their ¹³C cross peaks at δ 118.0 ppm and 132.8 ppm were, assigned for tyrosine (M11) (Figure S6). Tryptophan (M12) was characterized from its indole moiety two triplet signals at δ 7.05 and 6.66 (t, J = 7.5 Hz), two doublets at δ 7.37 ppm and 7.69 ppm (d, J = 7.5 Hz), in addition to a singlet at δ 7.20 ppm, showing HSQC cross-peak correlation with ¹³C at δ 121.3, 117.4, 113.7, 120.5, and 126.5, respectively (Figure S6).

2.4.4. Bufadienolides

A key feature of bufadienolides assignment in ¹H-NMR spectra are signals of α, β-unsaturated ketone of pyranone ring at δ 6.26, 7.93 ppm showing total correlation at δ 7.41 ppm, showing HSQC cross-peak with ¹³C at 116.7, 150.5 and 151.8 ppm, respectively. Quaternary carbons C-20 and C-23 at δ 125.9 and 164.8 ppm, respectively were identified from by HMBC distinct correlation and aiding in their assignment. A major bufadienolide “bufalin” (M13) was detected in both squill identified from CH signals at C-17 δ 2.54, C-5 2.16, and C-3 3.84 ppm showing HSQC cross-peak correlation with ¹³C at δ 42.5, 54.2 and 72.4 ppm, respectively (Figures S2, S5 and S6). Two methyl groups at δ 0.74 and 0.98 ppm with ¹³C at δ 18.5 and 23.2 ppm alongside a quaternary C-14 appearing at ¹³C δ 85.4 in HMBC spectrum (Figure S3), respectively. Furthermore, “scilliridin” (M14) another bufadienolide was recognized from two conjugated double bonds at δ 5.71, 5.75 and 5.88 ppm showing HSQC cross-peak correlation with ¹³C at 128.5, 129.3, and 129.8 ppm, respectively, (Figure S6). Further distinct HMBC cross peaks identified C-5 at δ 141.3 ppm (Figure S7).

2.4.5. Coumarins and Flavonoids

Hydroxy coumarin (M16) was identified based on α- and β-unsaturated ketone with signals at 6.18 and 7.85 ppm (d, J = 7.9 Hz), showing HSQC cross-peak correlations at 113.60 and 147.30 ppm, respectively (Figure S6). Moreover, an ABX benzene ring was revealed from signals at δ 6.71(d, J = 2.3 Hz), 6.80 (dd, J = 8.5, 2.3 Hz), and an overlapped peak at 7.4 ppm, showing HSQC cross-peak correlation at δ 104.63, 115.76, and 131.94 ppm, respectively (Figure S7).

Flavonoids were predicted primarily in RS and in accordance with UPLC-MS results are shown in Figure 2 and Table 2. An AABB system on chemical shift 6.98 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 8.4 Hz, 2H), and chroman ring as AB system, m-position were elucidated from δH 6.04 (d, J = 1.8 Hz, 0H), and 6.04 (d, J = 1.8 Hz, H) was assigned to dihydrokaempferol (M15), (Figure S8).

2.5. Quantification of Major Metabolites via ¹H-NMR

To aid in standardization of squill extract, ¹H-NMR was further used to determine absolute levels of major metabolites in squill varieties via integration of their well-resolved signals in NMR spectra [57]. The concentration of metabolites was calculated as µg/mg dry powder, as shown in Supplementary Material Table S1.

With regard to primary metabolites, unsaturated fatty acids were detected at much higher levels in WS at 89.6 ± 25.3 versus 2.84 ± 0.5 µg/mL in RS. In contrast, a comparable amino acid level was detected in both varieties exemplified by aspartic acid as major form at 43.1 ± 7.2 and 38.3 ± 4.9 µg/mL in WS and RS, respectively. Other less abundant amino acids, including glycine, alanine, and tryptophan, were detected at 19.8 ± 3.5; 8.5 ± 1.6; and 2.2 ± 0.18 µg/mL in WS versus lower levels in RS at 7.2 ± 1.0; 0.3 ± 0.1; and 1.0 ± 0.1 µg/mL in RS, respectively. Sugars were found at comparable levels in both WS and RS at 42.5 ± 1.2 and 36.6 ± 0.5 µg/mL, respectively.

With regard to secondary metabolites to influence squill health effects, higher levels of total bufadeinolides distinguished by α and β-unsaturated ketone of pyranone ring (H-22) were measured in WS at 17.5 ± 7.5 µg/mL while in RS, dihydrokampferol was predominated at 43.6 ± 2.3 µg/mL. Finally, coumarins detected at almost equal levels in both WS and RS at ca. 5–6 µg/mg.

2.6. Cytotoxic Screening Activity

Squill is recognized for its anticancer effect against various cancer cells and for its antioxidant and cytotoxic properties [58]. Consequently, a comparative cytotoxic assay of both WS and RS was evaluated on the different cell lines to include breast adenocarcinoma (MCF-7), lung (A-549) and ovarian cancer (SKOV-3) cell lines using Sulforhodamine B assay (SRB). The results revealed the potential cytotoxic activity of both varieties, with WS found to be more active against both cell lines A-549 and SKOV-3 than RS, as evidenced by its lower IC₅₀ values. The recorded IC₅₀ values of WS against A-549 and SKOV-3 cell lines were at 0.108 ± 0.003 and 0.690 ± 0.018 µg/mL versus RS’s IC₅₀ values at 0.271 ± 0.005 and 0.912 ± 0.021 µg/mL, respectively. In contrast, the RS extract showed a more potent effect than WS on the MCF-7 cell line, with IC₅₀ values of 0.165 ± 0.007 and 0.326 ± 0.005 µg/mL, respectively. The doxorubicin “positive control” IC₅₀ values recorded on MCF7, A-549, and SKOV-3 cancer cell lines were 0.2 ± 0.004, 0.56 ± 0.003 and 0.2 ± 0.01 µg/mL, respectively (Table 4 and Figure 8).

The potential cytotoxicity of WS compared to RS could be attributed to its abundance of bufadienolides, as revealed from both LC/MS and NMR reported for its cytotoxic action, and suggests that bufadieonolides are more determinant than flavonoids regarding cytotoxic action in squill, at least in case of A-549 and SKOV-3 cell lines.

Previous reports of hellebrigenin and bufatalin isolated from squill showed potential cytotoxic activity against leukemia, human colon carcinoma, human glioblastoma melanoma, and human liver carcinoma cells with IC₅₀ values ranging from 0.0007 to 0.16 μM [59]. Moreover, bufatalin induced apoptosis in human leukemia cells [60]. Scillarenin exhibited stronger cytotoxic action in the nanomolar range in comparison with bufatalin [61].

Proscillaridin A, a cardiac glycoside isolated from U. maritima, was reported to exert an cytotoxic and/or antiproliferative effect against human breast cancer. proscillaridin A anticancer properties are mediated via its ability to block Na+/K+ ATPase, leading to increase in Ca²⁺ levels, activating the AMPK pathway. Interestingly, on the opposite, the Ca²⁺ level was reduced by ca. 30% after an 18 h administration of proscillaridin A to the normal lung fibroblast cell line CCD19-LU, which suggests differential action mechanisms against normal and cancer cells [62].

In a previous report, bufadienolides recorded effective cytotoxic action on human cancer cells [63]. Although in WS, the most abundant bufadienolide glycoside “scilliroside” is suggested to mediate the observed toxic action of Urginea sp. The lethal dose (LD₅₀) of scilliroside was 0.7 and 0.43 mg/kg for male and female rats in vivo, respectively. Scilliroside and its aglycon, scillirosidin, exert more toxic effect than other bufadienolides, such as proscillaridin and desacetylscillirosidin. This would be attributed for the presence of an acetoxy group at the C-6 position of scilliroside [63].

3. Materials and Methods

3.1. Plant Material

Samples of U. maritima (Linn) Baker “Sea Squill” (RS and WS) were collected in summer 2018–2019 from the El-Arish desert, Sinai, Egypt. RS and WS were authenticated by Prof. Zaki Turki (Department of Botany, Faculty of Science, El-Menoufia University, Shebin El-Kom, Egypt) as U. maritima (L.) Baker, and voucher specimens Sp. No HRE139 have been deposited at the Herbarium of the Department of Botany, Faculty of Science, Menoufia University, Egypt.

3.2. Secondary Metabolites Extraction and Preparation of NMR and MS Analysis Sample

The extraction protocol for NMR and MS analysis followed that by Farag et al. [14]. Briefly, a freeze-dried squill sample was mixed with 5 mL methanol with umbelliferone “internal standard (10 μg/mL) for the quantification of metabolites using UPLC/MS”. The squill extract was vortexed and then centrifuged (3000× g) for half an hour to remove any plant wastes. NMR analysis was performed by 3 mL aliquot, then concentrated under “N₂” stream. The dried squill extract was resuspended with CD₃OD (700 μL) containing 0.94 mM HMDS, then centrifugation at 13,000× g for 1 min.

3.3. SPME/GC-MS

Solid phase micro extraction (SPME) technique was adopted for volatiles extraction as in Farag et al. [64]. Squill (5 g) was incubated at 50 °C for half an hour in a screw cap glass vial, through which the SPME fibers “stableflex fibers covered with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm), Supelco (Oakville, ON, Canada)” was placed for 15 min with the squill sample, then injected into the GC-MS injection port. The GC-MS specifications and the analysis method were previously explained in detail in a previous study by Farag et al. [64].

3.4. UHPLC/MS

The identification of secondary metabolites in the squill sample was following the specifications of the UPLC/MS as well as the procedure that was previously mentioned in Farag et al. [65]. Characterization of the secondary metabolites was carried by their UV-VIS spectra from 200–600 nm, the retention time (Rt.) relative to authentic, exact mass and upon comparing the mass spectra of those authentic, the natural products database dictionary (CRC), and the published literature [66].

Multivariate Data Analysis of UPLC-MS & GC-MS Dataset

Metabolites of U. maritima were identified using UHPLC-MS/MS-Orbitrap-HRMS. The quantification was followed what was mentioned in Farag et al. [65]. In brief, the quantification was done using XCMS analysis software downloaded from (http://137.131.20.83/download/, accessed on 14 March 2023) [67,68]. The data was then analyzed using both PCA and OPLS-DA (SIMCA-P 13.0 software package-Umetrics, Umea, Sweden).

3.5. Identification of Major Metabolites via NMR Analysis

All spectra were analyzed using VNMRS 600 NMR spectrometer. All the specifications were described in detail as mentioned in Farag et al. [69]. The 2D-NMR spectra were reported at 599.83 MHz frequency using CHEMPACK 6.2 pulse sequences as COSY, HSQC, and HMBC. The optimization of HSQC and HMBC experiments were documented previously in [68]. Similar to the previous work [15], WS was used as a reference to demonstrate the identification of U. maritima metabolites. Interpretation was achieved by chemical shifts of standards using 2D-NMR and ¹H-¹H-correlation spectroscopy COSY and TOCSY, ¹H-¹³C-HSQC, and HMBC.

Quantification of Major Metabolites via ¹H-NMR

16 metabolites were quantified by NMR spectroscopy Figure S1. The peak area of both target compounds and the internal standard (HMDS) specific protons were interpreted manually for both squill samples as described in [69].

3.6. Bioassays

3.6.1. Cell Culture

Different cancer cell lines have been tested, viz. breast adenocarcinoma (MCF-7), lung cancer (A-549), and ovarian cancer cells (SKOV-3). All cancer cells have been purchased from Nawah Scientific Inc., (Mokatam, Cairo, Egypt). Cells were kept in DMEM media supplemented with streptomycin (100 mg/mL), penicillin 100 (units/mL), and 10% of heat-inactivated fetal bovine serum in humid 5% (v/v) carbon dioxide at 37 °C.

3.6.2. Cytotoxic Screening Assay

The cell viability was conducted by SRB assay. A 100 μL of cell suspension aliquots (5 × 10³ cells) was placed in 96-well plates, then incubated for 1 day. 100 μL media with squill extracts (0.01, 0.1, 1, 10, 100 µg/mL) were added to the cell suspension. After 3 days of treatment, the cells were fixed by changing the media with 150 μL (10% Trichloroacetic acid (TCA)) and incubated at 4 °C for 1 h. The cells were then washed with distilled water. SRB (70 μL) aliquots (0.4% w/v) were added and incubated in a dark place for 10 min. The plates were washed with acetic acid (1%) and allowed to dry overnight. Then, 150 μL of tris (hydroxymethyl) aminomethane (TRIS) (10 mM) was added, and absorbance was measured at 540 nm using a BMG LABTECH^®-FLUOstar Omega microplate reader (Biotechnology company in Ortenberg, Germany).

3.6.3. Statistical Analysis

The cytotoxic screening results were represented as averages of 3 independent experiments with their standard deviation (mean ± SD). For statistical significance determination, results were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test to compare doxorubicin positive control with treatment groups of WS and RS extracts on different cancer cell lines tested in vitro using SRB assay. Graph pad prism version 5 was used where p was ≤ 0.05.

4. Conclusions

In this current study, the two squill varieties (red and white) were investigated using a metabolomic approach (PCA and OPLS-DA) coupled with different chromatographic (SPME-GC/MS and UPLC/MS) and spectroscopic techniques (1D and 2D-NMR) for the first time, where metabolites diversity was identified in the two varieties.

Volatiles assessment (SPME-GC/MS) resulted in identifying 27 volatiles in both red and white squills. Red squill was enriched with monoterpenes hydrocarbons (47.82%) than white one (25.90%), loading to more aroma profiling. The 1D and 2D-NMR spectroscopic technique was utilized to identify 16 major metabolites. The phenolic compounds were abundant in the red squill, whereas bufadienolides and fatty acids showed more intense peaks in the white one.

Secondary metabolites identification (UHPLC/MS) revealed 130 metabolites representing a myriad of classes. Bufadienolides class was the major one in white squill, whereas flavonoids were the major one in the red variety. The 1D and 2D-NMR spectroscopic technique was utilized to further identify 16 major metabolites. The phenolic compounds were abundant in the red squill, whereas bufadienolides and fatty acids showed more intense peaks in the white one.

Multivariate data analysis differentiated between both varieties and confirmed the abundance of flavonoids in red squill exemplified in dihydrokaempferol-O-hexoside, its aglycon, and taxifolin derivative, whereas fatty acids (oleic and linoleic acids) in addition to bufadienolides, viz. hydroxyscilliglaucosidin-O-rhamnoside, desacetylscillirosidin-O-rhamnoside, and bufotalidin-O-hexoside are more abundant in the white one. A cytotoxicity screening was implemented on both squills against different cell lines revealed the effectiveness of white squill over red one due to its enrichment with bufadienolides class, which has yet to be confirmed using isolated bufadienolides to be conclusive.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants12112078/s1, Figure S1: ESI-MS/MS spectrum of metabolites in the positive ion mode; Figure S2: Signal assignment of the ¹H-NMR markers for fatty acids (M1–M3), rhamnose (M4), alanine (M8), aspartic acid (M9), glycine (M10), and bufadienolides (M13 & M14) using ¹H-¹³C correlations observed in the HSQC spectrum of squill methanol extract; Figure S3: Signal assignment of the ¹H-NMR markers for fatty acids (M1-M3), rhamnose (M4), alanine (M8), aspartic acid (M9), glycine (M10), and bufadienolides (M13&M14) using ¹H-¹³C correlations observed in the HMBC spectrum of squill methanol extract; Figure S4: Signal assignment of the ¹H-NMR markers for fatty acids (M1-M3), rhamnose (M4), β, α glucose (M5 & M6), sucrose (M7), aspartic acid (M9), glycine (M10), tryptophan (M12), and bufalin (M13) using ¹H-¹³C correlations observed in the HMBC spectrum of squill methanol extract; Figure S5: Signal assignment of the ¹H-NMR markers for rhamnose (M4), β, α glucose (M5& M6), sucrose (M7), aspartic acid (M9), glycine (M10), tryptophan (M12), and bufalin (M13) using ¹H-¹³C correlations observed in the HSQC spectrum of squill methanol extract; Figure S6: Signal assignment of the ¹H-NMR markers for tyrosine (M11), tryptophan (M12), bufalin (M13), scilliridin (M14), dihydro kaempferol (M15), and coumarin (M16); ¹H-¹³C correlations observed in the HSQC spectrum of squill methanol extract; Figure S7: Signal assignment of the ¹H-NMR markers for tyrosine (M11), tryptophan (M12), bufalin (M13), scilliridin (M14), dihydro kaempferol (M15), and coumarin (M16); ¹H-¹³C correlations observed in the HMBC spectrum of squill methanol extract; Figure S8: Signal assignment of the proton markers for dihydro kaempferol (M15) observed in the ¹H-NMR spectrum of RS methanol extract; Table S1: ¹H-NMR quantification of most common primary and secondary metabolites detected in Urginea species, white squill (WS) and red squill (RS). Values are expressed as μg/mg dry powder ± S.D (n = 3). Chemical shifts used for metabolite quantification were determined in methanol-d₆ and expressed as relative values to HMDS (0.94 mM final concentration); Table S2: RS and WS extract inhibition and viability values in different concentrations against different cancer cell lines measured by SRB assay.

Author Contributions

M.A.F. and H.R.E.-S.; conceptualization, O.M.K.; methodology, O.M.K., N.Y. and M.A.F.; software, D.M.E.-K. and M.A.F.; validation, M.A.F.; formal analysis, O.M.K. and D.M.E.-K.; investigation, H.R.E.-S.; resources, M.A.F.; data curation, O.M.K. and N.Y.; writing—original draft preparation, D.M.E.-K., H.R.E.-S. and M.A.F.; writing—review and editing, S.A.M.K. and M.A.F.; supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data are available from omarkhattab500@gmail.com upon request.

Acknowledgments

Mohamed A. Farag wishes to thank the Alexander von Humboldt foundation, Germany and Science and Technology Development Fund (STDF), Egypt grant number 47051.

Conflicts of Interest

The authors declare no conflict of interest.

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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.

Table 1. Volatiles percentile of WS and RS using SPME coupled with GC-MS (n = 1).

Peak #	Rt (min)	KI	Name	Abundance %		Cas-No
Peak #	Rt (min)	KI	Name	WS	RS
Monoterpene hydrocarbon
G1	7.13	909.1	α-Pinene	2.09	8.04	7785-26-4
G2	8.12	965.6	β-Myrcene	6.74	8.73	123-35-3
G4	8.46	985.3	3-Carene	2.32	7.81	13466-78-9
G5	8.47	985.6	2-Thujene	2.32	7.29	28634-89-1
G6	8.75	1002.4	p-Cymene	1.96	_	99-87-6
G7	8.81	1005.9	Limonene	4.89	7.81	5989-27-5
G8	9.27	1035.2	α-Phellandrene	1.72	_	99-83-2
G9	9.70	1063	Isoterpinolene	1.91	8.15	586-63-0
G10	9.71	1063.3	4-Carene	1.74	_	29050-33-7
Total monoterpene hydrocarbon				25.69	47.82
Oxygenated monoterpene
G15	11.47	1182.9	Estragole	2.29	_	140-67-0
Total oxygenated monoterpene				2.29
Sesquiterpene hydrocarbon
G21	15.12	1471.1	β-Bisabolene	8.22	_	495-61-4
Total sesquiterpene hydrocarbon				8.22
Oxygenated hydrocarbon
G12	10.03	1084.1	Nonanal	2.64	_	124-19-6
G14	11.44	1181.4	Decanal	2.49	_	112-31-2
G26	21.50	1841.9	Palmitic acid, methyl ester	7.02	_	112-39-0
Total oxygenated hydrocarbon				12.15
Aromatic
G19	14.79	1444.4	2-Methyl-4-hydroxyacetophenone	1.75	_	875-59-2
Total aromatic				1.75
Heterocyclic
G3	8.17	968.8	Furan-2-pentyl-	4.57	_	3777-69-3
Total heterocyclic				4.57
Hydrocarbon
G11	9.81	1069.5	Decane	2.64	6.31	124-18-5
G13	11.22	1166	Tridecane	4.92	6.03	629-50-5
G16	12.16	1235.5	4,7-Dimethylundecane	2.84	5.97	17301-32-5
G20	14.87	1451	Pentadecane	5.97	6.43	544-76-3
G24	17.72	1644	Hexadecane	_	7.29	544-76-3
G25	19.83	1754.6	Heptadecane	3.39	5.22	629-50-5
Total hydrocarbon				19.76	37.26
Unknown
G17	12.52	1261.8	Unknown	4.78	6.66	_
G18	13.73	1357.6	Unknown	18.11	_	_
G22	15.23	1480.8	Unknown	1.86	_	624-24-8
G23	16.13	1546	Unknown	_	8.27	_
Total unknown				24.955	14.93
Total volatiles				100.0	100.0

Table 2. Secondary metabolites tentatively identified in two squill varieties (WS and RS) via UPLC/MS in positive (L) and negative (N) ionization modes.

Code	t_R (m.)	UV	Elemental Composition	Error ppm	Exact Mass (M+H)⁺/ (M−H)⁻	Identified Fragments (MS²)	Identified Metabolite	Class	WS	RS
L1	0.4	280–300	(C₂₁H₁₇O₆)⁺	8	365.1049	203, 185	Unknown glycoside	Unknown	+	−
L2	0.4	280–300	(C₂₇H₂₇O₁₁)⁺	3.7	527.1567	365, 347, 203, 185	Unknown Glycoside (dihexose)	Unknown	+	−
L3	0.4	280–300	(C₂₁H₃₁O₁₂)⁺	6.5	475.1779	133, 116	Hydroxycinnamyl-O-dihexoside	Phenolic	+	+
L4	0.4	280–300	(C₁₂H₂₁O₁₀)⁺	2.9	325.112	145, 127	Disacharide (cellobiosan)	Sugar	+	+
L5	0.5	280–300	(C₃₃H₃₇O₁₆)⁺	5	689.2041	527, 365, 347, 203, 185	Unknown glycoside (dihexoside)	Unknown	+	−
L6	0.6	280	(C₁₈H₃₁O₁₅)⁺	2.2	487.1668	325, 271, 163, 145, 127	Agarotriose	Sugar	+	+
L7	0.6	280	(C₂₄H₄₁O₂₀)⁺	4.4	649.2157	325, 289, 271, 253, 223,163, 145, 127	Polysaccharide (agarotriose dimer)	Sugar	−	+
L8	0.7	280	(C₉H₁₂NO₃)⁺	3.1	182.0806	165, 147, 136, 123	Tyrosine	Amino acid	+	+
L9	1.2	280	(C₉H₁₂NO₂)⁺	6.2	166.0852	120, 103	Phenylalanine	Amino acid	+	+
L10	1.6	280	(C₁₁H₁₆NO₂₎⁺	2.6	194.1171	148	Amino acid derivative	Amino acid	−	+
N1	1.7	280	(C₁₄H₁₉O₈)⁻	3.4	315.1075	153	Isorahmantin	Flavonol	−	+
L11	2.3	280	(C₁₁H₁₀NO₂)⁺	4.6	188.0697	170, 146	Tryptophan derivative	Amino acid	−	+
L12	2.5	280	(C₁₁H₁₃N₂O₂)⁺	6.1	205.0959	188, 170, 146, 118	Tryptophan	Amino acid	+	+
L13	2.8	282	(C₁₅H₁₅O₇)⁺	−1.8	307.0818	289, 271, 261, 243, 188, 151, 139	Leucocyanidin		−	+
N2	2.9	280	(C₂₀H₁₉O₁₀)⁻	0.7	419.0988	165	Juglanin	Flavonoid	−	+
N3	3.1	282–340	(C₂₁H₂₃O₁₁)⁻	1.9	451.1246	289, 271, 243, 227, 199, 177, 151	Catechin-O-hexoside	Flavanol	−	+
L14	3.3	283–340	(C₁₅H₁₅O₆)⁺	1.6	291.0859	273, 245, 151, 139	Catechin	Flavanol	−	+
N4	3.3	283–340	(C₂₁H₂₃O₁₁)⁻	1.9	451.1246	271, 243, 227, 199, 177, 151	Catechin-O-hexoside isomer	Flavanol	−	+
L15	3.5	283–340	(C₁₅H₁₁O₆)⁺	1.1	287.0547	259, 231, 149	Kaempferol	Flavone	−	+
N5	3.6	282–296–340	(C₂₀H₂₇O₁₄)⁻	0	491.1406	209, 191, 167, 123	Vanillic acid-O-dihexoside	Phenolic acid	−	+
L16	3.7	282–296–340	(C₂₇H₃₃O₁₆)⁺	5.1	613.1746	271, 243, 215	Dihydrokaempeferol-O-dihexoside	Flavanol	−	+
N6	3.8	282–296–340	(C₂₇H₃₁O₁₆)⁻	1.6	611.1608	287, 269, 259, 243, 215	Dihydrokaempeferol-O-dihexoside (aromadendrin-O-dihexoside)	Flavanol	−	+
N7	3.9	282–296–340	(C₃₃H₄₁O₂₁)⁻	0.4	773.2142	287, 269, 259, 243, 215	Dihydrokaempeferol-O-trihexoside (aromadendrin-O-trihexoside)	Flavanol	−	+
L17	4.2	291–330	(C₁₅H₁₃O₆)⁺	−0.9	289.0709	271, 253, 243, 215, 149	Dihydrokaempferol	Flavanol	−	+
N8	4.2	291–330	(C₃₆H₃₃O₁₈)⁻	0.3	753.167	465, 437, 315, 303, 285, 259, 245, 219	Dihydroquercitin-O-hexosidederivative (taxifolin-O-hexoside derivative)	Flavanol	−	+
L18	4.3	291–330	(C₂₁H₂₃O₁₁)⁺	2.2	451.1245	289, 271, 247	Dihydrokaempferol-O-hexoside	Flavanol	−	+
L19	4.3	291–330	(C₃₈H₄₅O₂₅⁾⁺	1.8	901.2228	451, 289, 271, 243	Dihydrokaempferol-O-hexoside dimer	Flavanol	−	+
L20	4.3	291–330	(C₁₅H₁₁O₅)⁺	−0.2	271.0601	243, 215, 149	Apigenin	Flavone	−	+
L21	4.3	291–330	(C₁₄H₁₁O4)⁺	2.2	243.0646	215, 149	Unknown	Unknown	−	+
N9	4.3	291–330	(C₄₂H₄₃O₂₂)⁻	−5.8	899.2304	449, 342, 287, 259	Dihydrokaempeferol-O-hexoside dimer	Flavanol	−	+
N10	4.4	291–330	(C₂₁H₂₁O₁₁)⁻	6.3	449.1122	287, 269, 259, 243, 225, 151	Dihydro kaempeferol-O-hexoside (aromadendrin-O-hexoside	Flavanol	−	+
L22	4.6	283–296–340	(C₁₅H₁₃O₇)⁺	1.8	305.065	287, 259, 231, 153	Dihydroquercitin	Flavanol	−	+
N11	4.6	283–296–340	(C₃₇H₄₅O₂₆)⁻	2.1	905.2205	450, 285, 261, 243, 191	Kampferol derivative	Flavonol	-	+
L23	4.7	283–296–340	(C₁₅H₁₁O₈)⁺	0	319.0812	301, 273, 269, 245	Myricetin	Flavonol	−	+
L24	4.7	283–296–340	(C₁₆H₁₅O₇)⁺	−0.3	319.0813	313, 307, 286, 273, 245, 217, 185, 149, 137	Dihydroisorhmentin or Methyltaxifolin	Flavonoid	−	+
N12	4.6	283–296–340	(C₂₂H₂₃O₁₂)⁻	2.9	479.1181	317, 306, 299, 289	Noidesol A/B	Flavonoid	−	+
L25	4.8	283–296-340	(C₂₀H₂₉O₁₃)⁺	1	477.1598	169, 151	Vanillin rhamnoglucoside	Phenolic acid	−	+
L25	4.9	283–296-340	(C₂₀H₂₇O₁₃)⁻	0.6	475.1457	209, 167, 123	Vanillin rhamnoglucoside	Phenolic acid	−	+
L26	4.8	283–296-340	(C₈H₉O₄)⁺	2.8	169.049	151, 127, 109	Vanillic acid	Phenolic acid	−	+
N13	4.8	283–296-340	(C₁₅H₁₁O₆)⁻	0.9	287.0559	201, 125	Dihydrokaempeferol	Dihydroflavonol	−	+
L27	4.9	283–296-340	(C₁₄H₁₉O₉)⁺	4.8	331.1008	169	Vanillic acid-O-hexoside	Phenolic acid	−	+
N14	4.9	283–296-340	(C₂₁H₂₉O₁₂)⁻	1.9	473.1651	209, 191, 167, 123	Dimethoxy hydrocinnamic acid (dimethoxyphenyl propionic acid)-dipentoside	Phenolic acid	−	+
L28	5.1	282	(C₂₁H₂₃O₁₀)⁺	3.2	435.1272	271, 255, 151, 119	(Apigenin-O-hexoside)	Flavanone	−	+
L29	5.1	282	(C₉H₇O₃)⁺	2.7	163.0385	135, 119, 107	Hydroxycoumarin	Coumarin	−	+
L30	5.1	282	(C₄₂H₃₇O₁₅)⁺	1.3	781.2117	409, 317, 287, 247, 169	Unknown glycoside of L31	Unknown	−	+
N15	5.1	280	(C₉H₅O₃)⁻	3.7	161.0244	133, 117, 105	Hydroxycoumarin	Coumarin	−	+
N16	5.4	283–340	(C₂₁H₂₁O₁₂)⁻	0.7	465.1035	285, 275, 259, 231, 217, 152, 125	Kaempferol-O-hexoside	Flavonol	−	+
L31	5.5	283–340	(C₃₆H₂₇O₁₀)⁺	3.6	619.1621	329, 317, 287, 271, 247, 229, 181, 169	Unknown aglycone of L30	Unknown	−	+
N17	5.5	283–340	(C₄₂H₄₃O₂₄)⁻	4.4	931.2208	465, 303, 285, 275, 259, 231, 217, 152, 125	Dihydroquercitin-O-hexoside (dimer)	Flavanol	−	+
N18	5.6	283–340	(C₂₇H₃₁O₁₇)⁻	0.2	627.1544	303,285, 217, 189, 151, 125	Dihydroquercitin-O- dihexoside	Flavanol	−	+
N19	5.6	283–340	(C₂₉H₂₉O₁₅)⁻	0.1	617.1497	314, 285, 221, 209, 167, 125	Kampferol derivative	Flavanol	−	+
L32	5.7	283–340	(C₂₇H₃₁O₁₇)⁺	6.3	627.1517	303	Quercitin-O-dihexoside	Flavonol	−	+
N20	5.7	283–340	(C₂₇H₂₉O₁₇)⁻	0.2	625.1411	463, 301, 125	Quercitin-O-dihexoside	Flavonol	−	+
L33	5.9	283–340	(C₂₇H₃₁O₁₅)⁺	6.7	595.1617	415, 397, 379, 361, 271	Apigenin-O-dihexoside	Flavanone	−	+
L34	6	299	(C₂₄H₂₇O₄)⁺	0.5	379.1902	351, 333, 315, 239	Monohydroxy-19-oxobufa-4,20,22-trienolide	Bufadienolide	+	−
L35	6.1	299	(C₂₄H₂₉O₅)⁺	0.3	397.2	381, 363, 345, 317	3-Dehydroscilliglaucosidin (scilliglaucosidine)	Bufadienolide	+	−
L36	6.2	299	(C₃₀H₄₁O₁₁)⁺	1.1	577.2637	417, 399, 381, 363, 345, 335	Desacetylscillirosidin-O-thevetoside	Bufadienolide	+	+
N21	6.2	280–298-340	(C₁₅H₁₁O₇)⁻	2.6	303.0502	294, 207, 181, 154, 99, 51	Dihydroquercitin	Flavanol	−	+
N22	6.3	280–298-340	(C₂₇H₃₁O₁₅)⁻	2.3	595.1654	271, 151, 125	Naringenin-O-dihexoside	Flavanoid	−	+
N23	6.3	280–298-340	(C₂₁H₁₉O₁₂)⁻	1.7	463.08	301, 300, 271, 151	Quercitin-O-hexoside	Flavonol	−	+
L37	6.5	299	(C₂₄H₃₁O₆)⁺	2	415.2107	397, 379, 351, 333	Trihydroxy-oxobufa-trienolide (hydroxy-scilliglaucosidin)	Bufadienolide	+	−
L38	6.6	298	(C₃₀H₄₅O₁₀)⁺	0.1	565.3008	403, 385, 367, 331, 272	Gammabufotalin-O-glucoside	Bufadienolide	+	−
L39	6.6	298	(C₂₄H₃₅O₅)⁺	3	403.247	385,367, 253	Gamabufotalin	Bufadienolide	+	+
L40	6.6	280–298–340	(C₁₅H₁₁O₇)⁺	0.8	303.05	191	Quercitin	Flavonol	−	+
L41	6.6	280–298–340	(C₂₁H₂₁O₁₂)⁺	2.8	465.1015	308, 303	Quercitin-O-hexoside	Flavonol	−	+
L42	6.8	298	(C₃₀H₄₃O₁₀)⁺	1.2	563.2844	545, 365, 347, 337, 323, 267, 252, 213	Scillirubroside	Bufadienolide	+	−
L43	6.9	298	(C₂₄H₃₁O₄)⁺	2.8	383.2228	365, 348, 251	Scillirubrosidin-H₂O(scillirubroside-hexoside-H₂O)	Bufadienolide	+	+
L44	7	298	(C₂₄H₂₉O₆)⁺	1.2	413.1954	395, 69, 351, 333	Trihydroxy-oxobufa-tetra-enolide	Bufadienolide	+	−
L45	7	298	(C₄₃H₄₅O₁₁)⁺	2.2	737.294	413, 395, 377, 359, 331	Trihydroxy-oxobufa-tetra-enolide-O-di-hexoside	Bufadienolide	+	−
L46	7.2	299	(C₃₀H₄₁O₁₁)⁺	3.8	577.2621	415, 397, 379, 351, 333	Hydroxyscilliglaucoside	Bufadienolide	+	+
N24	7.7	280–298–340	(C₂₁H₁₉O₁₁)⁻	3.7	447.0916	285, 255, 227	Kaempferol-O-glucoside (hexoside)	Flavonol	−	+
L47	7.8	299	(C₃₆H₅₁O₁₅)⁺	8.1	723.3164	561, 415, 397, 379, 361	Trihydroxy-oxobufa-trienolide-O-rhamnosdie-glucoside/Scilliglaucosidin-O-rhamnoside-glucoside	Bufadienolide	+	−
L48	7.9	299	(C₂₄H₃₃O₆)⁺	1.8	417.2279	399, 381, 363, 345, 335, 145	Desacetylscillirosidin/Hydroxyscilliphaeosidin/Bufotalidin	Bufadienolide	+	−
L49	7.9	299	(C₂₄H₃₃O₆)⁺	1.8	417.2279	399, 381, 363, 335,	Desacetylscillirosidin/Hydroxyscilliphaeosidin/Bufotalidin isomer	Bufadienolide	+	−
L50	8	299	(C₃₀H₄₁O₁₀)⁺	1.1	561.269	415, 379, 361, 351	Trihydroxy-oxobufa-trienolide-O-rhamoside/Hydroxy-scilliglaucosidin-O-rhamnoside	Bufadienolide	+	+
N25	8.1	280–298–340	(C₂₂H₂₁O₁₂)⁻	1.9	477.103	315, 314, 299, 285, 271, 243	Isorhamnetin 3-hexoside	Flavonol	−	+
L51	8.3	299	(C₃₀H₄₃O₁₀)⁺	2.1	563.2839	417, 399, 381, 363, 345, 315, 278	Desacetylscillirosidin-O-rhamnoside/Hydroxyscilliphaeosidin-O-rhamnoside	Bufadienolide	+	−
L52	8.3	299	(C₃₀H₄₃O₁₀)⁺	2.1	563.2839	417, 399, 381, 363, 333	Bufotalidin-O-rhamnoside	Bufadienolide	+	−
L53	8.5	299	(C₂₄H₃₁O₅)⁺	0.9	399.2169	381, 363, 345, 223.,157	Scilliglaucosidin	Bufadienolide	+	+
L54	8.7	298	(C₂₆H₃₇O₈)⁺	0.1	477.248	417, 399, 381, 363, 345	Hydroxy-scillirosidin+2H	Bufadienolide	+	−
L55	8.8	298	(C₃₀H₄₅O₉)⁺	0.8	549.3053	403, 385, 367, 349, 193, 179	Gamabufotalin-O-rhamnoside	Bufadienolide	+	−
L56	8.9	298	(C₂₄H₃₃O₅)⁺	1.9	401.2315	383, 347, 197	Scillirubrosidin or scilliphosidin	Bufadienolide	+	+
L57	8.9	298	(C₃₀H₄₃O₉)⁺	2.9	547.2886	401,383, 347	Scilliphaeoside “Scillipheosidin-O-rhamnoside”	Bufadienolide	+	+
L58	8.9	299	(C₃₆H₅₃O₁₄)⁺	2.6	709.3411	547, 417, 367, 349, 287	Scillipheoside-O-glucoside	Bufadienolide	+	+
L59	8.9	285	(C₃₄H₂₇O₁₂⁾⁺	0.1	627.1498	401, 383, 365, 303, 269, 193	Scillirubrosidin-O-hexoside or scilliphosidin-o-hexoside	Bufadienolide	−	+
N26	8.9	299	(C₃₆H₆₇O₂₆)⁻	1.5	915.3912	869, 707, 545, 399, 355, 221, 161, 113	Unknown	Unknown	−	+
L60	9.3	283	(C₃₆H₅₅O₁₄)⁺	4.6	711.3554	549, 531, 403, 367, 349, 253, 199	Gamabufotalin-rhamnoglucoside	Bufadienolide	−	+
L61	9.6	298	(C₂₄H₂₇O₃)⁺	2.5	363.1946	345, 335, 317, 273	Unknown	Unknown	+	−
L62	10	299	(C₃₀H₄₁O₁₀)⁺	1.1	561.269	399, 381, 363, 345, 223, 157	Scilliglaucoside	Bufadienolide	+	+
L63	10	399	(C₃₃H₄₉O₁₂)⁺	2.7	637.3201	477, 417, 399, 381, 363, 345	Hydroxyscilliroside	Bufadienolide	+	−
L64	10	299	(C₂₄H₃₁O₃)⁺	4.1	367.2253	349, 287, 175, 133	Scillaridin A	Bufadienolide	+	+
L65	11	298	(C₃₉H₅₉O₁₇)⁺	7.2	799.3651	477, 399, 381, 363, 345	Hydroxyscilliphaeosidin-O-thevetoside-glucoside-Ac	Bufadienolide	+	−
L66	11	282	(C₃₆H₄₅O₁₉)⁺	4.5	781.2514	325, 241, 163, 145, 115	Unknown	Unknown	−	+
L67	12	298	(C₃₃H₅₅O₈)⁺	3.1	579.387	417, 237, 255	Bufotalidin-O-hexoside	Bufadienolide	+	+
L68	12	298	(C₃₀H₄₁O₉)⁺	4.6	545.272	399, 381, 363, 353, 345, 335, 317	Scilliglaucosidin-O-rhmnoside	Bufadienolide	+	−
L69	12	299	(C₃₆H₅₁O₁₄)⁺	8.4	707.3214	545,399, 381, 363, 335, 317, 275, 223	Scilliglaucosidin-O-rhmnoside-O-hexoside	Bufadienolide	+	−
L70	12	299	(C₂₄H₃₃O₄)⁺	0	385.2373	367, 349, 289, 253	Scillarenin	Bufadienolide	+	+
L71	12	283	(C₂₄H₂₉O₃)⁺	−1.9	365.2118	349, 287, 175, 147	Unknown aglycone of L42	Bufadienolide	−	+
L72	12	296	(C₂₆H₃₅O₆)⁺	2.9	443.2415	425, 383, 365, 347, 319, 269, 239, 225, 197	Cinobufagin or acetylmarinobufogenin	Bufadienolide	−	+
L73	12	296	(C₄₅H₅₁O₁₀)⁺	0.6	751.3472	589, 443, 425, 365, 347, 285, 225, 173	Cinobufagin or acetylmarinobufogenin-O-rhamnoside-glucoside	Bufadienolide	−	+
L74	12	299	(C₁₇H₂₉O₇)⁺	0.4	345.1906	281, 263, 253, 193	Unknown	Unknown	+	−
L75	12	283	(C₁₉H₃₈NO₅)⁺	3.8	360.2731	342, 324, 306, 278, 260, 240, 222	3-Hydroxydodecanoylcarnitine	Acylcarnitine	−	+
L76	12	298	(C₃₃H₄₉O₁₁)⁺	3.2	621.3249	461, 401, 383, 365, 319, 251, 213	Scilliroside	Bufadienolide	+	−
L77	12	296	(C₃₂H₄₅O₁₁)⁺	5.8	605.2921	591, 572, 537, 529, 462, 443, 417, 337, 256, 237, 207, 165, 145, 135, 108	Cinobufagin-O-hexoside or acetylmarinobufogenin-O-hexoside	Bufadienolide	−	+
L78	12	298	(C₂₄H₃₅O₆)⁺	4.5	419.2409	401, 383, 365, 347, 213	Hellebrigenol (19-hydroxytelocinobufagin)	Bufadienolide	+	−
L79	12	298	(C₃₈H₅₇O₁₆)⁺	5.3	769.36	607, 461, 401 383, 365, 347, 305	Scillirosidin-rhamnoside-glucoside+2H	Bufadienolide	+	−
L80	12	299	(C₃₉H₆₅O₁₃)⁺	12.8	741.4325	579, 461, 419, 401, 383, 365	Hellebrigenin-3-O-D-diglucopyranoside.	Bufadienolide	+	+
L81	12	300	(C₃₈H₅₉O₁₉)⁺	-1.4	819.3692	367, 349, 273, 255, 237	Unknown	Unknown	−	+
L82	12	299	(C₂₄H₃₅O₄)⁺	7.6	387.25	385, 367, 349, 331, 199	Bufalin	Bufadienolide	−	+
L83	12	300	(C₃₀H₄₅O₈)⁺	1.4	533.3102	515, 387, 367, 349, 274, 255, 199	Dihydro-Proscillaridin (rhamnosylbufalin)	Bufadienolide	−	+
L84	12	298	(C₃₉H₅₉O₁₆)⁺	5.3	783.3697	621, 543, 461, 401, 383, 365, 347,251	Scilliroside-O-glucoside	Bufadienolide	+	−
L85	12	299	(C₃₈H₆₁O₂₀)⁺	1.3	837.3761	515, 387, 349, 255, 237	Bufalin derivative	Bufadienolide	−	+
L86	12	299	(C₃₆H₅₃O₁₃)⁺	4	693.3394	531, 385, 367, 349, 287	Proscillaridin A-O-glucoside	Bufadienolide	+	+
L87	12	299		7	362.2876	344, 308, 224	Unknown fat	Fat	−	+
L88	12	299	(C₂₆H₃₇O₇)⁺	3.3	461.2549	401, 383, 365, 337, 329, 305, 285	Dihydroscillirosidin	Bufadienolide	+	−
L89	12	298	(C₃₈H₅₅O₁₄)⁺	10.7	735.351	573, 461, 385, 367, 349, 331, 287	Scillaren A acetate-O-rhamnoside-hexoside	Bufadienolide	+	−
L90	12	299	(C₃₂H₄₇O₁₁)⁺	3.4	607.3092	547, 401, 383, 365, 347	Acetyl-scilliphaeoside-rhamnoside	Bufadienolide	+	−
L91	13	298	(C₃₀H₄₃O₈)⁺	1.7	531.2943	513, 385, 367, 349, 321, 303, 253, 215	Proscillaridin A	Bufadienolide	+	+
L92	13	298	C₄₄H₆₅O₁₉	13.7	897.3992	735, 573, 385, 367, 349, 331, 287	Scillaren A acetate-O-rhamnoside-dihexoside	Bufadienolide	+	−
L93	13	282	(C₁₆H₁₃O₅)⁺	4.9	285.0744	191	Acacetin or prunetin	Flavonoid	−	+
L94	13	282	(C₁₇H₁₇O₆)⁺	2.8	317.1011	299, 271, 121	Dihydroxy-dimethoxyflavanone	Flavonoid	−	+
L95	13	286	(C₂₀H₄₂NO₆)⁺	2.3	392.2997	356, 338, 278, 261, 232	N-tetradecyl-D-gluconamide	Amide	−	+
L96	13	280	(C₂₀H₄₂NO₅)⁺	1.7	376.3051	340, 262, 245, 219	2-(14-Aminotetradecyl)-6-(hydroxymethyl) oxane-3,4,5-triol	Fatty alcohol	−	+
L97	13	280	(C₁₉H₃₈NO₄)⁺	1.6	344.279	326, 308, 280, 224	19-(hydroxyamino)-19-oxo-nonadecanoic acid	Fatty acid	−	+
L98	13	280	(C₁₉H₃₆NO₃)⁺	3.5	326.2678	308, 252	Dodecadienyl carnitine	Fatty acyl-L-carnitine	−	+
L99	13	280	(C₂₀H₄₀NO₄)⁺	2.1	358.2944	340, 322, 294	Tridecanoyl carnitine	Fatty acyl-L-carnitine	−	+
L100	13	280	(C₁₈H₄₀NO₄)⁺	3.7	334.294	316, 298, 280, 251, 238	1-(hydroperoxyamino) octadecane-1,18-diol	Fatty alcohol	−	+
L101	14	299	(C₃₂H₄₅O₉)⁺	4.4	573.3033	367, 349, 331, 253, 133	Scillaridin-acetate-O-rhamnoside	Bufadienolide	+	−
L102	14	298	(C₁₆H₂₇O₄)⁺	3.9	283.1893	270, 265	Fumagillol	Sesquiterpenoid	+	−
L103	14	298	(C₁₇H₂₇O₅)⁺ (C16H27O3)+	2.1	311.1847 (−46 Formate)	265, 247, 209	(4E,6Z)-3-Hydroxy-4,6,15-hexadecatrienoic acid	Acid	+	−
L104	14	282	(C₁₉H₄₀NO₄)⁺	5.1	346.2934	328, 310, 282, 264, 226	Monomethyl phytosphingosine	Sphingolipid	−	+
L105	14	298	(C₁₇H₂₉O₆)⁺	3.7	329.1946	265, 237, 209, 191,	Spiculisporic acid	Acid	+	−
L106	14	282	(C₂₀H₄₂NO₄)⁺	1.3	360.3104	342, 324, 296	N-acetyl phytosphingosine	Sphingolipid	−	+
L107	14	282	(C₁₇H₁₇O₅)⁺	4.6	301.1057	282, 267	Unknown	Unknown	−	+
L108	14	289	(C₁₈H₄₀NO₃)⁺	4.2	318.2989	300, 282, 270, 264	Phytosphingosine	Sphingolipid	−	+
L109	14	289	(C₁₉H₂₁O₅)^+F	2.5	329.1375	207, 121	Hirsutanone		−	+
L110	14	-	(C₃₀H₂₃O₁₀)⁺	1.8	543.1296	273, 255, 213	Unknown	Unknown	+	−
L111	14	-	(C₁₈H₄₀NO₂)⁺	4.7	302.3039	284, 266, 254	Sphinganine	Sphingolipid	+	+
L112	14	-	(C₁₇H₃₁O₅)⁺	5.1	315.215	265	Unknown	Unknown	+	−
L113	14	280	(C₂₇H₄₅O₃)⁺	2.3	417.3354	273, 255, 161	24-Hydroperoxycholesta-5,25-dien-3beta-ol	Fatty acid	−	+
L114	15	-	(C₁₈H₃₁O₂)⁺	0.3	279.2318	261, 223, 173	Octadecatrienoic acid	Fatty acid	+	+
N27	15	-	(C₁₈H₃₁O₃)⁻	2.2	295.2279	195, 277	Coriolic acid	Fatty acid	−	+
L116	16	-	(C₄₈H₈₁O₈)⁺	11.5	733.4379	367,253	Dimer of unknown fatty acid	Fatty acid	+	+
L117	16	-	(C₁₈H₂₉O₃)⁺	5.2	293.2096	275, 223, 95	Licanic acid	Fatty acid	+	−
L118	17	-	(C₂₁H₃₉O₄)⁺	0.1	355.2843	337, 263	Glyceryl 2-linoleate	Fatty acid	+	−
N28	17	-	(C₁₈H₂₉O₂)⁻	3.1	277.2164	250, 226, 171, 150, 109, 77, 53	Linolenic acid	Fatty acid	−	+
L119	17	-	(C₁₈H₂₇O)⁺	1.1	259.2054	175	Unknown	Unknown	+	−
L120	17	-	(C₁₈H₃₃O₂)⁺	0.4	281.2476	263, 245, 189,	Linoleic acid	Fatty acid	+	+
L121	17	-	(C₂₀H₃₃O₃)⁺	1.7	321.243	305, 265, 245, 179	8-Hydroxyicosa-5,9,11,14-tetraenoic acid	Fatty acid	+	−
L122	17	-	(C₃₆H₆₅O₄)⁺	3.9	561.4899	543, 307, 245, 175	Linoleic acid dimer	Fatty acid	+	−
L123	17	-	(C₂₁H₄₁O₄)⁺	5.4	357.298	339, 265, 247, 205, 135, 124, 112, 75	Glyceryl Monooleate	Fatty acid	+	+
N29	17	-	(C₁₈H₃₁O₂)⁻	0.9	279.2327	201, 167, 141, 127, 89, 70, 54	Linoleic acid	Fatty acid	+	+
L124	17.7	-	(C₁₆H₃₁O)⁺	6.7	239.2353	109, 95	Hexadeca-10,12-dien-1-ol	Fatty acid	+	+
L125	17.7	-	(C₁₆H₃₃O₂)⁺	3.3	257.2467	237, 120, 103	Hexadecanoic acid	Fatty acid	+	+
L126	17.8	-	(C₁₈H₃₅O₂)⁺	5.9	283.2615	265, 247, 191, 153, 137, 121	Palmitic acid	Fatty acid	+	−
L127	17.8	-	(C₁₈H₃₃O)⁺	6.1	265.251	247, 205, 191, 149	Linolenyl alcohol	Fatty alcohol	+	+
N30	17.8	-	(C₁₈H₃₃O₂)⁻	0.8	281.24	185, 155, 95, 58	Oleic acid	Fatty acid	+	+
N31	17.8	-	C19H35O4	10	327.2505	281, 185, 95	Chaetomellic acid A	Fatty acid	+	−
L128	18	-	(C₁₄H₂₃O₁₆)⁺	1.6	447.0988	359, 341, 324, 225, 207, 149	Unknown	Unknown	−	+
L129	18	-	(C₂₂H₃₉O₄)⁺	0.1	367.2843	349, 331,293, 251, 205, 179, 133	16,17-dihydroxydocosa-7,10,13-trienoic acid	Fatty acid	+	+
L130	18	-	(C₂₂H₃₇O₃)⁺	2.6	349.2728	331, 293, 183, 165	Anacardic acid	Phenolic lipid	+	+

Table 3. Assignment data from 1D and 2D NMR on WS and RS extracts.

Metabolite	Assignment	δ ¹H in ppm	δ ¹H COSEY (ppm)	δ ¹³C in ppm	HMBC Correlations δ ¹³C in ppm
Fatty acid (M1–M3)	Olefinic carbons	5.23–5.34	2.05	128.0–131.3	29.4 (bis allylic CH₂), 27.9 allylic CH₂
	allylic CH₂	2.07 m	5.34	27.9	Olefinic 128.0–131.0, (CH₂)_n 31.5
ω-9 Fatty acid (M1)	t-CH₃	0.91 (t, J = 6.9 Hz)	1.3 (CH₂)n	14.1	23.9 C-2, 31.5(CH₂)_n
	(CH2)n	1.3 (br. s)	0.89 (t-CH3), 1.61 (H-3), 2.09 (allylic CH2)	30.4	32.0 (CH₂)_n
	C-2	2.27 (t, J = 7.4 Hz)	1.59	34.7	C-1 178.8, C-32.4, (CH₂)n 31.6
	C-3	1.59 m	2.27	25.8	C-1 177.8, (CH₂)_n 30.4
ω-6 Fatty acid (M2)	Bis allylic CH₂	2.77 (t, J = 6.9 Hz)	5.33	24.7	Olefinic carbons 131.2,
ω-3 Fatty acid (M3)	Bis allylic CH₂	2.87	5.33	26.3	Olefinic carbons 131.2,
	t-CH₃	0.97	2.07 (allylic CH₂)	19.5	21.6 C-2
Sugars
Rhamnoside (M4)	C-6	1.23	3.77	19.5	(C-4) 69.8, (C-5) 72.8
	C-1	5.44	-	93. 8	(C-2) 73.8
	C-5	3.77	-	72.7	-
	C-4	3.78	-	69.1	-
β-Glucose (M5)	C-1	4.48 (d, J = 7.8 Hz)	3.11, 3.27, 3.32	97.9	-
	C-2	3.11		76.1	(C-3) 77.8, (C-1)97.9
	C-3	3.35		77.8	-
	C-5	3.28		77.7	(C-6) 62.2
	C-6	3.65		62.1	-
α-Glucose (M6)	C-1	5.10 (d, J = 3.7 Hz)	3.35	93.7	(C-4) 72.7, (C-3)74.61
	C-2	3.35		73.5	74.2 (C-3)
	C-3	3.84		74.2	-
	C-4	3.77		72.7	(C-6) 62.5
	C-6	3.86		62.5	-
Sucarose (M7)	C-1	5.48 (t, J = 3.6 Hz)	3.35, 3.42, 3.69	93.3	(C-3) 74.3, (C-1′) 105.0
	C-2	3.42	3.69	72.9	(C-3) 74.3
	C-3	3.69	3.35	74.2	(C-2) 72.9
	C-4	3.35		72.8	(C-3) 74.3
	C-2′	4.08	-	87.9	(C-1′) 105.0
	C-3′	4.03	-	76.8	(C-5′) 63.1, (C-1′) 105.0
	C-4′	3.75	-	83.6	(C-6′) 63.9
	C-5′	3.76	-	63.1	(C-1′) 105.0, (C-4′) 83.6
	C-6′	3.62	-	63.9	(C-1′) 105.0
Amino acids
Alanine (M8)	C-3	1.47 (d, J = 7.2)	3.64	16.9	51.5, 174.9
	C-2	3.64	1.47	51.4	-
Aaspartic acid (M9)	C-3a	2.96	2.71, 3.85	35.3	(C-2) 52.6, (C-4) 172.9, (C-1)174.9
	C-3b	2.71	-	35.3	(C-2) 52.6, (C-4) 172.9, (C-1)174.9
	C-2	3.85	-	52.6	(C-4) 172.9, (C-1) 174.9
Glycine (M10)	C-2a	3.88	-	43.6
	C-2b	4.01	-	43.6	(C-1) 174.7
Tyrosine (M11)	C-3	2.98–3.31		40.0	(C-4) 127.2
	C-2	4.14		50.6
	C-6, C-8	6.77	7.13	116.5	(C-4) 127.2
	C-5, C-9	7.13	6.77	131.3	(C-7) 157.6
Tryptophan (M12)	C-2	3.89	-	56.5	(C-3) 24.9, (C-1) 173.4
	C-8	7.05 (t, J = 7.5 Hz)	7.12, 7.37	119.8	(C-7) 112.2, (C-11) 128.3
	C-9	7.12 (overlap)	7.69	122.5	(C-6) 138.2, (C-8) 119.0
	C-5	7.20 (s)	-	124.9	(C-4) 102.1, (C-6) 138.2
	C-10	7.69 (d, J = 7.9 Hz)	7.37	118.9	(C-9) 122.5, (C-6)138.2
	C-7	7.37 (d, J = 8.2 Hz)	-	112.2	-
Bufadienolides
Bufalin and Scilliridin (M13, M14)	C-21	7.98 (d, J = 2.6 Hz)	6.25	149.9
	C-22	6.27 (d, J = 3.0 Hz)	7.97	115.2	122.4, 164.3 (C-20, 23)
	C-24	7.43 (s)		150.3	122.4, 164.2 (C-20, 23)
	C-17	2.59		51.7	124.2 (C-20)
	C-8	1.83	1.68	42.8	84.0, 49.0 (C-14, 13)
	C-9	1.68	1.83	43.1
Bufalin (M13)	C-18	0.714		12.1	(C-13) 49.0, (C-17) 51.0, (C-14) 84.0
	C-19	0.98		19.0	(C-1) 35.0, (C-2) 37.0, (C-9) 57.0
	C-3	3.93		73.5	(C-1) 35.0
Scilliridin (M14)
	C-3	5.96		128.6	(C-5)141.0
	C-4	5.75		127.3	-
	C-6	5.71		127.5	(C-5) 141.0
	C-19	1.02 3H		19.2	(C-10) 38.3, (C-9) 51.9, (C-6) 141.4
	C-18	0.81		17.7	(C-13) 49.0, (C-17) 51.0, (C-14) 84.0
Flavanoids
kaempeferol derv. (M15)	C (3′, 5′)	δ 6.98 (d, J = 8.5 Hz, 2H)		-	-
	C (2′, 6′)	6.56 (d, J = 8.4 Hz, 2H)		-	-
	C-8	6.04 (d, J = 1.8 Hz, H)		-	-
	C-6	6.04 (d, J = 1.8 Hz, H)		-	-
Coumarins
6-Hydroxy coumarin (M16)	C-3	6.19 (d, J = 9.4 Hz)	7.84	112.1	(C-10) 112.9, 157.0
	C-4	7.84 (d, J = 9.4 Hz)	6.19	145.8	(C-2) 157.0
	C-8	6.72 (d, J = 2.3 Hz)		103.1	(C-6) 157.6
	C-6	6.80 (dd, J = 8.5, 2.3 Hz)	7.47	114.25	(C-7) 154
	C-5	7.47 overlap (d, J = 8.5 Hz)	6.80	130.43	(C-4) 145.79

Table 4. The IC₅₀ values (mean ± SD) of RS and WS extract against different cancer cell lines measured by SRB assay versus dox. (doxorubicin) positive control.

RS (MCF7) IC50	WS (MCF7) IC50	Dox.(MCF7) IC50	RS (A-549) IC50	WS (A-549) IC50	Dox. (A-549) IC50	RS (SKOV-3) IC50	WS (SKOV-3) IC50	Dox. (SKOV-3)IC50
0.165 ± 0.007 *	0.326 ± 0.005	0.2 ± 0.004 *	0.271 ± 0.005 *	0.108 ± 0.003 *	0.56 ± 0.003 *	0.912 ± 0.021	0.690 ± 0.018	0.2 ± 0.01

* p < 0.05 comparing dox. positive control with each treatment group of RS and WS extracts on different cancer cell lines using one way ANOVA followed by Dunnett’s test.

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Khattab, O.M.; El-Kersh, D.M.; Khalifa, S.A.M.; Yosri, N.; El-Seedi, H.R.; Farag, M.A. Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect. Plants 2023, 12, 2078. https://doi.org/10.3390/plants12112078

AMA Style

Khattab OM, El-Kersh DM, Khalifa SAM, Yosri N, El-Seedi HR, Farag MA. Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect. Plants. 2023; 12(11):2078. https://doi.org/10.3390/plants12112078

Chicago/Turabian Style

Khattab, Omar M., Dina M. El-Kersh, Shaden A. M. Khalifa, Nermeen Yosri, Hesham R. El-Seedi, and Mohamed A. Farag. 2023. "Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect" Plants 12, no. 11: 2078. https://doi.org/10.3390/plants12112078

APA Style

Khattab, O. M., El-Kersh, D. M., Khalifa, S. A. M., Yosri, N., El-Seedi, H. R., & Farag, M. A. (2023). Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect. Plants, 12(11), 2078. https://doi.org/10.3390/plants12112078

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

Abstract

1. Introduction

2. Results and Discussion

2.1. Identification of Volatile Organic Compounds (VOCs)

2.2. Secondary Metabolites Profiling via UHPLC/MS

2.2.1. Bufadienolides

Hydroxyoxobufaenolide

2.2.2. Flavonoids

Identification of Flavanols/Flavonols

Identification of Flavones/Flavanones

Identification of Coumarins

2.2.3. Phenolic Acids

2.2.4. Amino Acids and Fatty Acids

2.3. Multivariate Data Analysis of UPLC-MS Dataset

2.4. NMR Metabolites Fingerprinting

2.4.1. Fatty Acids

2.4.2. Sugars

2.4.3. Amino Acids

2.4.4. Bufadienolides

2.4.5. Coumarins and Flavonoids

2.5. Quantification of Major Metabolites via 1H-NMR

2.6. Cytotoxic Screening Activity

3. Materials and Methods

3.1. Plant Material

3.2. Secondary Metabolites Extraction and Preparation of NMR and MS Analysis Sample

3.3. SPME/GC-MS

3.4. UHPLC/MS

Multivariate Data Analysis of UPLC-MS & GC-MS Dataset

3.5. Identification of Major Metabolites via NMR Analysis

Quantification of Major Metabolites via 1H-NMR

3.6. Bioassays

3.6.1. Cell Culture

3.6.2. Cytotoxic Screening Assay

3.6.3. Statistical Analysis

4. Conclusions

Supplementary Materials

Author Contributions

Funding

Data Availability Statement

Acknowledgments

Conflicts of Interest

References

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2.5. Quantification of Major Metabolites via ¹H-NMR

Quantification of Major Metabolites via ¹H-NMR