Discriminative Metabolomics Analysis and Cytotoxic Evaluation of Flowers, Leaves, and Roots Extracts of Matthiola longipetala subsp. livida

Mona M Marzouk; Nesrine M Hegazi; Mona O A El Shabrawy; Mai M Farid; Salwa A Kawashty; Sameh R Hussein; Nabiel A M Saleh

doi:10.3390/metabo13080909

. 2023 Aug 3;13(8):909. doi: 10.3390/metabo13080909

Discriminative Metabolomics Analysis and Cytotoxic Evaluation of Flowers, Leaves, and Roots Extracts of Matthiola longipetala subsp. livida

Mona M Marzouk ^1,^*, Nesrine M Hegazi ¹, Mona O A El Shabrawy ¹, Mai M Farid ¹, Salwa A Kawashty ¹, Sameh R Hussein ¹, Nabiel A M Saleh ¹

Editor: Wolfgang Eisenreich¹

PMCID: PMC10456503 PMID: 37623853

Abstract

Matthiola longipetala subsp. livida is an annual herb in Brassicaceae that has received little attention despite the family’s high reputation for health benefits, particularly cancer prevention. In this study, UPLC-HRMS-MS analysis was used for mapping the chemical constituents of different plant parts (i.e., flowers, leaves, and roots). Also, spectral similarity networks via the Global Natural Products Social Molecular Networking (GNPS) were employed to visualize their chemical differences and similarities. Additionally, the cytotoxic activity on HCT-116, HeLa, and HepG2 cell lines was evaluated. Throughout the current analysis, 154 compounds were annotated, with the prevalence of phenolic acids, glucosinolates, flavonol glucosides, lipids, peptides, and others. Predictably, secondary metabolites (phenolic acids, flavonoids, and glucosinolates) were predominant in flowers and leaves, while the roots were characterized by primary metabolites (peptides and fatty acids). Four diacetyl derivatives tentatively assigned as O-acetyl O-malonyl glucoside of quercetin (103), kaempferol (108 and 112), and isorhamnetin (114) were detected for the first time in nature. The flowers and leaves extracts showed significant inhibition of HeLa cell line propagation with LC₅₀ values of 18.1 ± 0.42 and 29.6 ± 0.35 µg/mL, respectively, whereas the flowers extract inhibited HCT-116 with LC₅₀ 24.8 ± 0.45 µg/mL, compared to those of Doxorubicin (26.1 ± 0.27 and 37.6 ± 0.21 µg/mL), respectively. In conclusion, the flowers of M. longipetala are responsible for the abundance of bioactive compounds with cytotoxic properties.

Keywords: cytotoxicity, HeLa cell line, Matthiola longipetala, molecular networking, UPLC-HRMS-MS

1. Introduction

Brassicaceae (=Cruciferae) is one of the economically important angiosperm families, commonly known as the crucifers, cabbage, or mustard family, containing over 372 genera and approximately 4636 species [1]. Plants of the family Brassicaceae have been an interesting research subject for years due to their economic and agricultural importance. Many species have been valued as food crops; some are vegetables, others are sources of industrial and cooking oils, forage, and condiments and others are grown as ornamental species for their showy flowers and significant numbers as medicinal herbs [2]. Additionally, certain wild cruciferous plants are rich in secondary metabolites, especially glucosinolates, phenolic acids, and flavonoids, which have many biological activities and, therefore, numerous nutritional and medicinal benefits [3,4]. Matthiola longipetala subsp. livida (Delile) Maire is one of the common wild medicinal cruciferous herbs growing mainly in the Egyptian Mediterranean region, and it is locally known as “Manthor” [5]. Although some previous phytochemical studies have been conducted on M. longipetala subsp. livida [6,7,8,9], the reported compounds represent only a small portion of the species’ chemical composition. Similarly, certain biological activities such as antibacterial, antifungal, and anticancer effects have been reported for the investigated species [8,9,10].

Lately, metabolomics platforms have been widely used to map the metabolome of plants, among which ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS) as the most extensively adopted for mapping the secondary metabolome space. UPLC-HRMS/MS offers the advantages of high efficiency, reproducibility, and shorter analysis [11]. Additionally, advances in the data analysis tools, such as molecular networks through the Global Natural Products Social Molecular Networking (GNPS) [12], allow for the visual display of the constitutive metabolome among samples and the propagation of metabolites annotation [13].

Over the last few decades, most new therapeutic interventions involving plant secondary metabolites and their derivatives have been aimed at combating cancer. In this regard, cruciferous plants have been previously reported to lower the risk of developing various cancers [14]. Our previous research reported the moderate cytotoxic potential of the alcoholic extract of the aerial part of M. longipetala subsp. livida against cervix (HeLa) and colon (HCT116) cell lines [8]. Moreover, another report assessed the extract’s activity against HepG2 cells in vitro using MTT, DNA fragmentation, and cell proliferation cycle measurements, and it demonstrated significant activity [15].

In continuation of our previous study, the present work aimed to map the under-explored chemistries of different organs (i.e., flowers, leaves, and roots) of M. longipetala using UPLC–HRMS-MS analysis that recruited for a holistic overview of the plant’s constitutive chemistries, coupled with spectral similarity networks through the GNPS [12]; this was in addition to evaluating the cytotoxic activities of the three organs on HCT-116, HeLa, and HepG2 cell lines to suggest the one responsible for this potential.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals for chemical analysis were obtained from Sigma-Aldrich (Merck, Kenilworth, NJ, USA).

2.2. Plant Material and Preparation of the Extracts

M. longipetala subsp. livida (650 g fresh weight) was collected from Alexandria-Marsa Matruh Road, 31°04′15.3″ N 27°58′10.4″ E, Egypt, in February 2018. The identity of the plant was authenticated by Prof. Dr. Mona M. Marzouk. A voucher specimen (ML_28_2_18) was placed in the herbarium of the National Research Centre (CAIRC), Cairo, Egypt. The flowers, leaves, and roots (117, 175, and 152 g fresh weight, respectively) were washed thoroughly with bi-distilled H₂O, dried in shade, and ground finely. Fifteen grams of each dried powdered organ was separately extracted using 70% methanol (500 mL) by sonication (2 h, 60 °C) and filtered over charcoal to yield three aqueous methanolic extracts [16,17]. The flowers, leaves, and roots extracts were concentrated under reduced pressure at 50 °C to produce three dried extracts (2.761, 1.140, and 1.832 g, respectively).

2.3. Sample Preparation for UPLC-HRMS-MS Measurement

The dried extracts were prepared for UPLC-HRMS/MS analyses following a previously described protocol [16]. The extracts (50 mg each) were dissolved in 70% MeOH (HPLC-grade) with sonication (10 min), then centrifuged. Aliquots were then evaporated under reduced pressure, followed by freeze-drying for 48 h. For MS analysis, 1 mg in 250 µL MeOH (MS-grade) were prepared consuming 5 µL as an injection volume in the UPLC-MS analysis.

2.4. UPLC-HRMS-MS Analysis

The HRMS/MS analysis was conducted on a MaXis 4G instrument (Bruker Daltonics^®, Bremen, Germany) coupled with an Ultimate 3000 UPLC (Thermo Fisher Scientific^®, Waltham, MA, USA). A UPLC method was applied as described by [17] as follows: (with 0.1% formic acid in H₂O as solvent A and 100% ACN as solvent B), an isocratic gradient of 10% B for 10 min, 10% to 100% B in 30 min, 100% B for an additional 10 min, using a flow rate of 0.3 mL/min; 5 µL injection volume and UV detector (UV/VIS) wavelength monitoring at 210, 254, 280, and 360 nm. The separation was conducted on a Nucleoshell RP 18 column, 2.7 µm, 150 × 2 mm (Macherey-Nagel^®, Düren, Germany), and the range for MS acquisition was 50–1800 Daltons (Da). A capillary voltage of 4500 V, nebulizer gas pressure (nitrogen) of 2 (1.6) bar, ion source temperature of 200 °C, dry gas flow of 9 L/min, and spectral rates of 3 Hz for MS¹ and 10 Hz for MS², were utilized. For acquiring MS/MS fragmentation, the 10 most intense ions per MS¹ were selected for subsequent CID, with stepped CID energy applied. The employed parameters for tandem MS were applied as previously detailed [18].

2.5. Data Analysis and Preprocessing

Raw data inspection was performed using Compass Data Analysis 4.4 (Bruker Daltonics^®). A Metaboscape 3.0 (Bruker Daltonics^®) was utilized for feature detection, grouping, and alignment, employing the T-ReX 3D (Time aligned Region Complete eXtraction) algorithm [19]. Bucketing was performed with an intensity threshold of 1 × 10⁵ and a retention time range from 0 to 40 min with a restricted mass range m/z from 130 to 1800.

2.6. Feature-Based Molecular Networking (FBMN) and Metabolites Dereplication

The produced MGF file and the feature quantification table (CSV file) were used in the feature-based molecular networking (FBMN) following the online workflow in GNPS platform (http://gnps.ucsd.edu), accessed on 28 December 2019 [20]. The parameters applied for the construction of the FBMN via the GNPS platform as follows: a parent mass tolerance (0.05 Da), a fragment ion tolerance (0.05 Da), a cosine score (0.7), and minimum shared fragments (6). To avoid misinterpretation of artifacts, the blank run was uploaded as a distinct sample on GNPS workflow and excluded from the networks. Cytoscape version 3.9.1 (https://cytoscape.org/), accessed on 28 February 2022, was used for the network visualization.

The metabolites’ dereplication was based on the chromatographic performance, chemical formula, and fragmentation pattern compared to those of MS² data from literature and spectra from MS reference database (MoNA, NIST14, and Respect) (Table 1). Sirius plus CSI:FingerID 5.5.4 were used for the manual putative structures identification [21], assisted by the molecular formula prediction (C, H, N, O, S, and P) and candidate search with m/z tolerance set to 20 ppm connected to online Pubchem. The proposed in silico fragmentation trees are the impetus for further support for identification.

Table 1.

Metabolites identified in the aqueous methanol extracts of flowers, leaves, and roots from Matthiola longipetala subsp. livida via UPLC-HRMS-MS in negative and positive ionization modes.

No.	Chemical Class (Sub-Class)	RT	(M − H)⁻	(M + H)⁺	MS²		Root	Leaf	Flower	Tentatively Identification	Molecular Formula (Error in ppm)	Ref.
No.	Chemical Class (Sub-Class)	RT	(M − H)⁻	(M + H)⁺	Negative	Positive	Root	Leaf	Flower	Tentatively Identification	Molecular Formula (Error in ppm)	Ref.
1	Amino acids	2.21	-	175.1193	-	158.0928, 128.0200, 116.0708, 86.9928 70.0651	+	+	++	Arginine	C₆H₁₄N₄O₂ (1.9)	[22]
2	Amino acids	2.41	-	116.0713	-	70.0660	+	+	++	Proline	C₅H₉NO₂ (5.3)	[22]
3	Saccharides	2.45	341.1090	-	179.0565, 161.0469, 149.0471, 119.0348, 89.0245	-	+	+		Dihexoside	C₁₂H₂₂O₁₁ (0.4)	[23]
4	Alkaloids and derivatives (alkaloids)	2.54	-	138.0505	-	122.0648, 110.0594, 96.0445	-	+	-	Trigonelline *	C₇H₇NO₂ (−2.7)	[24]
5	Amino acids and derivatives	2.58	-	130.0861	-	86.0965, 84.0807, 70.0651	++	+	+	Methyl proline *	C₆H₁₁NO₂ (1.0)	[22]
6	Amino acids and derivatives	2.59	-	144.1019	-	130.0502, 104.0294, 98.0602, 86.0965, 84.0807, 70.0651	+	++	+	Dimethyl proline	C₇H₁₃NO₂ (0.3)	[22]
7	Glucosinolates (alkylglucosinolates)	2.60	434.0253	-	419.0020, 274.9899, 259.0128, 240.9670, 195.0335, 168.9510, 129.0253, 96.9602, 74.9912	-	+	+	++	Glucoraphenin	C₁₂H₂₁NO₁₀S₃ (0.4)	[25]
8	Organic acids (hydroxy acids)	2.70	147.0298	-	129.0193, 101.0244, 85.0295	-	t	++	++	Hydroxyglutaric acid	C₅H₈O₅ (0.6)	[23]
9	Flavonoids (flavonol-O-glycoside)	2.72	-	873.2667	-	595.1675, 449.1086, 287.0556	-	+	+++	Km-O-rhamnosyl-arabinoside-O- rhamnosyl glucoside	C₃₈H₄₈O₂₃ (0.2)	[8]
10	Glucosinolates (alkylglucosinolates)	2.83	-	176.0201	-	149.0595, 117.0335, 96.0443, 70.9950	-	+	+++	Raphenin *	C₆H₉NOS₂ (−1.6)	[26]
11	Glucosinolates (alkylglucosinolates)	2.93	418.0299	-	259.0127, 224.9726, 176.0208, 96.9601, 74.9911	-	+++	+	-	Methylthiobutenyl- glucosinolate	C₁₂H₂₁NO₉S₃ (0.8)	[27]
12	Phenolic acids and derivatives (hydroxycinnamic acid glycosides)	3.02	487.1458	-	163.0422, 145.0295	-	-	-	+	Coumaric acid-O- dihexoside	C₂₁H₂₈O₁₃ (0.4)	[28]
13	Organic acids (hydroxy acids)	3.60	133.0143	-	115.0039, 72.9935	-	t	+	+++	Malic acid	C₄H₆O₅ (0.4)	[29]
14	Glucosinolates (alkylglucosinolates)	3.67	434.0254	-	419.0020, 274.9899, 259.0129, 240.9671, 195.0333, 168.9509, 129.0251, 96.9602, 74.9912	-	+	+	++	Glucoraphenin isomer	C₁₂H₂₁NO₁₀S₃ (0.2)	[30]
15	Organic acids (dicarboxylic acids)	3.75	-	131.0342	-	116.9329, 108.9868, 85.0282, 71.0153, 62.9826	+	+	+	Citraconic acid (methyl maleic acid)	C₅H₆O₄ (−2.3)	[31]
16	Organic acids (carboxylic acids)	3.76	-	130.0868	-	86.0992, 84.0807, 85.0845, 70.0653	+	+	+	Pipecolic acid	C₆H₁₁NO₂ (1.9)	[31]
17	Amino acids	3.79	-	132.1019	-	86.0965, 84.0809, 73.0648, 71.0128, 69.0699	+	+	+	Leucine/isoleucine	C₆H₁₃NO₂ (0.1)	[22]
18	Glucosinolates (alkylglucosinolates)	3.92	418.0299	-	274.9898, 259.0127, 195.0335, 96.9602, 74.9912	-	+	-	-	Methylthiobutenyl-glucosinolate isomer	C₁₂H₂₁NO₉S₃ (0.2)	[27]
19	Phenolic acids and derivatives (hydroxybenzoic acid glucoside)	4.21	299.0770	-	137.0244, 93.0345	-	+	+	++	Hydroxy benzoic acid-O-hexoside	C₁₃H₁₆O₈ (0.8)	[31]
20	Glucosinolates (alkylglucosinolates)	4.31	374.0582	-	259.0134, 195.0338, 96.9601, 74.9909	-	+	+	++	Butyl glucosinolate	C₁₁H₂₁NO₉S₂ (0.9)	[27]
21	Organic acids (cinnamic acids)	4.36	-	149.0600		105.0444, 104.0543, 79.0536	+	+	+	Cinnamic acid	C₉H₈O₂ (−1.8)	[29]
22	Amino acids	4.37	164.0715	166.0630	147.0457, 103.0558, 72.0092	120.080, 103.0543, 93.0699	+	+	+	Phenylalanine *	C₉H₁₁NO₂ (0.3)	[22]
23	Glucosinolates (alkylglucosinolates)	4.53	418.0299	-	274.9805, 259.0129, 224.9700, 195.0330, 176.0207, 96.9602, 74.9911	-	-	-	+	Methylthio-butenyl-glucosinolate isomer	C₁₂H₂₁NO₉S₃ (0.1)	[27]
24	Organic acids (carboxylic acids)	4.54	218.1036	220.1184	146.0823, 88.0406, 71.0501	202.1084, 184.0974, 172.1327, 158.0603, 142.0860, 124.0760, 98.0238, 72.0443	+	+	++	Pantothenic acid (vitamin B5)	C₉H₁₇NO₅ (0.7)	[32]
25	Phenolic acids and derivatives (hydroxybenzoic acid glycosides)	4.70	315.0716	-	153.0186, 152.0113, 109.0297, 108.0225	-	+	+	+	Dihydroxybenzoic acid-O-hexoside	C₁₃H₁₆O₉ (0.8)	[17]
26	Phenolic acids and derivatives (hydroxycinnamic acid glycosides)	4.81	517.1558	-	397.1158, 193.0508, 175.0401, 119.0345	-	t	-	+	Ferulic acid-O-dihexoside *	C₂₂H₃₀O₁₄ (0.0)	[33]
27	Flavonoids (flavonol-O- glycosides)	5.01	-	773.2147	-	611.1624, 449.1084, 287.0555	t	+	+++	Km 3-O-sophroside- 7-O-glucoside	C₃₃H₄₀O₂₁ (1.5)	[34]
28	Phenols (methoxyphenols)	5.02	-	151.0757	-	136.0605, 119.0496, 91.0543, 79.0550, 68.9826	t	+	+++	Methoxy- vinylphenol	C₉H₁₀O₂ (−2.1)	[35]
29	Glucosinolates (alkylglucosinolates)	5.11	386.0582	-	274.9892, 259.0134, 195.0335, 96.9602, 74.9912	-	-	+	+++	Glucobrassicanapin	C₁₂H₂₁NO₉S₂ (0.5)	[28]
30	Iridoids (iridoids-O-glycosides)	5.13	375.1297	-	167.0709, 152.077	-	t	+	+++	Loganic acid	C₁₆H₂₄O₁₀ (0.1)	[36]
31	Amino acids and derivatives	5.20	-	367.1504	-	349.1397, 332.1128, 303.1349, 276.1241, 258.1131, 229.0976, 202.064, 188.0713, 146.0603	+	+	+	Tryptophan N-hexoside	C₁₇H₂₂N₂O₇ (0.8)	[37]
32	Phenolic acids and derivatives (hydroxybenzoic acid glycosides)	5.30	329.0853	-	209.0445, 167.0350, 119.0342, 89.0245	-	t	+	+++	Vanillic acid- O-hexoside	C₁₄H₁₈O₉ (0.2)	[17]
33	Flavonoids (flavonol-O- glycosides)	5.50	917.2648 ^a	-	871.2510, 709.1991, 563.1405, 431.0940, 285.0369	-	-	-	+	Km -O-arabinosyl rhamnoside-O- rhamnoside-O- glucoside ^b	C₃₈H₄₈O₂₃ (0.5)	[8]
34	Amino acids	5.54	203.0824	205.0975	142.0664, 116.0503, 74.0248	188.0711, 170.0606, 159.0916, 146.0602, 132.0810, 118.0653	+	+	+	Tryptophan *	C₁₁H₁₂N₂O₂ (−0.2)	[22]
35	Glucosinolates (alkylglucosinolates)	5.65	418.0299	420.0457	274.9805, 259.0129, 224.9700, 195.0330, 176.0207, 96.9602, 74.9911	178.0360, 130.0324, 85.0282	+	+	++	Methylthio-butenyl-glucosinolate isomer	C₁₂H₂₁NO₉S₃ (0.6)	[27]
36	Phenolic acids and derivatives (hydroxybenzoic acid derivative)	5.81	246.9919	-	167.0350, 152.0116, 153.0452, 108.0219	-	+	+	+	Vanillic acid-sulfate	C₈H₈O₇S (0.5)
37	Phenolic acids and derivatives (hydroxybenzoic acid glycosides)	5.81	285.0614	-	153.019, 152.0112, 109.0292, 108.0220	-	+	+	+	Dihydroxybenzoic acid-O-pentoside	C₁₂H₁₄O₈ (0.5)	[38]
38	Coumarins and derivatives (hydroxycoumarins)	5.91	-	163.0605	-	147.0446, 131.0497, 119.0494, 103.0544, 91.0541	t	+	+++	Hydroxycoumarin	C₉H₆O₃ (−2.0)	[31]
39	Phenolic acids and derivatives (hydroxycinnamic acid glycosides)	5.94	487.1452	-	367.1031, 163.0397, 145.0293	-	t	+	+++	Coumaric acid- O-dihexoside	C₂₁H₂₈O₁₃ (0.5)	[28]
40	Flavonoids (flavonol-O- glycosides)	6.10	609.1459	-	447.0927, 429.0825, 285.0404	-	t	++	++	Km 3-O- sophoroside *	C₂₇H₃₀O₁₆ (0.3)	[39]
41	Phenolic acids and derivatives	6.11	395.0649	-	241.0023, 152.9863, 96.9602	-	+++	+	t	Dihydroxyphenylethanol-O-sulfoglucoside	C₁₄H₂₀O₁₁S (0.9)	[40]
42	Flavonoids (flavonol-O- glycosides)	6.14	-	757.2199	-	449.1087, 287.0557	t	+	+++	Km-O-rutinoside- O-glucoside	C₃₃H₄₀O₂₀ (1.1)	[41]
43	Phenolic acids and derivatives (hydroxycinnamic acid glycosides)	6.24	517.1556	-	397.115, 193.051, 175.039	-	t	+	+++	Ferulic acid-O- dihexoside isomer *	C₂₂H₃₀O₁₄ (0.4)	[33]
44	Fatty acids	6.25	-	163.0601	-	131.0496, 119.0494, 103.0544, 91.0544, 77.0385	t	+	+++	Hydroxylhexanedioic acid * (Hydroxyadipic acid)	C₆H₁₀O₅ (−2.7)	[42]
45	Coumarins and derivatives	6.31	-	207.065	-	193.0544, 179.0701, 147.0436, 119.0494, 91.0546	t	+	+++	Dimethoxycoumarin	C₁₁H₁₀O₄ (2.1)	[43]
46	Phenolic acids and derivatives (hydroxycinnamic acid glycosides)	6.31	547.167	-	427.1245, 223.0618, 205.051, 190.0268, 179.0564	-	t	+	+++	Sinapic acid-O- dihexoside *	C₂₃H₃₂O₁₅ (4.5)	[44]
47	Flavonoids (flavonol-O- glycosides)	6.39	871.2510	873.2667	709.1976, 563.1404, 447.0923, 431.0975, 285.105	711.1985, 595.1675, 449.1086, 433.1137, 287.0556	-	+	+++	Km 3-O-(2″-rhamnosyl)-arab-inoside-7-O-rhamnoside-4′-O-β-glucoside ^b	C₃₈H₄₈O₂₃ (0.7)	[8]
48	Flavonoids (Flavonol-O- glycosides)	6.39	593.1511	595.1665	285.0397, 284.0323	449.1088, 287.0559, 229.0868, 207.0658	t	+	+++	Km 3-O- rutinoside	C₂₇H₃₀O₁₅ (0.4)	[31]
49	Flavonoids (flavonol-O- glycosides)	6.40	-	727.2088	-	449.1093, 287.0559	-	+	+++	Km O-arabinosyl rhamnoside-O- glucoside	C₃₂H₃₈O₁₉ (1.1)	[31]
50	Flavonoids (flavonol-O- glycosides)	6.44	-	711.2146	-	433.1137, 287.0557	t	+	+++	Km-O-arabinosyl rhamnoside-O- rhamnoside	C₃₂H₃₈O₁₈ (1.3)	[8]
51	Flavonoids (flavonol-O- glycosides)	6.54	741.1881	743.2038	609.1461, 579.1348, 463.0872, 447.0924, 301.0351	611.1608, 465.1035, 303.0504	t	+	+++	Qn-O-arabinosyl rhamnoside-O- glucoside	C₃₂H₃₈O₂₀ (1.9)	[45]
52	Flavonoids (flavonol-O- glycosides)	6.74	739.2087	741.2247	593.1511, 447.0923, 431.0952, 285.0397, 284.0323	465.1035, 433.1137, 287.0556	t	+	+++	Km 3-O-rhamnoside 7-O-rutinoside *	C₃₃H₄₀O₁₉ (0.5)	[46]
53	Flavonoids (flavonol-O- glycoside)	6.83	-	757.2199	-	479.1195, 463.1243, 317.0661	-	+	+++	Is O-arabinosyl rhamnoside-O- glucoside	C₃₃H₄₀O₂₀ (1.8)	[31]
54	Phenolic derivatives (benzoyl derivatives)	6.88	-	137.0600	-	123.0394, 122.0365, 107.0500, 95.0415, 94.0415, 79.0544, 77.0387	t	+	+++	Methoxybenzaldehyde	C₈H₈O₂ (2.5)	[47]
55	Flavonoids (flavonol-O- glycoside)	6.94	709.1973	-	563.1407, 431.0960, 285.0397, 284.0324	-	-	-	+	Km-O-rhamnosyl arabinoside-O- rhamnoside	C₃₂H₃₈O₁₈ (0.3)	[8]
56	Coumarin and derivatives	6.98	-	207.0657	-	193.0544, 179.0710, 147.0447, 119.0494, 91.0543, 83.0495	t	+	+++	Dimethoxycoumarin isomer	C₁₁H₁₀O₄ (2.0)	[43]
57	Phenolic acids and derivatives (hydroxycinnamic acid glycosides)	6.98	385.1141	-	223.0617, 205.0505, 190.0271, 179.0715, 164.0478	-	t	+	+++	Sinapic acid-O- hexoside *	C₁₇H₂₂O₁₀ (0.1)	[17]
58	Flavonoids (flavonol-O- glycosides)	7.22	725.1937	727.2094	579.1357, 447.0898, 446.0850, 301.035	449.1086, 303.0507	t	t	+	Qn-O-rhamnosyl- arabinoside-O- rhamnoside	C₃₂H₃₈O₁₉ (0.7)	[46]
59	Flavonoids (flavonol-O- glycosides)	7.29	609.1464	611.1615	463.0878, 447.0852, 301.0352, 285.0399	303.0505	t	+	+++	Qn 3-O-rhamnoside 7-O-glucoside	C₂₇H₃₀O₁₆ (0.5)	[47,48]
60	Flavonoids (flavonol-O- glycosides)	7.41	609.1462	-	447.0927, 285.0404	-	t	+	+++	Km 3,7 di-O-glucoside *	C₂₇H₃₀O₁₆ (0.3)	[39,49]
61	Phenolic derivatives (Benzoyl derivatives)	7.55	-	137.0601	-	123.0394, 122.0365, 107.0510, 95.0508, 94.0415	t	+	+++	Methoxybenzaldehyde isomer	C₈H₈O₂ (−1.9)	[50]
62	Flavonoids (flavonol-O- glycosides)	7.65	579.1349	581.1510	447.0906, 446.0854, 433.0779, 301.0347	303.0506	t	t	+++	Qn 3-O-rhamnoside 7-O-arabinoside	C₂₆H₂₈O₁₅ (1.3)	[51]
63	Flavonoids (flavonol-O- glycosides)	7.72	-	565.1552	-	287.0555	t	+	+++	Km 3-O-arabinoside-7-O rhamnoside	C₂₆H₂₈O₁₄ (1.4)	[48]
64	Flavonoids (flavonol-O- glycosides)	7.73	709.1991	-	563.1407, 431.0960, 285.0397	-	-	-	+	Km O-rhamnosyl arabinoside-O- rhamnoside	C₃₂H₃₈O₁₈	[8]
65	Flavonoids (flavonol-O- glycosides)	7.71	431.0960	433.1133	285.0397, 284.0324	287.0557	-	+	+++	Km 7-O- rhamnoside *	C₂₁H₂₀O₁₀ (0.8)	[31]
66	Flavonoids (flavonol-O- glycosides)	7.82	709.1992	711.2139	563.1403, 431.0986, 285.0400	433.1138, 287.0556	t	+	+++	Km O-arabinosyl rhamnoside-O- rhamnoside ^b	C₃₂H₃₈O₁₈ (1.3)	[8]
67	Phenolic derivatives (Benzoyl derivatives)	7.86	357.1559	-	195.1032, 180.0784, 101.0236	-	t	+	+++	Trimethoxy benzaldehyde-O- hexoside	C₁₇H₂₆O₈ (0.9)	[50]
68	Flavonoids (flavonol-O- glycosides)	7.91	739.2094	741.2243	285.0402	287.0557	t	+	+++	Km 3-O-(di-O- rhamnosyl) glucoside *	C₃₃H₄₀O₁₉ (0.5)	[51]
69	Phenolic derivatives (benzaldehydes)	7.93	-	197.0813	-	137.0597, 105.0338, 79.0541	+	+	++	Trimethoxy benzaldehyde	C₁₀H₁₂O₄ (−2.6)	[50]
70	Fatty acid derivatives (fatty acyl glycosides)	8.00		-	387.2039, 225.1504, 161.0458, 113.0258, 101.0247	-	t	++	++	Hydroxyjasmonic acid-O-hexoside (tuberonic acid- O-hexoside)	C₁₈H₂₈ O₉ (2.0)	[52]
71	Flavonoids (flavonol-O- glycosides)	8.04	623.1633	625.1774	477.1031, 461.1071, 315.0510	317.0662	-	-	+	Is 3-O-rhamnoside 7-O-glucoside	C₂₈H₃₂O₁₆ (1.5)	[46]
72	Flavonoids (flavonol-O- glycosides)	8.14	563.1398	-	431.0903, 417.08, 285.0403	-	-	t	+	Km 3-O-rhamnoside 7-O-arabinoside	C₂₆H₂₈O₁₄ (0.5)	[48]
73	Flavonoids (flavonol-O- glycosides)	8.19	609.1446	-	447.0918, 285.0396, 284.0318	-	-	+	+++	Km 3-O- gentobiosde	C₂₇H₃₀O₁₆ (0.8)	[39]
74	Flavonoids (flavonol-O- glycosides)	8.39	461.1069	463.1240	315.0509, 314.0429	317.0661	+	+	++	Is 7-O- rhamnoside	C₂₂H₂₂O₁₁ (1.8)	[47]
75	Benzoic acid	8.41	153.0192	-	109.0296, 81.0350	-	t	+	+++	Dihydroxybenzoic acid	C₇H₆O₄ (0.9)	[53]
76	Flavonoids (flavonol-O- glycosides)	8.43	593.1509	595.1663	461.1069, 447.0928, 315.0509	317.0661	t	++	++	Is 3-O-rhamnoside 7-O-arabinoside	C₂₇H₃₀O₁₅ (0.5)	[28]
77	Flavonoids (flavonol-O- glycosides)	8.45	609.1468	611.1614	301.0346	303.0511, 287.0552, 229.0500, 129.0554, 85.0289	t	+	+++	Qn 3-O-rutinoside (rutin) *	C₂₇H₃₀O₁₆ (0.3)	[46]
78	Benzoic acids and derivatives	8.61	223.0246	-	179.0354, 135.0461, 109.0297	-	t	+	+++	Carboxyvinyl benzoic acid	C₁₀H₈O₆ (0.9)	[23]
79	Coumarins and derivatives	8.63	-	455.1164	-	411.1268, 369.1162, 207.0938, 179.0701, 147.0494, 79.0283, 69.0334	-	+	+++	Dimethoxycoumarin-O-malonyl glucoside	C₂₀H₂₂O₁₂ (4.4)	[43]
80	Phenolic derivatives	8.92	355.1401	-	221.0442, 161.0453, 139.0222, 119.0345, 101.0245, 89.0247, 71.0140, 59.0141	-	-	-	+	Hydroxy phenyl pentanoic acid-O- glucoside	C₁₇H₂₄O₈ (0.6)	[54]
81	Flavonoids (flavonol-O- glycosides)	9.02	463.0878	-	301.0277, 300.0276	-	t	+	+++	Qn 3-O- glucoside *	C₂₁H₂₀O₁₂ (0.2)	[34]
82	Flavonoids (flavonol-O- glycosides)	9.08	593.1580	595.1665	285.0396, 284.0325	287.0552	t	+++	+++	Km-O- rutinoside *	C₂₇H₃₀O₁₅ (0.6)	[55]
83	Flavonoids (flavonol-O- glycosides)	9.26	623.1622	625.1770	315.0510	317.0661	t	++	+++	Is 3-O- rutinoside *	C₂₈H₃₂O₁₆ (0.7)	[51]
84	Phenolic derivatives (Cinnamaldehyde)	9.4	207.0665	-	192.0429, 179.0536	-	t	+	+++	Sinapaldehyde	C₁₁H₁₂O₄ (0.7)	[17]
85	Flavonoids (flavonol-O- acylglycosides)	9.45	549.0881	551.1040	505.0988, 463.0882, 301.0331, 300.0275, 271.0242	303.0507	t	+	+++	Qn 3-O- malonylglucoside *	C₂₄H₂₂O₁₅ (1.8)	[56]
86	Flavonoids (flavonol-O- acylglycosides)	9.54	505.0979	-	463.0886, 301.0332, 300.0272, 271.0247, 255.0306	-	t	+	+++	Qn 3-O-acetyl- glucoside *	C₂₃H₂₂O₁₃ (1.7)	[57]
87	Flavonoids (flavonoid aglycones)	9.59	-	287.0505	-	257.0450, 229.0114, 149.0140, 97.0287	t	++	++	Km *^,b	C₁₅H₁₀O₆ (0.2)	[57]
88	Flavonoids (flavonol-O- glycosides)	9.60	447.0931	449.1083	327.0528, 285.0387, 284.0325, 255.0299, 227.0345	287.0555	t	++	++	Km 3-O-glucoside (astragalin) *	C₂₁H₂₀O₁₁ (0.8)	[47]
89	Flavonoids (flavonol-O- glycosides)	9.71	433.0755	-	301.0332, 300.0276	-	t	+	+++	Qn 3-O- arabinoside	C₂₀H₁₈O₁₁ (0.4)	[58]
90	Flavonoids (flavonol-O- glycosides)	9.83	477.1035	479.1195	315.0481, 314.0430, 299.0199, 285.0397, 271.0249, 243.0299	317.0664	t	-	+	Is 3-O- glucoside *	C₂₂H₂₂O₁₂ (0.8)	[49,58]
91	Phenolics and derivatives	9.92	223.0251	-	179.0351, 153.0194, 135.0452, 109.0297	-	t	+	+++	Carboxyvinyl benzoic acid isomer	C₁₀H₈O₆ (0.6)	[23]
92	Flavonoids (flavone-O- glycosides)	10.07	431.0981	433.1128	269.0447	271.060	+++	t	t	Apigenin 7-O- glucoside *	C₂₁H₂₀O₁₀ (0.3)	[52]
93	Phenolic acids and derivatives (hydroxycinnamic acids)	10.11	179.0354	-	135.0461, 109.0297	-	t	+	+++	Caffeic acid *	C₉H₈O₄ (0.4)	[52]
94	Flavonoids (flavonol-O- acylglycosides)	10.15	505.0979	507.1142	463.0886, 301.0332, 300.0275	303.0507	t	+	+++	Qn 3-O-acetyl glucoside isomer	C₂₃H₂₂O₁₃ (1.7)	[56]
95	Flavonoids (flavonol-O- acylglycosides)	10.25	533.0936	535.1091	285.0385, 284.0323	287.0557	t	++	++	Km 3-O- malonylglucoside	C₂₄H₂₂O₁₄ (1.6)	[59,60]
96	Flavonoids (flavonol-O- acylglycosides)	10.46	-	565.1198	-	317.0662	t	+	-	Is 3-O- malonylglucoside	C₂₅H₂₄O₁₅ (1.8)	[61]
97	Flavonoids (flavonol-O- acylglycosides)	10.47	519.1143	-	477.0989, 315.0502, 314.0429	-	t	+	+	Is 3-O-acetyl glucoside	C₂₄H₂₄O₁₃ (2.1)	[62]
98	Flavonoids (flavonol-O- acylglycosides)	10.51	505.0979	507.1142	463.0886, 301.0338, 300.0275	303.0505	t	+	+++	Qn 3-O-acetyl glucoside isomer	C₂₃H₂₂O₁₃ (1.7)	[56]
99	Phenolic derivatives (phenylpropanoid glycosides)	10.58	341.1242	-	161.0428, 133.0660, 101.0254	-	t	+	+++	Coniferyl alcohol-O- glucoside (coniferin)	C₁₆H₂₂O₈ (0.2)	[63]
100	Flavonoids (flavonol-O- glycosides)	10.65	447.0925	-	315.0477, 314.0430, 299.0209	-	t	+	+++	Is 3-O- arabinoside	C₂₁H₂₀O₁₁ (2.2)	[46]
101	Phenolic derivatives (phenylpropanoid glycosides)	10.91	371.1350	-	209.0441, 163.0767, 148.0531	-	t	+	+++	Sinapoyl alcohol-O-glucoside (syringin)	C₁₇H₂₄O₉ (−0.6)	[64]
102	Flavonoids (flavonol-O- glycosides)	10.99	431.0980	-	285.0400, 284.0323, 155.0303, 227.0347	-	-	+	+++	Km 3-O- rhamnoside (afzelin) *	C₂₁H₁₉O₁₀ (0.9)	[65]
103	Flavonoids (flavonol-O- acylglycosides)	11.01	591.0995	593.1148	547.1082, 505.0982, 301.0333, 300.0277	303.0506	t	+	+++	Qn 3-O-X₁ acetyl-X₂ malonyl glucoside	C₂₆H₂₄O₁₆ (0.3)
104	Flavonoids (flavonol-O- acylglycosides)	11.11	519.1121	521.1297	477.1016, 315.0428, 314.0430, 299.0192	317.0664, 287.0547	t	++	++	Is 3-O-acetyl glucoside isomer	C₂₄H₂₄O₁₃ (1.5)	[62]
105	Flavonoids (flavonol-O- acylglycosides)	11.23	489.1035	491.1192	285.0385, 284.0327	287.0555	t	++	++	Km 3-O-acetyl glucoside	C₂₃H₂₂O₁₂ (0.7)	[56]
106	Saccharides	11.33	405.09	-	241.0022, 152.9869, 96.9604	-	+	+	+	Thioglucose-penta acetate	C₁₆H₂₂O₁₀S (1.4)
107	Flavonoids (flavonol-O- acylglycosides)	11.44		521.1296		317.0661, 127.0391	t	++	+++	Is 3-O-acetyl glucoside isomer	C₂₄H₂₅O₁₃ (0.8)	[62]
108	Flavonoids (flavonol-O- acylglycosides)	12.01	575.1044	577.1191	531.1144, 489.1029, 285.0385, 284.0321	287.0555, 255.0441, 127.0391	t	++	+++	Km 3-O-X₁ acetyl-X₂ malonyl glucoside	C₂₆H₂₄O₁₅ (0.5)
109	Flavonoids (flavonol-O- acylglycosides)	12.02	531.1138	-	489.1031, 285.0385, 284.325, 255.0295	-	t	+	+++	Km 3-O-diacetyl glucoside	C₂₅H₂₄O₁₃ (0.7)	[62]
110	Flavonoids (flavonol-O- acylglycosides)	12.23	561.1236	-	519.1152, 477.1016 315.0428, 314.0427, 299.0198	-	t	+	+++	Is 3-O-diacetyl glucoside	C₂₆H₂₆O₁₄ (0.7)	[62]
111	Flavonoids (flavonol-O- glycosides)	12.31	461.1085	-	299.0554, 298.0485, 283.0250	-	+	++	+	Rh 3-O- glucoside ^b	C₂₂H₂₂O₁₁ (1.9)	[8]
112	Flavonoids (flavonol-O- acylglycosides)	12.68	-	577.1201	-	449.0908, 287.0555	t	+	+++	Km 3-O-X₁ acetyl-X_{2 -}malonyl glucoside isomer	C₂₆H₂₄O₁₅ (−2.2)
113	Flavonoids (flavonol-O- acylglycosides)	12.86	547.1095	549.1246	505.0963, 299.0239, 298.0280, 271.0250, 163.0770	301.0713, 231.0520, 159.0292, 127.0396	+	+++	+	Rh 3-O-malonoyl glucoside	C₂₅H₂₄O₁₄ (−1.5)	[61]
114	Flavonoids (flavonol-O- acylglycosides)	12.88	-	607.1295	-	317.0662, 302.0427, 287.0546, 255.0512, 231.0500, 127.0392, 109.0287	t	+	+++	Is 3-O-X₁ acetyl X₂ malonyl glucoside	C₂₇H₂₆O₁₆ (0.3)
115	Flavonoids (flavonol-O- acylglycosides)	12.90	561.1223	-	519.1147, 477.0989, 315.0502, 314.0430, 299.0205	-	-	-	+	Is 3-O-diacetyl glucoside (isomer II)	C₂₆H₂₆O₁₄ (0.5)	[62]
116	Fatty acid	13.70	327.2178	-	291.1951, 229.1452, 211.1384, 171.1026, 85.0297	-	++	+	+	Trihydroxy- octadecadienoic acid *	C₁₈H₃₂O₅ (0.8)	[66]
117	Fatty acid	14.64	329.2328	-	311.2220, 229.1442, 211.1342, 171.1025	-	+++	+	+	Trihydroxy- octadecanoic acid*	C₁₈H₃₆O₅ (0.4)	[67]
118	Flavonoids (flavonoid aglycones)	15.87	-	373.1285	358.1051, 343.0819, 329.1048, 315.0864, 229.0576	-	t	+++	+	Pentamethoxyflavone (tangeretin) *	C₂₀H₂₀O₇ (0.9)	[68]
119	Flavonoids (flavonoid aglycones)	17.21	-	373.1287	359.1180, 343.0821, 329.1024, 312.1006, 297.0771, 283.0981, 229.0553	-	t	+++	+	Pentamethoxyflavone isomer (sinensetin) *	C₂₀H₂₀O₇ (1.2)	[68]
120	Flavonoids (biflavone)	17.08	551.098	-	519.0691, 457.0573, 431.0760, 389.0667	-	t	+++	+	Amentoflavone methyl ether	C₃₁H₂₀O₁₀ (0.0)	[69]
121	Flavonoids (biflavone)	18.05	551.099	-	519.0727, 457.0560, 431.0769, 389.0660	-	t	+++	+	Amentoflavone methyl ether isomer	C₃₁H₂₀O₁₀ (0.1)	[69]
122	Flavonoids (flavonoid aglycones)	18.47	-	403.1393	373.0923, 359.1129, 343.0824, 329.1024, 313.0703	-	t	+++	+	Hexamethoxyflavone (Irigenin trimethyl ether) *	C₂₁H₂₂O₈ (1.3)	[68]
123	Flavonoids (flavonoid aglycones)	19.29	-	433.1492	419.1299, 418.1265, 403.1029, 385.0914	-	t	+++	+	Heptamethoxyflavone (Nobiletin) *	C₂₂H₂₄O₉ (0.8)	[68]
124	Flavonoids (flavonoid aglycones)	19.88	-	373.1286	358.1057, 343.0822, 325.0715, 312.0995, 271.0609, 211.0249, 183.0300	-	t	+++	+	Pentamethoxyflavone *	C₂₀H₂₀O₇ (1.1)	[68]
125	Phospholipids (lysoglycerophos- phoinositol)	19.92	593.2724	595.2888	413.2085, 315.0483, 277.2171, 241.0119, 152.9966	335.2586, 261.2222, 243.2124, 184.0707, 155.0107, 81.0697	++	+	++	Octadecatrienoyl-glycero-phosphoinositol	C₂₇H₄₇O₁₂P (0.4)	[70]
126	Phospholipids (lysoglycerophos- phoinositol)	21.71	595.2885	-	415.2244, 315.0475, 279.2329, 241.0116, 152.9960	-	++	+	++	Linoleoyl- glycero- phosphoinositol	C₂₇H₄₉O₁₂P (0.2)	[70]
127	Diterpenes	21.82	-	283.1698	-	265.1586, 223.1485, 197.1330, 183.1205	+	+	++	Miltirone *	C₁₉H₂₂O₂ (−1.0)	[71]
128	Fatty amides	22.41	-	298.346	-	281.0533, 245.1075, 227.0968, 74.0965	t	+	+++	N-hydroxyoleylamide *	C₁₈H₃₅NO₂ (1.6)
129	Phospholipids (lysoglycerophos- phoinositol)	22.88	571.2884	-	391.2254, 315.0487, 255.2329, 241.0119, 152.9959	-	+++	+	+	Palmitoyl- glycero-phosphoinositol *	C₂₅H₄₉O₁₂P (−1.7)	[70]
130	Peptides	23.12	-	643.2734	-	586.2621, 583.2526, 529.2143, 523.2311, 381.2094, 311.1647, 293.1540, 265.1591, 247.1489, 205.1966, 182.1016, 147.0811, 133.1029, 89.0603	+	-	-	Glycyl-glycyl- phenylalanyl- alanyl- glutamyl-tyrosine	C₃₀H₃₈N₆O₁₀ (−1.8)	[72]
131	Peptides	23.94	-	657.2866	-	597.2677, 537.2455, 507.2379, 343.132, 311.1642, 247.1480, 205.1966, 181.1016 166.0754, 147.0811 133.1029	+	-	-	Serinyl- serinyl- glycyl- tyrosyl- phenylalanyl- proline	C₃₁H₄₀N₆O₁₀ (−1.4)	[72]
132	Peptides	24.00	-	691.2720	-	631.2520, 571.2318, 541.2204, 495.1999, 453.1890, 393.1681, 353.1750, 311.1641, 293.1539, 265.1591, 247.1485, 223.1123, 133.0858, 91.0540	+	-	-	Serinyl- phenylalanyl- glycyl- glutamyl- aspartyl- histidine	C₃₄H₃₈N₆O₁₀ (−1.5)	[72]
133	Fatty amides	24.43	-	322.2751	-	304.2645, 135.0326, 107.0862, 95.0860	+++	t	+	α-Linolenoyl ethanolamide *	C₂₀H₃₅NO₂ (0.5)	[73]
134	Fatty acids	24.52	297.243		279.2334, 183.0120	-	+++	t	+	Methyl-oxo-heptadecanoic acid (lichesterylic acid) *	C₁₈H₃₄O₃ (0.6)
135	Peptides	24.61	-	685.29	-	625.2624, 565.2402, 353.1726, 293.1542, 247.1489, 181.1005, 182.0998, 147.0851, 119.0874, 106.0743	+	-	-	Tryptophyl-glutamyl-tyrosyl-serinyl-threonine	C₃₂H₄₀N₆O₁₁ (−1.8)	[72]
136	Lipid (sulfoglycolipids)	24.79	555.2844	-	299.0446, 255.2331, 206.9963, 80.9655	-	++	+++	+	Hexadecanoyl glycerol-O- sulfo-rhamnoside	C₂₅H₄₈O₁₁S (0.2)	[74]
137	Peptides	24.99	-	627.3956	-	369.1955, 351.1849, 333.1749, 313.2087, 277.1588, 182.1230, 166.1178, 106.4462, 91.0543, 97.1052, 75.0725	+	-	-	Tyrosyl-glycyl-glycyl phenylalanyl- serinyl-proline	C₃₀H₃₈N₆O₉ (−1.6)	[72]
138	Fatty acids	25.05	295.2282	-	279.2331	-	+++	t	t	Hydroxyoctadecadienoic acid *	C₁₈H₃₂O₃ (1.0)
139	Phospholipids (lysophosphatidylglycerols)	25.16	481.2568	-	253.2174, 245.0430, 227.0324, 152.9959	-	t	+++	+	Hexadecenoyl glycero-phospho- sn-glycerol *	C₂₂H₄₃O₉P (0.6)	[70]
140	Peptides	25.36	-	699.2995	-	639.2786, 579.2571, 519.2348, 495.1999, 453.1890, 393.1681, 353.1750, 311.1641 293.1539, 265.1591, 247.1485, 223.1123, 133.0858, 91.0540	+	-	-	Tyrosyl- threonyl-valinyl-methionyl-tryptophan	C₃₄H₄₆N₆O₈S (−1.6)	[72]
141	Peptides	25.72	-	675.2624	-	618.2624, 455.2040, 421.1996, 295.1699, 277.1592, 267.1749, 249.1642, 205.1903, 107.0875, 91.0543	+	-	-	Glycyl-Serinyl-tyrosyl-tryptophyl-tyrosine	C₃₄H₃₈N₆O₉ (−1.3)	[72]
142	Peptides	25.75	-	593.2754	-	397.2016, 355.1903, 295.1695, 277.1592, 267.1747, 249.1640, 207.1173, 165.0915	+	-	-	Acetyl-tryptophyl-methyl-alanyl-aspartyl-phenylalaninamide	C₃₀H₃₆N₆O₇ (−1.8)	[72]
143	Peptides	25.82	-	641.2942	-	581.2734, 521.2517, 461.2302, 313.1501, 295.1700, 277.1592, 249.1640, 173.0965, 106.0743, 91.0543	+	-	-	Serinyl-serinyl- glycyl-prolinyl- phenylalanyl-phenylalanine	C₃₁H₄₀N₆O₉ (−1.6)	[72]
144	Peptides	26.03	-	633.2679	-	576.2636, 523.2266, 437.1942, 377.1728, 267.1752, 253.1588, 239.1456, 107.0864, 91.0544	+	-	-	Nicotinoyl- alanyl- alanyl tyrosyl- glycyl- phenylalanine	C₃₂H₃₆N₆O₈ (−1.8)	[72]
145	Fatty amides	26.26	-	324.29	306.2793, 263.2363 245.2256, 147.1161, 109.1010, 95.0857, 81.0698, 62.0599	-	+++	t	+	Linoleoyl ethanolamide *	C₂₀H₃₇NO₂ (0.2)	[73]
146	Phospholipids (lysophosphatidylglycerols)	26.60	483.2718		227.0324, 152.9955	-	+	+	+	Hexadecanoyl glycerophospho- glycerol	C₂₂H₄₅O₉P (0.2)	[70]
147	Peptides	27.16	-	617.2724	-	557.2543, 497.298, 421.1988, 361.1785, 321.2406, 297.1849, 279.1747, 251.1804, 209.1325, 91.0538	+	-	-	Tryptophyl-glutamyl-prolyl-tryptophan	C₁₈H₃₇NO₂ (0.5)	[72]
148	Fatty amides	27.70	-	300.2902	283.2642, 242.2482, 109.1012, 95.0857 85.1013, 71.0855, 67.0545, 62.0598	-	+++	t	+	Palmitoyl ethanolamide *	C₁₈H₃₇NO₂ (0.5)	[75]
149	Fatty acids	28.07	-	347.2610	-	275.1620, 235.1318, 195.1004, 179.9946, 95.0865	t	t	+++	Hydroxy- docosa-pentaenoic acid *	C₂₂H₃₄O₃ (0.5)	[76]
150	Fatty acids	28.34	-	326.3796	-	308.2959, 107.0847, 95.0857, 81.0889, 62.0599	+++	t	+	N-Oleoylethanolamine *	C₂₀H₃₉NO₂ (0.2)	[77]
151	Fatty acids	28.68	-	347.2560	-	-	t	t	+++	Hydroxy- docosa-pentaenoic acid isomer *	C₂₂H₃₄O₃ (0.6)	[76]
152	Fatty esters	30.21	-	325.274	-	265.2527, 247.2421, 135.1169, 121.1013, 109.1013, 95.0856, 81.0700, 67.0540	t	+	+++	Octadecenoic acid methyl ethyl ester	C₂₀H₃₆O₃ (1.2)	[78]
153	Fatty acids	32.10	355.3217	-	337.3118, 309.3161, 297.1527	-	+++	t	t	Hydroxyl docosanoate	C₂₂H₄₄O₃ (0.2)	[79]
154	Fatty amides	33.72	-	310.3111	293.2853, 275.2741, 97.1015, 83.0857, 69.0700	-	+	+	+	Dimethyl-octadecenamide	C₂₀H₃₉NO (−2.1)	[77]

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^a; [M + FA − H]⁻, ^b; compound reported before from M. longipetala subsp. livida, *; tentatively identified compounds reported by GNPS libraries, +++; very strong, ++; strong, +, present, t; trace, -; absent, Km; kaempferol, Qn; quercetin, Is; isorhamnetin, Rh; rhamnocitrin.

2.7. Cytotoxic Activity

2.7.1. Cell Lines

Human tumor cell lines: the colon carcinoma (HCT116), cervix carcinoma (HeLa), and hepatocellular carcinoma (HepG2) cell lines were supplied by Vacsera (Giza, Egypt) and maintained at the Bioassay-Cell Culture Laboratory, National Research Centre, El-Bohouth St., Dokki, Cairo 12622, Egypt.

2.7.2. Cell Viability by MTT Assay

The samples were prepared by dissolving stock solution in DMSO to give operating concentrations of each sample range from 100 to 0.78 µg/mL, and the cells were incubated with these concentrations in triplicate (37 °C, 72 h) in a CO₂ environment. Control wells were treated with the same amount of complete growth media only. For all treatments and untreated control groups, complete growth media without cells were added as a blank to reduce the background absorbance values. Separately, each experiment were conducted three times. MTT assay was performed by removing the medium quietly and adding MTT solution (10 μL) with a last concentration (5 mg/mL) per well then incubating (37 °C, 4 h) until the purple crystals were shaped. Then, the MTT solution was discarded from every well and DMSO (100 μL) was subjected to dissolve the crystals. The 96-well plate was shaken (15 min) using a microplate shaker until totally dissolved of the crystals. For each well, the absorbance value was assayed (595 nm wavelength) using a microplate multi-well reader [63]. The cell viability (CV) percentage after treatment with M. longipetala subsp. livida extracts were considered as follows: CV (%) = (absorbance of the treated cells − absorbance of blanks)/(absorbance of control cell − absorbance of blanks) × 100. The lethal concentration of the samples caused the death of 50% of cells (LC₅₀) which was also calculated at 48 h. Doxorubicin, the anticancer drug, was used as a positive control.

3. Results and Discussion

3.1. UPLC-HRMS/MS Metabolites Profiling of the Extracts

The current study aimed to comparably chart the metabolic composition of different organs (i.e., flowers, leaves, and roots) of M. longipetala via UPLC-PDA-ESI-HRMS/MS analysis in both positive and negative ionization modes. The overlaid BPC (base peak chromatograms) of the three extracts exhibited some differences, especially at the Rt range of (10–25 min) in the positive ionization mode and (6–15 min) in the negative ionization mode (Supplementary Figure S1), suggesting that the three extracts could be of different biological relevance.

3.2. UPLC-HRMS/MS Metabolite Annotation Aided with Molecular Networking

The UPLC-HRMS/MS data were mined employing the GNPS platform, in which feature-based molecular networks (FBMNs) were generated to visually display the existing chemical space and the metabolites distribution in the different plant parts of M. longipetala.

Two FBMNs were laid out from the acquired MS/MS data for both ionization modes. The negative FBMN constituted 188 nodes grouped into 19 clusters (with a minimum of two connected nodes) and 130 singletons. The significant dereplicated sets of the negative FBMN were the secondary metabolites clusters: A (flavonoid glycosides), B (glucosinolates), C (hydroxycinnamic acid derivatives), D (hydroxybenzoic acid derivatives), and E (biflavones) (Figure 1). These metabolites are distributed in the flowers and leaves organs with their abundance in flowers, which could be responsible for the current cytotoxic assessment and guidance for further biological activities. Similarly, the positive FBMN constituted 257 nodes in 41 clusters and 104 discrete nodes, in which the classes of interest are cluster A (flavonoid glycosides and hydroxylated flavonoid aglycones) and B (methoxylated flavonoid aglycones); besides, cluster C (peptides) is presented as a primary metabolites class which characterized the roots organ and ionized in the positive ionization mode only (Figure 2). In general, nodes were portrayed as a pie chart to reflect the relative abundance of each ion in the three plant parts.

FBMN created using MS/MS data of *Matthiola longipetala* subsp. *livida* extracts (negative ionization mode): flowers (purple), leaves (green), and roots (yellow) extracts. Cluster A (flavonoid glucosides), Cluster B (glucosinolates), Cluster C (hydroxycinnamic acids), Cluster D (hydroxybenzoic acids), and Cluster E (biflavones). *; the substitution position may vary.

FBMN created using MS/MS data of *Matthiola longipetala* subsp. *livida* extracts (positive ionization mode): flowers (purple), leaves (green), and roots (yellow) extracts. Cluster A (flavonoid glycosides and hydroxylated flavonoid aglycones), Cluster B (methoxylated flavonoid aglycones), and Cluster C (peptides). *; the substitution position may vary. The red circles explained the enlarged clusters.

In total, 154 compounds were annotated belonging to different chemical classes (i.e., glucosinolates, phenolic acids, flavonoids, etc.). Almost all the annotated features are reported for the first time to exist in M. longipetala subsp. livida (Table 1). The classes and/or subclasses of compounds were preformed manually guided by the literature [2,28,33,80,81] and automatically through the ClassyFire webserver at http://classyfire.wishartlab.com/ (accessed on 27 June 2023) [82]. Following is a detailed discussion of the detected metabolites according to their chemical class.

3.2.1. Glucosinolates

Glucosinolates are one of the main bioactive metabolites of the Brassicaceae species and are thought to play a significant role in the health benefits of such species [2,28,80]. Their fragmentation behavior involves the cleavage of the sugar–sulfur bond, giving the fragment ion m/z 259 and the sulfur-aglycone showing fragment ions at m/z 195 and m/z 275. The intramolecular rearrangements of the attachment of aglycone and sulfate to the glucose moiety give the fragment ion m/z 241 after water cleavage from m/z 259 [28,80].

Eight of the nine identified glucosinolates are grouped in cluster B of the negative FBMN, occurring in the three plant parts (Figure 1). This includes isomers of glucoraphanin (7 and 14, m/z 434.0253 [M − H]⁻), together with isomers of methylthio-butenyl-glucosinolate (11, 18, 23 and 35, m/z 418.0299 [M − H]⁻), butyl glucosinolate (20, m/z 374.0582 [M − H]⁻), and glucobrassicanapin (29, m/z 386.0582 [M − H]⁻). Lastly, one glucosinolate was observed in the positive FBMN as a self-looped node and was identified as raphenin (10, m/z 176.0201 [M − H]⁺) (Table 1).

3.2.2. Phenolics

Besides the glucosinolates, members of the Brassicaceae are well recognized for their high content of phenolic metabolites, with qualitative and quantitative differences among species and varieties, within the same species, and plant parts [33]. In the present study, phenolic metabolites showed the highest accumulation in the flowers extract and the least in the roots. The major phenolic classes identified were phenolic acids and flavonoids.

Phenolic Acids and Derivatives

Detected phenolic acids included hydroxybenzoic acid and hydroxycinnamic acid (coumaric, ferulic, and sinapic acids) derivatives, which are widely distributed in numerous members of the Brassicaceae family, commonly as glycosylated descendants [2,33].

The negative FBMN delineated the abundance of glycosylated hydroxycinnamic acids in the flowers and grouped in cluster C (Figure 1), including isomers of coumaric acid-O-dihexoside (12 and 39, m/z 487.145 [M − H]⁻), isomers of ferulic acid-O-dihexoside (26 and 43, m/z 517.155 [M − H]⁻), sinapic acid-O-dihexoside (46, m/z 547.167 [M − H]⁻), and later sinapic acid-O-hexoside (57, m/z 385.1141 [M − H]⁻). Caffeic acid (93, m/z 179.0354 [M − H]⁻) was also observed in the negative FBMN, but as a self-looped node and also accumulated in the flowers organ.

Similarly, glycosylated hydroxybenzoic acids were distributed in the three organs, and were observed in the negative FBMN (Figure 1). Hydroxybenzoic acid-O-hexoside (19; m/z 299.0770 [M − H]⁻), and vanillic acid-O-hexoside (32; m/z 329.0853 [M − H]⁻) were viewed as a cluster of two connected nodes (D). Other glycosides were observed as self-looped nodes and identified as dihydroxybenzoic acid-O-hexoside (25; m/z 315.0716 [M − H]⁻) and dihydroxybenzoic acid-O-pentoside (37; m/z 285.0614). Lastly was dihydroxybenzoic acid (75; m/z 153.0192 [M − H]⁻) which existed only in the roots extract. Additionally, one sulfo-hydroxybenzoic acid (36; m/z 246.9917 [M − H]⁻) was noted as a self-looped node in negative FBMN, showing the characteristic loss of a sulfate group (−80 Da) and was assigned as vanillic acid-sulfate.

Other phenolic derivatives were also observed as self-looped nodes either in the positive or negative FBMN. They also distributed in the three extracts with richness in the flowers and tentatively identified as mono-hydroxy benzaldehyde isomers (54 and 61; m/z 137.0601 [M + H]⁺), trimethoxy benzaldehyde (69; m/z 197.0813 [M + H]⁺), trimethoxy benzaldehyde-O-hexoside (67; m/z 357.1559 [M − H]⁻), sinapaldehyde (84; m/z 207.0665 [M − H]⁻), coniferin (99; m/z 341.1242 [M − H]⁻), and syringin (101; m/z 371.1350 [M − H]⁻).

Flavonoids

Flavonoids protect plants from various biotic and abiotic stresses by acting as natural antioxidants, unique UV filters, signal molecules, allelopathic compounds, and antimicrobial defensive compounds [81]. Additionally, their impressive biological effects have made them excellent candidates as nutraceutical supplements for human intake, disease prevention, and health promotion [2,81].

Throughout the current analysis, around 40% of the detected constituents are flavonoids (64 metabolites) (Table 1) delivered as cluster A and some as self-looped nodes in FBMN of the negative ionization mode (Figure 1) and clusters A and B in the positive one (Figure 2), being more abundant in the flowers.

Flavonoid-O-glycosides

The flavonoid-O-glycosides (56 compounds) were represented in cluster A in both FBMNs (Figure 1 and Figure 2), mainly as flavonol-O-glycosides, which have been previously reported in various species of Brassicaceae [2,3,4,28,31,39,45,46,48,65,83]. They were mostly distributed among the three investigated plant organs, with more abundance in the flowers. Only one flavone-O-glycoside was detected exclusively in the roots and was assigned as apigenin 7-O-glucoside (92, m/z 431.0981 [M − H]⁻) based on the main fragment ion at m/z 269 which corresponds to apigenin aglycone and the loss of a glucose moiety [M − H–162]⁻.

Flavonol-O-glycosides

The predominant annotated flavonol glycosides were mainly glycosides of kaempferol, isorhamnetin, and quercetin with little presence of rhamnocitrin, based on our former studies through acid hydrolysis and NMR data [8,46]. The quercetin glycosides in both FBMN were directly linked to their kaempferol correspondences by a difference of 16 Da (–O–), and with the isorhamnetin correspondences by a difference of 14 Da (–CH₂). The direct attachment of the isorhamnetin glycosides to those of the kaempferol correspondents with a mass difference of 30 Da suggests possible OCH₃ expansion.

The flavonol-O-glycosides showed the typical fragmentation patterns corresponding to the respective sugar moiety, such as deoxyhexose (−146 Da), hexose (−162 Da), and pentose (−132 Da). Mostly, the sugar moieties were tentatively assigned as rhamnose, glucose, and arabinose based on previous studies with acid hydrolysis and NMR data of the investigated species [8,46] and several members of the family Brassicaceae [3,28,46,65,83]. Some glycosides were acylated by acetic acid (−42 Da) and/or malonic acid (−86 Da).

Twelve flavonol mono-glycosides were observed and reported previously in some crucifers [46,83]. The 3-O-glucoside of quercetin (81, m/z 463.0878 [M − H]⁻), kaempferol (88, m/z 447.0931 [M − H]⁻), isorhamnetin (90, m/z 477.1035 [M − H]⁻), and rhamnocitrin (111, m/z 461.1085 [M − H]⁻), the 3-O-rhamnoside of kaempferol (102, m/z 431.0980 [M − H]⁻), as well as the 3-O-arabinoside of quercetin (89, m/z 433.0755 [M − H]⁻), which were tentatively identified according to Ablajan et al. [49], and Qin et al. [47]. In this case (3-O-glycosides), the intensity of the anion radical fragment [Agl–H–H]⁻ is higher than the anion one [Agl–H]⁻ and vis versus for 65 (kaempferol 7-O-rhamnoside) and 74 (isorhamnetin 7-O-rhamnoside).

Additionally, 12 flavonol di-O-glycoside structures were annotated and were grouped within the same cluster (Figure 1, cluster A). Compounds (40, 60, and 73) were assigned as kaempferol di-O-glycosides, showing the same molecular ion peak at m/z 609 [M − H]⁻ and common MS fragments at m/z 447 and 285. The MS fragmentation pattern of compound 40 is typical for kaempferol 3-O-sophroside. This compound revealed the deprotonated base peak at m/z 285 [M − H–324]⁻, a fragment ion at 429 [M − H–180]⁻, and a fragment ion at 447 [M − H–162]⁻, suggesting a sophoroside at the 3-O position [39]. Conversely, the absence of the fragment ion [M – H–180]⁻ in compound 73, indicates a kaempferol 3-O-gentobioside structure [39,49]. In contrast, the appearance of the fragment ion m/z 447 as the base peak confirmed the identification of compound 60 as kaempferol-3,7-di-O-glucoside [49]. Additionally, two O-rutinoside isomers of kaempferol (48, m/z 593.1511 [M − H]⁻, 82, m/z 593.1580 [M − H]⁻, 595.1665 [M + H]⁺) were confirmed. Sophoroside and rutinoside substitution have been observed in several cruciferous species as predominant disaccharide moieties [39].

Flavonol di-O-glycosides with sugar units at different hydroxyl positions of the aglycone nucleus provide two flavonol monoglycoside fragment ions with different intensities, where the higher fragment represents the 3-O-substitution while the lower one indicates the occupation of position 7 [39,47,49]. Consequently, compounds 62, 72, and 76 could be identified as 3-O-rhamnoside 7-O-arabinoside of quercetin (m/z 579.1349 [M − H]⁻), kaempferol (m/z 563.1398 [M − H]⁻), and isorhamnetin (m/z 593.1509 [M − H]⁻), respectively. Likewise, the MS fragmentation of compounds 59 and 71 is typical for the 3-O-rhamnoside 7-O-glucoside of quercetin (m/z 609.1464 [M − H]⁻) and isorhamnetin (m/z 623.1633 [M − H]⁻), respectively.

Furthermore, different triglycosides of kaempferol (27, 42, 49, 50, 55, 64, 66, and 68), quercetin (51 and 58), and isorhamnetin (53) were also grouped in cluster A of both FBMNs (Figure 1 and Figure 2). The MS fragmentation of compound 27 (m/z 773.2147 [M + H]⁺) was characteristic of kaempferol-3-O-sophroside-7-O-glucoside [39]. Four kaempferol triglycosides isomers (50, 55, 64, and 66) showed a common molecular formula (C₃₂H₃₈O₁₈), molecular ion peaks (m/z 709 [M − H]^−- and 711 [M + H]⁺), and MS fragments at m/z (431 [M − H–296]⁻ and 433 [M + H–296]⁺), after the neural loss of a disaccharide residue (rhamnosyl arabinoside) and (285 [Agl − H]⁻, 287 [Agl + H]⁺). These compounds were directly connected with compound 72 (m/z 563.1398) in the negative MN with a mass difference 146 Da (rhamnosyl) (Figure 1), therefore, they could be annotated as kaempferol O-rhamnosyl arabinoside-O-rhamnoside isomers, one of them is recommended to be kaempferol 3-O-(2″-α-rhamnopyranosyl)-β-arabinopyranoside-7-O-α-rhamnopyranoside which was isolated before from the investigated plant by Marzouk et al. [8]. Likewise, compound (58, m/z 725.1937 [M − H]⁻ and 727.2094 [M + H]⁺) was linked with compound 62 (m/z 579. 1349 [M − H]⁻ and 581.1510 [M + H]⁺) in both FBMNs and could be identified as quercetin O-rhamnosyl arabinoside-O-rhamnoside. Also, the O-rhamnosyl arabinoside-O-glucoside derivatives of kaempferol (49, m/z 727.2088 [M + H]⁺), quercetin (51, m/z 741.1881 [M − H]⁻ and 743.2038 [M + H]⁺), and isorhamnetin (53, m/z 757.2199 [M + H]⁺) were identified. Based on previous studies, three kaempferol triglycosides 42 (m/z 757.2199 [M + H]⁺), 52 and 68 (m/z 739.2087 [M − H]⁻, 741.2243 [M + H]⁺) were confirmed as kaempferol-O-glucoside-O-rutinoside, kaempferol-O-rhamnoside-O-rutinoside, and kaempferol-O-rhamnosyl rutinoside, respectively [46,51].

Lastly, the highest glycosylation pattern was found in two tetra glycosides of kaempferol (33 and 47) which are concentrated in the flowers extract. Compound (33) appeared at m/z 917.2648 [M + FA − H]⁻, while 47 appeared at m/z 871.2510 [M − H]⁻, they have the same molecular formula (C₃₈H₄₈O₂₃) and MS fragments (m/z 709 [M − H − 162 (glucose)]−, 563 [Agl − H + 278 (arabinosyl rhamnoside)]⁻, 447 [Agl − H + 162 (glucoside)]⁻, 431 [Agl − H + 146 (rhamnoside)]⁻, 285 [Agl − H]⁻). Thus, they tentatively identified as kaempferol-O-arabinosyl-rhamnoside-O-rhamnoside-O-glucoside isomers, one of them could be identified as kaempferol 3-O-(2″-rhamnopyranosyl)-arabinopyranoside-7-O-rhamnopyranoside-4′-O-glucopyranoside which was isolated before from the current species [8].

Acylated flavonol-O-glycosides

A total of 16 acylated flavonol mono-glycosides were also observed in group A of the positive and negative FBMNs and connected with their O-glucoside analogs with MS differences of either 42 Da (acetyl) and/or 86 Da (malonyl). Whereas the acetylated and malonylated counterparts were correlated with each other with a 44 Da (CO₂) difference (Figure 1 and Figure 2). The 3-O-malonyl glucoside of quercetin (85, m/z 549.0881 [M − H]⁻), kaempferol (95, m/z 533.0936 [M − H]⁻), isorhamnetin (96, m/z 565.1198 [M + H]⁺), and rhamnocitrin (113, m/z 549.1246 [M + H]⁺) were characterized by the neutral loss −86 Da (malonyl), then −162 Da (glucoside). Other mono-acylated flavonol glycosides were annotated as kaempferol 3-O-acetyl glucoside (105, m/z 489.1035 [M − H]⁻, m/z 491.1192 [M + H]⁺), two isomers of quercetin 3-O-acetyl glucoside (86 and 94, m/z 505.0979 [M − H]⁻, m/z 507.1142 [M + H]⁺), and three isomers of isorhamnetin 3-O-acetyl glucoside (97, 104, and 107, m/z 519.1143 [M − H]⁻, m/z 521.129 [M + H]⁺) (Supplementary Figure S2). Acylated monoglycoside derivatives of quercetin, kaempferol, and isorhamnetin have already been found in some cruciferous species [3,39], while reported for the first time from the genus Matthiola.

Similarly, the diacylated flavonol glycosides were represented as 3-O-diacetyl glucoside of kaempferol (109, m/z 531.1138 [M − H]⁻), and isorhamnetin (110 and 115, m/z 561.12 [M − H]⁻) (Supplementary Figure S2), connected with their 3-O-glucoside analogs with MS difference of 84 Da (2 acetyl residues) in negative FBMN (Figure 1). Four additional diacylated flavonol glycosides were 3-O-acetyl malonyl glucoside of quercetin (103, m/z 591.0995 [M − H]⁻, m/z 593.1148 [M + H]⁺), kaempferol (108 and 112, m/z 575.1044 [M − H]⁻, m/z 577.1201 [M + H]⁺), and isorhamnetin (114, m/z 607.1295 [M + H]⁺). They were linked with their 3-O-acetyl glucoside or 3-O-malonyl glucoside derivatives with a difference of 86 Da (malonyl) or 42 Da (acetyl), respectively, in either the negative or positive FBMNs (Figure 1 and Figure 2). For instance, compound 103 showed a deprotonated molecular ion peak at m/z 591.0995 [M − H]⁻ and produced fragment ions at m/z 547 [M − H − 44]⁻ after the neutral loss of CO₂ then m/z 505 [M − H − 86]⁻, for malonyl elimination then m/z 301 [M − H − 86 − 42]⁻ and m/z 300 [M − H − H − 86 − 42]⁻, after the loss of the acetyl group (Supplementary Figure S3). Therefore, compound 103 could be identified as quercetin 3-O-X₁ acetyl X₂ malonyl glucoside. Similarly, compounds (108 and 112) were identified as kaempferol 3-O-X₁ acetyl X₂ malonyl glucoside and 114 as isorhamnetin 3-O-X₁ acetyl X₂ malonyl glucoside (Supplementary Figures S4 and S5). These four structural proposals were not found before in nature.

Flavonoid aglycones

Five polymethoxylated flavone-type aglycones were mainly observed in the positive ionization mode and represented as a cluster (B) of the FBMN (Figure 2). On the bases of GNPS libraries, they could be annotated as tangeretin (118), sinensetin (119), and 3,5,7,3′,4′pentatamethylflavone (124), all at m/z 373 [M + H]⁺, irigenin trimethyl ether (122) at m/z 403.1393 [M + H]⁺, and 3,5,6,7,3′,4′,5′heptamethylflavone (123). The polymethoxylated flavone aglycones were reported before from some species of the family Brassicaceae [27,33]. Likewise, one flavonol-type aglycone was linked in cluster (A) of the positive FBMN (Figure 2) and annotated as kaempferol (87, m/z 287.0505 [M + H]⁺) that was a predominant structure for all family members [39,46,47,83].

Additionally, two biflavone-structure were detected as a cluster (E) in a negative FBMN and elucidated as two isomers of methylamentoflavone (120 and 121, at m/z 551.09 [M − H]⁻), confirmed by their fragmentation pattern and GNPS library (Figure 1). Rare biflavone derivatives were reported before for some species of Brassicaceae [48].

3.2.3. Iridoids and Diterpenes

Only one iridoid compound was found for the first time in the investigated species and concentrated in the flower parts. The iridoid is identified as loganic acid (30) and has a molecular ion peak m/z 375.1297 [M − H]⁻ and fragment ions at m/z 213 [M − H − 162]⁻. Similarly, one diterpene structure was identified as miltirone (127, m/z 283.1698 [M − H]⁺) and produced fragment ions at m/z 265 ([M + H − H₂O]⁺) and m/z 223 ([M + H − H₂O − C₃H₆]⁺). Both compounds showed a wide range of activities including anti-cancer, anti-inflammatory, and antioxidant effects [36,71].

3.2.4. Coumarin

Coumarins are another vital class of secondary metabolites and were mainly observed in the positive ionization mode (Table 1). Hydroxy coumarin (38, m/z 163.0605 [M + H]⁺) revealed ions at m/z 147 [M + H–O]⁺, and 119 [M + H–CO₂]⁺. Two isomers of dimethoxycoumarin (45, 56, m/z 207.065 [M + H]⁺) exhibited two characteristic fragments at m/z 193 [M + H−CH₂]⁺, and 179 [M + H−2CH₂]⁺, after loss of 14 Da. Additionally, compound 79 (m/z 455.1164 [M + H]⁺) was directly connected to 45 and 56, with a mass difference (248 Da). It produced fragment ions at 411, 369, and 207 after the loss of 42, 44, and then 162 Da, respectively, suggesting the presence of -O-malonyl glucoside dimethoxy coumarin.

3.2.5. Amino Acids, Organic Acids, and Derivatives

The annotation of the amino acids was derived from the abundant fragments of the protonated ions and their corresponding derivatives arising from either losing H₂O (−18 Da) yielding their residue mass or the loss of (H₂O + CO) (−46 Da) producing their immonium ions [84] leading to the detection of five amino acids including arginine (1), proline (2), leucine/isoleucine (17), phenylalanine (22), tryptophan (34), and three amino acids derivatives; methyl proline (5), dimethyl proline (6) and tryptophan N-glucoside (31), mainly distributed among the three plant organs. Similarly, five organic acids were detected as self-looped nodes either in the negative or positive FBMNs and identified as hydroxyl glutaric acid (8), malic acid (13), citraconic acid (methyl maleic acid) (15), dimethyl malate (38), and cinnamic acid (21) (Table 1).

3.2.6. Fatty Acids and Derivatives

Eight fatty acids and one fatty acid ester were detected, in the case of compound (116), the fragmentation patterns were matched with 9,12,13- trihydroxy-octadecadienoic acid, the molecular ion at m/z 327.2178 [M − H]⁻ and the fragments at m/z 229 and m/z 171 pointed to the positions of hydroxyl groups of fatty acids (that is, at 12 and/or 13, 9 and/or 10th carbon) but it was not easy to assign the functional groups and double bonds depending on our data. Therefore, this compound was identified as trihydroxy-octadecadienoic acid. Similarly, trihydroxy-octadecanoic acid (117) has a molecular ion at m/z 329.2328 [M − H]⁻ and the base peak at m/z 211.1342, other fragments were detected at m/z 311, 229,171 which confirmed the skeleton of trihydroxy-octadecanoic acid. Other fatty acids were detected in the negative ionization mode as lichesterylic acid (methyl-oxo-heptadecanoic acid) (134) at m/z 297.243 [M − H]⁻, 10-hydroxyoctadeca-12,15-dienoic acid (138) at m/z 295.2282 [M − H]⁻, and hydroxyl docosanoate (153) at m/z 355.3217 [M − H]⁻. While, MS/MS fragmentation of compound (150) gave molecular ion at m/z 326.3796 [M − H]⁺ and characteristic fragment ions m/z 62.05 ([ethanolamine + H]⁺); m/z 308.2 ([M-H₂O + H]⁺) and identified as N-oleoylethanolamine. In addition, two isomers of (17s)-hydroxy-docosa-pentaenoic acid were tentatively identified in all the examined M. longipetala plant parts (149; m/z 347.261 and 151; m/z 347.256 [M + H]⁺). Moreover, one fatty acid ester was assigned (152 at m/z 325.274 [M + H]⁺) as octadecenoic acid methyl ethyl ester.

3.2.7. Lipids

Five phospholipids were detected in M. longipetala extracts; three glycerophosphoinositol lipids were identified mainly in the leaves (negative ion mode) and identified as octadecatrienoyl-glycerophosphoinositol (125, m/z 593.2724 [M − H]⁻), linoleoyl-glycerophosphoinositol (126, m/z 595.2885 [M − H]⁻), and palmitoyl-glycerophosphoinositol (129, m/z 571.2884 [M − H]⁻). In addition, two glycerophosphoglycerol lipids were identified in the root extract as hexadecenoyl-glycerophosphoglycerol at m/z 481.2568 [M − H]⁻ (139) and hexadecanoyl-glycerophosphoglycerol at m/z 483.2718 [M − H]⁻ (146).

Linoleoyl ethanolamide isomers (133 and 145; 322.2751 [M − H]⁻, 324.2901 [M + H]⁺) and palmitoyl ethanolamide (148; 300.2902 [M + H]⁺) are fatty amides that belong to the class of organic compounds known as N-acylethanolamines, in addition to dimethyl octadecenamide (154; 300.2902 [M + H]⁺). Lastly, one sulfoglycolipids was identified as hexadecanoyl glycerol-O-sulfo-rhamnoside at m/z 555.2844 [M − H]⁻ (136).

3.2.8. Peptides

Eleven polypeptides were detected in the positive ionization mode (cluster D), thoroughly characterized for the root organ (Table 1, Figure 2). They were tentatively identified according to MS differences and fragmentation patterns, then further sequenced corresponding to [72].

3.3. Cytotoxicity

As expected, flower extract that showed the highest abundance of secondary metabolites revealed a significant cell viability inhibition of HCT-116 and HeLa cell lines growth, with LC₅₀ values (24.8 ± 0.45 and 18.1 ± 0.42 µg/mL), compared to those of Doxorubicin (37.6 ± 0.21 and 26.1 ± 0.27 µg/mL), respectively. Similarly, the leaf extract inhibited the propagation of the HeLa cell line with an LC₅₀ value of 29.6 ± 0.35 µg/mL. The three methanolic extracts did not show any cytotoxic effect on the HepG2 cell line (Supplementary Table S1). These findings summarize the relationships between the cytotoxic assessment of the three examined organs and the concentration of secondary metabolites, particularly flavonoids. Consequently, the present data indicates that the flower organ is responsible for activities reported before for aerial parts on the same species [8].

4. Conclusions

The current study provided a holistic overview of the constitutive metabolome of M. longipetala, an under-explored member of Brassicaceae. UPLC-HRMS/MS coupled to FBMN, and in silico fragmentation trees allowed for the annotation of 154 metabolites, belonging to phenolic acids, glucosinolates, flavonoids, lipids, peptides, and others. Furthermore, four previously unknown compounds were tentatively assigned as O-acetyl O-malonyl glucosides of quercetin (103), kaempferol (108 and 112), and isorhamnetin (114) based on their fragmentation pattern and their connectivity to their known analogs. Yet their full structure elucidation requires other spectroscopic techniques (i.e., NMR) after their isolation. Lastly, cytotoxicity assessment of the plant parts revealed that the flowers are effective against HeLa and HCT-116 cell lines suggesting that they are a potential resource of bioactive cytotoxic compounds. Future in vivo research should focus on the chemical modification and targeted delivery of these promising bioactive molecules to maximize their anticancer potential.

Acknowledgments

The authors thank Hamada Saad, of the Department of Pharmaceutical Biology, the Pharmaceutical Institute, the Eberhard Karls University of Tübingen, Germany, for the UPLC-LC-HRMS/MS measurements.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/metabo13080909/s1, Figure S1: The base peak chromatograms of Matthiola longipetala subsp. livida extracts: flowers (purple), leaves (green), and roots (yellow) in the negative ionization mode (A) and the positive ionization mode (B); Figure S2: Proposed fragmentation scheme and MS2 spectra (negative ionization mode) of A (isorhamnetin 3-O-glucoside, 90), B (isorhamnetin 3-O-acetyl glucoside 96 and 104), and C (isorhamnetin 3-O-diacetyl glucoside, 110 and 115); Figure S3: Proposed fragmentation scheme and MS² spectrum (negative ionization mode) of quercetin 3-O-X₁ acetyl -X₂ malonyl glucoside, 103; Figure S4: Proposed fragmentation scheme and MS² spectrum (negative ionization mode) of kaempferol 3-O-X₁ acetyl -X₂ malonyl glucoside, 108; Figure S5: Proposed fragmentation scheme and MS² spectrum (positive ionization mode) of isorhamnetin 3-O-X₁ acetyl -X₂ malonyl glucoside, 114. Table S1: LC₅₀ values (µg/mL) of the cell viability inhibition of Matthiola longipetala subsp. livida extracts on different cell lines.

Click here for additional data file.^{(617.3KB, zip)}

Author Contributions

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

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The designed MNs and parameters can be retrieved via the following links: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=a88f28d3d0514197b8a14b54e60e9a13 accessed on 28 December 2019 for the positive network and https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c8bb90ad81564de9b0d8032c016cc140 accessed on 28 December 2019 for the negative network.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

The chemical extraction and the cytotoxic approach were funded by the National Research Centre, Cairo, Egypt (grant number: M120201).

Footnotes

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PERMALINK

Discriminative Metabolomics Analysis and Cytotoxic Evaluation of Flowers, Leaves, and Roots Extracts of Matthiola longipetala subsp. livida

Mona M Marzouk

Nesrine M Hegazi

Mona O A El Shabrawy

Mai M Farid

Salwa A Kawashty

Sameh R Hussein

Nabiel A M Saleh

Roles

Abstract

1. Introduction

2. Materials and Methods

2.1. Chemicals and Reagents

2.2. Plant Material and Preparation of the Extracts

2.3. Sample Preparation for UPLC-HRMS-MS Measurement

2.4. UPLC-HRMS-MS Analysis

2.5. Data Analysis and Preprocessing

2.6. Feature-Based Molecular Networking (FBMN) and Metabolites Dereplication

Table 1.

2.7. Cytotoxic Activity

2.7.1. Cell Lines

2.7.2. Cell Viability by MTT Assay

3. Results and Discussion

3.1. UPLC-HRMS/MS Metabolites Profiling of the Extracts

3.2. UPLC-HRMS/MS Metabolite Annotation Aided with Molecular Networking

Figure 1.

Figure 2.

3.2.1. Glucosinolates

3.2.2. Phenolics

Phenolic Acids and Derivatives

Flavonoids

3.2.3. Iridoids and Diterpenes

3.2.4. Coumarin

3.2.5. Amino Acids, Organic Acids, and Derivatives

3.2.6. Fatty Acids and Derivatives

3.2.7. Lipids

3.2.8. Peptides

3.3. Cytotoxicity

4. Conclusions

Acknowledgments

Supplementary Materials

Author Contributions

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Conflicts of Interest

Funding Statement

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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