How Does Lagenaria siceraria (Bottle Gourd) Metabolome Compare to Cucumis sativus (Cucumber) F. Cucurbitaceae? A Multiplex Approach of HR-UPLC/MS/MS and GC/MS Using Molecular Networking and Chemometrics

Abstract

Cucurbitaceae comprises 800 species, the majority of which are known for their nutritive, economic, and health-promoting effects. This study aims at the metabolome profiling of cucumber (Cucumis sativus) and bottle gourd (Lagenaria siceraria) fruits in a comparative manner for the first time, considering that both species are reported to exhibit several in-common phytochemical classes and bioactivities. Nevertheless, bottle gourd is far less known and/or consumed than cucumber, which is famous worldwide. A multiplex approach, including HR-UPLC/MS/MS, GNPS networking, SPME, and GC/MS, was employed to profile primary and secondary metabolites in both species that could mediate for new health and nutritive aspects, in addition to their aroma profiling, which affects the consumers’ preferences. Spectroscopic datasets were analyzed using multivariate data analyses (PCA and OPLS) for assigning biomarkers that distinguish each fruit. Herein, 107 metabolites were annotated in cucumber and bottle gourd fruits via HR-UPLC/MS/MS analysis in both modes, aided by GNPS networking. Metabolites belong to amino acids, organic acids, cinnamates, alkaloids, flavonoids, pterocarpans, alkyl glycosides, sesquiterpenes, saponins, lignans, fatty acids/amides, and lysophospholipids, including several first-time reported metabolites and classes in Cucurbitaceae. Aroma profiling detected 93 volatiles presented at comparable levels in both species, from which it can be inferred that bottle gourds possess a consumer-pleasant aroma, although data analyses detected further enrichment of bottle gourd with ketones and esters versus aldehydes in cucumber. GC/MS analysis of silylated compounds detected 49 peaks in both species, including alcohols, amino acids, fatty acids/esters, nitrogenous compounds, organic acids, phenolic acids, steroids, and sugars, from which data analyses recognized that the bottle gourd was further enriched with fatty acids in contrast to higher sugar levels in cucumber. This study provides new possible attributes for both species in nutrition and health-care fields based on the newly detected metabolites, and further highlights the potential of the less famous fruit “bottle gourd”, recommending its propagation.

1. Introduction

Family Cucurbitaceae comprises 130 genera and 800 species, including several crucial crops of high nutritive and medicinal values and increasing economic interest [1,2]. Roots and seeds of most Cucurbitaceae members share the presence of toxic triterpenes, cucurbitacins. In contrast, their fruits are safe, edible, nutritious, and possess health-promoting effects [1,3]. Cucumis sativus (cucumber) is a well-known species of Cucurbitaceae that is cultivated worldwide, and its fruit is edible either fresh or cooked [1,4]; in contrast, the less famous Lagenaria siceraria species (bottle gourd) is commonly used on the Indo-Pakistan subcontinent and few other countries in Europe and Africa for its nutritive and therapeutic values [5]. Aside from the macromorphological resemblance, both species are reported to exhibit anti-inflammatory, antioxidant, antimicrobial, anticancer, antihyperlipidemic, and cardioprotective activities, and are used in treatment of GIT disorders [1,4]; additionally, fruits’ extracts are involved in the treatment of skin disorders [3,4,6]. Previous phytochemical screening of L. siceraria and C. sativus indicated the presence of flavonoids, sterols, terpenes, phenolic acids, saponins, and carbohydrates in both fruits, whereas alkaloids were detected in C. sativus [3,4,7].

Despite that both bottle gourd and cucumber fruits share several common nutritional and medicinal purposes as well as their involvement in Ayurvedic and folk medicines [4,8], few studies have reported on their metabolome heterogeneity and how the bottle gourd compares to the most famous fruit in F. Cucurbitaceae, the cucumber, as analyzed using metabolomics and chemometrics. In this study, the first comparative metabolome profiling of L. siceraria and C. sativus fruit crude extracts is presented as measured via high resolution-ultrahigh performance liquid chromatography coupled to mass spectrometry (HR-UPLC/MS/MS) in both ionization modes for a comprehensive overview of metabolites. HR-UPLC/MS/MS is the analytical tool of choice for rapid and sensitive monitoring of secondary metabolites due to its robustness and selectivity [9]. Identification of metabolites was aided by Global Natural Products Social molecular networking (GNPS), that allowed rapid dereplication of compounds and extrapolating tentative identification into the unknown [9,10].

Solid phase microextraction (SPME) of volatile constituents alongside silylation of nonvolatile compounds followed by GC/MS analyses provided insight of their aroma and nutrients profile, respectively, which affect food value and consumer preferences, likewise for the first time [11]. Owing to the complexity of the obtained datasets via GC/MS analyses, multivariate data analyses using principal component analysis (PCA) and orthogonal projection to latent structures-discriminant supervised data analysis (OPLS) were applied for the first time for samples classification and assigning discriminating metabolites for each fruit.

The study provides new insights into the chemical composition of cucumber and the less investigated species, bottle gourd, in a comparative approach leading to the identification of several first-time-reported metabolites and classes in both species and a better rationalization of their health effects and potential application as nutraceuticals in the future, based on such chemical profiling.

2. Materials and Methods

2.1. Plant Material

The fresh fruits of cucumber (Cucumis sativus) and bottle gourd (Lagenaria siceraria var. siceraria) were obtained from Barrage Experimental Farm of Horticulture Research Station, El-Kanater El-Khyreia, Qalubia Governorate, Egypt. They were identified by Prof. Dr. Mahmoud Kotb Hatem; Breeding Research Department for Vegetable Crops, Medicinal, and Aromatic Plants, Horticulture Research Institute, Agricultural Research Center.

2.2. Chemicals and Fibers

SPME fiber of stableflex coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 µm) was purchased by Supelco (Oakville, ON, Canada). All chemicals and standards were purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile and formic acid (LC–MS grade) were obtained from J. T. Baker (The Netherlands), and milliQ water was used for UPLC/PDA/ESI–qTOF-MS analysis.

2.3. Extracts Preparation for UPLC-MS Analysis

Freeze-dried samples of C. sativus and L. siceraria fruits (n = 3 each) were ground in liquid nitrogen (Sigma-Aldrich, St. Louis, MO, USA, purity ≥ 99.998%) using pestle and mortar. The extraction procedure was conducted as previously described in Hegazi, N.M. et al., [9]. About 150 of the powdered samples was mixed with 6 mL methanol containing 1 g/mL umbelliferone as an internal standard (Sigma-Aldrich, St. Louis, MO, USA, purity ≥ 98.0%) and homogenized with an Ultra-Turrax (IKA, Staufen, Germany) at 11,000 rpm, 5 × 60 s with 1 min break intervals. Extracts were then vortexed for 1 min, centrifuged at 3000× g for 30 min, and filtered through a 22 m pore size filter.

2.4. UPLC-MS Analysis

Chromatographic separation was performed on an ACQUITY UPLC system (Waters, Milford, MA, USA) equipped with a HSS T3 column (100 × 1.0 mm, particle size 1.8 µm; Waters). The analysis was carried out under exact conditions described in Hegazi, N.M. et.al., [9]. Characterization of compounds was performed by generation of the elemental formula (mass accuracy < 5 ppm) and considering RT, MS2 data and reference literature.

2.5. Molecular Networking

The molecular network (MN) was constructed using the UPLC-HRMS/MS data (in the negative and positive ion mode) from both fruit extracts as prepared in Section 2.3 following exact conditions mentioned in Hegazi, N.M. et al. [9]. The MN parameters were as follows: minimum cosine score 0.70; 0.1 Da parent mass tolerance, 0.5 Da as fragment ion tolerance to create consensus spectra, more than 6 matched peaks, and a minimum cluster size of 2. All of the matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 4 matched peaks. Cytoscape (ver. 3.8.2.) was used for network visualization and analysis.

2.6. Headspace Volatiles Analysis of C. sativus and L. siceraria

The sample was prepared and analyzed following the same procedure and conditions reported in Farag et al. [12]. GC–MS analysis was adopted on an Agilent 5977B GC/MSD (Santa Clara, CA, USA) equipped with a DB-5 column (30 m × 0.25 mm i.d. × 0.25 µm film thickness; Supelco, Bellefonte, PA, USA) and coupled to a quadrupole mass spectrometer following the exact conditions mentioned in Farag, M.A. et al., [12]. For assessment of replicates, three different samples for each fruit were analyzed under the same conditions. Blank runs were conducted during sample analyses. The mass spectrometer was adjusted to EI mode at 70 eV with a scan range set at m/z 40–500.

2.7. GC–MS Analysis of Silylated Primary Metabolites in C. sativus and L. siceraria Fruits

100 Mg of finely freeze-dried powdered sample (for both fruits) was extracted with 5 mL 100% methanol with sonication for 30 min using Branson CPX-952-518R set at 36 °C, (Branson Ultrasonics, Carouge, SA Switzerland.) and with regular shaking, followed by centrifugation (LC-04C 80-2C regen lab prp centrifuge, Zhejiang, China) at 12,000× g for 10 min to eliminate debris. For evaluation of biological replicates, 3 independent samples for each fruit was analyzed under the same conditions. Then, 100 µL of the methanol extract was kept in opened screw-cap vials and left to evaporate under stream of nitrogen gas until full dryness. For derivatization, 150 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA), previously diluted 1/1 with anhydrous pyridine, was mixed with the dried methanol extract and incubated (Yamato Scientific DGS400 Oven, QTE TECHNOLOGIES, Hanoi, Vietnam) for 45 min at 60 °C previous analysis using GC–MS. Separation of silylated derivatives was completed on a Rtx-5MS Restek, Bellefonte, PA, USA (30-m length, 0.25-mm inner diameter and 0.25-m film). Analysis of these primary metabolites followed the exact protocol detailed in Sedeek, M.S. et al., and Farag, M.A. et al. [11,12].

2.8. Metabolites Identification and Multivariate Data Analyses of Volatile and Non-Volatile Silylated Components Analyzed Using GC/MS

Identification was performed by comparing their retention indices (RI) in relation to n-alkanes (C8-C30), mass matching to NIST, WILEY library database and with standards if available. Peaks were first deconvoluted using AMDIS software (www.amdis.net, accessed on 23 April 2022) before mass spectral matching. Peak abundance data were exported for multivariate data analysis by extraction using MET-IDEA software (Broeckling, Reddy, Duran, Zhao, and Sumner, 2006). Data were then normalized to the amount of spiked internal standard (Z)-3-hexenyl acetate, pareto scaled and then subjected to principal component analysis (PCA), hierarchical clustering analysis (HCA) and partial least squares discriminant analysis (OPLS-DA) using SIMCA-P version 13.0 software package (Umetrics, Umeå, Sweden). All variables were mean-centered and scaled to Pareto variance.

3. Results and Discussion

3.1. Metabolome Profiling of L. siceraria and C. sativus Fruit Extracts via HR-UPLC/PDA/ESI-MS Based Molecular Networking

The comparative profiling of metabolites in bottle gourd and cucumber crude fruit methanol extracts was conducted via HR-UPLC/MS/MS in both negative and positive ionization modes (Section 2.4) for a comprehensive overview of metabolites that belong to different phytochemical classes. Gradient elution system of formic acid in water (0.1%): acetonitrile allowed for metabolites’ elution in order of their decreasing polarity. The obtained base peak chromatograms (BPCs) of each fruit extract in both ionization modes are presented in Figure 1.

Furthermore, molecular networking was applied herein for the in-depth exploration and discrimination of samples guided by the Global Natural Products Social (GNPS) networking software and its spectral library that aided in peaks’ assignment and annotation based on analyzing the HR-tandem MS/MS data [9,10]. Two molecular networks (MN) were constructed from both ionization mode analyses that provided visual discrimination of samples based on metabolites’ abundance in each node, presented as a pie chart, allowing for rapid dereplication of known compounds (Figure 2 and Figure S1). Samples were coded with orange and green colors for bottle gourd and cucumber, respectively. All nodes were labelled with parent mass and edges were labelled with neutral loss values.

Identification was based on determining retention time (Rt. min.) for each metabolite, its mass spectral data—including molecular ion, daughter ions, their respective formulae and fragmentation pattern—and comparing the collective data with the reported literature and databases such as HMDB, PubChem, FooDB, the Phytochemical dictionary of natural products, and others, combined with GNPS library annotations. The clustering of metabolites in MN (minimum two connected nodes) was based on shared fragments and their fragmentation pattern; this allowed us to extrapolate the identification of annotated compounds to the unknown peaks aided by the generated formulae [10].

Overall, 107 peaks were annotated in both modes (

Table 1. Metabolome profiling of Cucumis sativus and Lagenaria siceraria crude fruit extracts via HR-UPLC/MS/MS in both negative and positive modes of analyses.

Peak	Identification	[M-H]-	[M+H]+	Elemental Composition	Error (ppm)	MSn ions	Rt (min)	C. sativus	L. siceraria
Sugars
1	Disaccharide	377.0853		C18H17O9−	4	341, 215, 179	5.1	+	+
Amino acids/amines
2	Lysine-O-hexosyl	307.1508		C12H23N2O7−	1	217, 187, 145, 127	4.6	+	+
3	N-trimethyl-lysine		189.1597	C9H21N2O2+	0.3	144, 130, 84	4.7	+	+
4	Pyroglutamic acid		130.499	C5H8NO3+	2	84	5	+	+
5	N-hexosyl-pyroglutamate	290.0895		C11H16NO8−	4.8	128	6.8	+	+
6	Pyrrolidine		72.0806	C4H10N+	1.9	56, 55	5.4	+	+
7	Nicotinamide		123.0555	C6H7N2O+	1.7	96, 80	6.6	+	+
8	Piperidine		86.0962	C5H12N+	2	70, 57, 56	6.7	+	+
9	Cyclo(leucylprolyl)		211.1437	C11H19N2O2+	1.7	192, 183, 154, 138, 114, 98, 86, 70	7	+	+
10	N-(deoxyhexosyl) phenylalanine		328.1395	C15H22NO7+	1.3	310, 292, 264, 246, 198, 178, 166, 132, 120	7.3	+	+
11	N-(deoxy-fructosyl)-iso/leucine	292.1402		C12H22NO7−	0	172, 130, 128, 101, 73	7.8	+	+
12	Pantothenic acid (Vit. B5)	218.1032		C9H16NO5−	1.1	146, 116, 99, 88	9.1	+	+
13	Methyl-pyroglutamic acid		144.066	C6H10NO3+	2	98, 84	9.5	+	+
14	N-(methoxybenzyl)glutamine		267.1337	C13H19N2O4+	0.7	250, 237, 232, 221, 211, 203, 166, 136, 121, 101, 86, 74	9.9	-	+
15	Iso/Leucyl-Iso/Leucine		245.1859	C12H25N2O3+	0.4	228, 210, 132, 120, 86, 69	11.3	-	+
Alkaloids
16	N,N,N-Trimethyltryptophan betaine (Lenticin alkaloid)		247.1446	C14H19N2O2+	2	188, 170, 146, 144, 118, 60	11.4	+	-
Organic acids
17	Shikimic acid	173.0455		C7H9O5−	0.3	137, 93	5.7	-	+
18	Homocitric acid	205.0352		C7H9O7−	0.8	161, 125, 117, 81, 73, 59	5.75	-	+
19	Malic acid	133.0139		C4H5O5−	2.6	115, 71	5.8	+	+
20	Citric acid	191.0195		C6H7O7−	1	173, 129	7.5	-	+
21	Citramalic acid	147.0304		C5H7O5−	3	129, 103, 87	7.9	+	-
22	Hydroxy-methylglutaric acid	161.0453		C6H9O5−	1.5	99, 57	8	-	+
Phenolic and cinnamic acids derivatives
23	Dihydroxy-methoxybenzene-O-hexoside (Methoxycatechol-O-hexoside)	301.0928		C13H17O8−	0.1	139, 121, 93	7.9	-	+
24	Caffeoyl-O-hexoside	341.0878		C15H17O9−	0.1	179	10.1		+
25	Sinapic acid		225.0763	C11H13O5+	2	207, 192, 175, 157, 123, 119	11.6	+
26	Sinapoyl-O-hexoside	385.1138		C17H21O10−	0.6	223, 179, 164	11.7	+
27	Feruloyl-O-hexoside	355.1035		C16H19O9−	1	265, 235,193, 175, 161, 149, 134	12.2	+	+
28	Dihydroxy-methoxybenzene -O-hexosylferuloyl-O-hexoside	639.1918		C29H35O16−	1.9	499, 463, 445, 431, 337, 193, 175, 139, 121,	12.9	-	+
29	Hydroxymethylphenyl -O-hexosylferuloyl-O-hexoside	623.1981		C29H35O15−	0	499, 461, 337, 193, 175, 161, 123, 105	13.1	-	+
30	Caffeic acid	179.035		C9H7O−	1	135, 107	10	-	+
31	Hydroxymethylphenyl -O-Caffeoyl-O-hexoside	447.1295		C22H23O10−	0.5	323, 179, 161, 135, 123, 105	13.4	-	+
32	Hydroxymethylphenyl -O-hexosylferuloyl-O-malonylhexoside	709.1985		C32H37O18−	0	665, 623, 499, 337, 193, 175, 119, 113	14.3	-	+
33	Dihydroxy-methoxybenzene -O-Feruloyl-O-hexoside	477.1398		C23H25O11−	1	337, 314, 193, 175, 139, 121, 103, 73	14.5	-	+
34	Ferulic acid		195.0648	C10H11O4+	0.4	177, 163	14.6	-	+
35	Hydroxymethylphenyl -O-Feruloyl-O-hexoside	461.1449		C23H25O10−	0.1	337, 193, 175, 160, 123, 105	14.7	-	+
36	Hydroxybenzoic acid -O-Feruloyl-O-hexoside	475.1237		C23H23O11−	1.9	337, 193, 175, 161, 137, 93	14.9	-	+
37	Dihydroxy-methoxybenzene -O-Feruloyl-O-malonylhexoside	563.1401		C26H27O14−	1	337, 193, 175, 121	15.7	-	+
38	Hydroxymethylphenyl -O-Feruloyl-O-malonylhexoside	547.1452		C26H27O13−	1	503, 461, 337, 193, 175, 160	16.1	-	+
Sesquiterpenes
39	Cynaroside A	443.1918		C21H31O10−	0.7	281, 237, 219, 189, 161, 131, 119, 113, 89, 71, 59	10	+	+
40	Unknown	429.1394		C19H25O11−	1.8	205, 119, 113, 89, 87, 73, 71	13	+	-
41	Unknown	407.1553		C17H27O11−	1.3	243, 183, 119, 101, 89, 87, 73, 59		+	-
42	Unknown	409.1716		C17H29O11−	0.2	269, 225, 89, 87, 59	14.2	+	-
43	Unknown	461.2021		C21H33O11−	1.6	343, 161, 153, 101, 87, 73, 71	16.5	+	-
Alkyl glycosides
44	Butanol-O-pentosyl-hexoside	367.1608		C15H27O10−	0.5	235, 203, 161, 131, 113, 101, 85, 73 71, 59	10.2	+	-
45	Benzyl-O-pentosyl-hexoside	401.1448		C18H25O10−	1.8	269, 161, 131, 113, 101, 85, 71	11.1	+	-
46	Hexanol-O-pentosyl-hexoside	395.1928		C17H31O10−	1.4	263, 161, 131, 101, 71	13.2	+	-
47	Unknown	529.2643		C26H41O11−	2	397, 347, 89	15.1	+	-
Flavonoids
48	Isovitexin-O-hexoside (Saponarin)		595.167	C27H31O15+	0.1	577, 475, 433, 397, 313, 283	11.2	+	+
49	Iso/orientin	447.0933		C21H19O11−	1	429, 369, 357, 327, 297, 285	12.1	-	+
50	Iso/vitexin	431.0974		C21H19O10−	2.2	341, 311, 283, 269	13.3	+	+
51	Quercetin-O-hexoside	463.0878		C21H19O12−	0.8	300, 271, 255, 179, 151	13.5	-	+
52	Iso/vitexin-O-feruloylhexoside		771.2141	C37H39O18+	1.3	753, 595, 433, 415, 397, 337, 313, 283, 271, 195, 177, 145	13.5	+	-
53	Kaempferol -O-(2′-rhamnosyl)-hexoside		595.1652	C27H31O15+	0.9	327, 285, 151	13.58	-	+
54	Iso/vitexin-O-coumaroylhexoside		741.2041	C36H37O17+	2.1	595, 579, 433, 415, 397, 337, 313, 283, 271, 165, 147, 145	13.6	+	-
55	Isorhamentin-O-rutinoside		625.1756	C28H33O16+	1	479, 317, 302, 286,	13.65	-	+
56	Quercetin		303.0493	C15H11O7+	2	253, 121, 107	13.9		+
57	Kaempferol-O-hexoside	447.0927		C21H19O11−	1.3	284	14.3	+	+
58	Isorhamnetin-O-hexoside	477.1031		C22H21O12−	1.5	314, 153	14.4	+	+
59	Luteolin		287.0549	C15H11O6+	0.3	255, 151, 133	14.5	-	+
60	Isorhamnetin		317.0653	C16H13O7+	0.7	302, 270	14.6	-	+
61	Luteolin-O-malonylhexoside		535.1083	C24H23O14+	0.1	431, 287, 231, 159	15.1	-	+
62	Isorhamnetin-O-malonylhexoside		565.1186	C25H25O15+	0.4	529, 479, 317, 302, 285, 245, 127	15.2	+	+
63	Chryseriol-O-hexoside		463.1233	C22H23O11+	0.4	301, 286, 258, 227, 211, 153, 145, 85	17.2	+	+
64	Dimethoxy-dihydroxyflavone-O-hexoside	491.1189		C23H23O12−	1.3	329, 313, 299, 271	17.3	-	+
65	Methylapigenin-O-hexoside		447.1295	C22H23O10+	2.2	285, 270, 229, 184, 159, 119	17.7	+	+
66	Methoxy-trihydroxyflavone-O-malonylhexoside		549.1237	C25H25O14+	0.3	301, 286, 231, 159, 85	17.75	-	+
67	Dimethoxy-trihydroxyflavone-O-malonylhexoside		579.1344	C26H27O15+	0	331, 316, 299, 271, 184, 159	17.85	-	+
68	* Dimethoxy-dihydroxyflavone-O-acetylhexoside	533.1297		C25H25O13−	0.6	329, 314, 299, 255	17.9	-	+
69	* Dimethoxy-trihydroxyflavone-O-hexoside		507.1497	C24H27O12+	0	345, 330, 315, 159	18.3	-	+
70	Dihydroxy-methoxy-isoflavone	283.061		C16H11O5−	0.6	268, 239, 211	18.7	+	-
71	* Dimethoxy-dihydroxyflavone-O-malonylhexoside		563.1396	C26H27O14+	0.1	315, 300, 184, 159	20	-	+
Pterocarpans
72	Hedysarimpterocarpene A-O-hexoside (HPA-O-hexoside)	475.124		C23H23O11−	1.2	313, 298, 283, 255, 225	19.5	-	+
73	Hedysarimpterocarpene A-O-acetylhexoside (HPA-O-acetylhexoside)	517.1345		C25H25O12−	1.3	313, 298, 283, 255	19.8	-	+
74	Hedysarimpterocarpene A (HPA)	313.0715		C17H13O6−	0.7	298, 283, 270	20.3	-	+
Lignans
75	Pentahydroxy-dimethoxylignan (Carinol)		379.1747	C20H27O7+	1.1	361, 347, 343, 315, 297, 285, 269, 253, 251, 225, 207, 197, 181, 137, 119, 105	13.1	-	+
76	Secoisolariciresinol		363.1795	C20H27O6+	1.9	327, 299, 281, 269, 253, 237, 223, 209, 195, 181, 143, 137, 121, 107, 103, 93	17.5	-	+
77	Trihydroxy-dimethoxylignan		347.1829	C20H27O5+	5	329, 311, 283, 255, 237, 207, 195, 183, 163, 137, 123, 107, 105	20.5	-	+
Saponins
78	Unknown O-pentosyl-O-hexoside saponin derivative	917.5112		C46H77O18−	0.3	785, 397, 179	16.7	+	-
79	* Soyasapogenol E-O-dihexosyl-O-glucuronide (Soyasaponin Bd)	955.4889		C48H75O19−	2	397, 113	17.3	+	-
80	Unknown O-pentosyl saponin derivative	1059.5385		C52H83O22−	0.3	927, 397, 113	17.8	+	-
81	* Soyasapogenol B-O-rhamnosyl-O-pentosyl-O-hexosyl-O-glucuronide (Melilotussaponin O1)	1073.5534		C53H85O22−	0.4	927, 397, 341	18	+	-
82	Soyasaponin I	941.5107		C48H77O18	0.9	923, 397, 341, 113	19.9	+	-
			943.5275	C48H79O18+	1.5	797, 635, 617, 599, 581, 459, 441, 423, 405, 383
Fatty acids/amides
83	Trihydoxy-butanoic acid	135.0295		C4H7O5−	1.8	89, 75, 59	5.5	+	-
84	Undecane-tricarboxylic acid	287.15		C14H23O6−	0	269, 227, 209, 199, 59	14.8	+	-
85	Tetrahydroxy-octadecynoic acid	343.2124		C18H31O6−	0.6	325, 307, 289, 273, 229, 209, 171, 135, 83	15.5	+	+
86	Azelaic acid	187.0975		C9H15O4−	0.2	169, 143, 125, 97, 57	16.3	+	+
87	Octanedicarboxylic acid	201.1132		C10H17O4−	0.3	183, 139, 111, 57	18.1	+	-
88	Trihydroxy-octadecadienoic acid	327.2176		C18H31O5−	0.2	291, 211, 183	18.8	+	-
89	Octadecenamide		282.2796	C18H36NO+	1.7	265	18.9	+	+
90	Trihydroxyoctadecenoic acid	329.2332		C18H33O5−	0.6	311, 293, 229, 211, 171	19.7	+	+
91	Unsautrated fatty acid		275.2011	C18H27O2+	2.1	251, 219,185, 147, 133, 119, 105, 91, 81, 79, 67, 55	22	+	+
92	Dihydroxyoctadecadienoic acid	311.2227		C18H31O4−	0.2	293, 275, 199	25.2	+	+
93	Unsaturated fatty acid		277.2161	C18H29O2+	0.4	207, 133, 107, 93, 79, 67	25.4	+	+
94	Octadecatrienoic acid		279.2318	C18H31O2+	0.2	255, 149, 135, 107, 81,69	26	+	+
95	Linolenic acid derivative	559.3119		C28H47O11−	0.8	277	26.7	+	-
96	* Stearidonic acid	275.2017		C18H27O2−	0.2		28	+	+
97	Oxo-octadecatrienoic acid		293.2111	C18H29O3+	0	275, 257, 133, 107, 93, 81, 67	28.5	+	+
98	* Oxo-octadecanoic acid	297.2434		C18H33O3−	0.3	279, 171, 155	30.9	+	+
99	Hydroxy-tetracosanoic acid	383.3529		C24H47O3−	0.3	365, 309, 112	32.7	+	+
Lysophospholipids
100	Lysophosphatidic acid derivative	481.2203		C21H38O10P−	1	255, 171, 153, 79	19.2	+	-
101	Lysophosphatidic acid derivative	497.2156		C21H38O11P−	0.3	171, 153, 79	19.8	+	-
102	Lysophosphatidic acid derivative	463.21		C21H36O9P−	0.5	335, 171, 153, 79	22.6	+	-
103	Palmitoyl-sn-glycero-phosphoglycerol	483.2725		C22H44O9P−	0.6	255	23	-	+
104	Hexosyl LPE 16:0	614.3308		C27H53NO12P−	0.4	452, 255	25.8	+	+
105	Palmitoyl-GPE	452.278		C21H43NO7P−	0.6	255, 196	27.7	+	+
106	LPE 18:2	476.278		C23H43NO7P−	0.6	280, 79	28.8	+	+
107	Lysophosphatidic acid derivative	431.2202		C21H36O7P−	0.3	277, 153, 79	30	+	-

* Metabolites identified based on GNPS networking via clustering with known compounds, (+) and (-) indicate presence and absence of a metabolite, respectively.

), belonging to amino acids, organic acids, cinnamates, alkaloids, flavonoids, pterocarpans, alkyl glycosides, sesquiterpenes, saponins, lignans, fatty acids/amides, and lysophospholipids, including several first-time reported metabolites and classes, as explained in detail in the following subsections. The constructed MN from the HR- negative ESI-MS/MS analysis was composed of 351 nodes and 449 edges, in which the clusters of interest included cluster A: fatty acids/amides, cluster B: lysophospholipids, cluster C: flavonoids and pterocarpans, cluster D: alkyl glycosides, cluster E: saponins, cluster F: organic acids, cluster G: amino acids derivatives, cluster H: sesquiterpenes, cluster I: lysophosphatidic acid derivatives, and cluster J: cinnamates (Figure 2). The positive MN was composed of 1002 nodes and 1451 edges, in which the clusters of interest included cluster A: lignans, cluster B: flavonoids, cluster C: amino acid derivatives, singleton D: alkaloid, and cluster E: fatty acids/amides (Figure S1).

The detection of alkaloids in positive mode versus phenolic acids in negative mode is expected considering the improved sensitivity for each class in respective mode and highlighting the importance of acquiring in different ionization types. Glycosides were detected based on the neutral loss of the attached O-sugar moieties at 162 amu (C₆H₁₀O₅) for hexoses, 146 amu (C₆H₁₀O₄) for rhamnose, 176 amu (C₆H₈O₆) for glucuronide and 132 amu (C₅H₈O₄) for pentoses. In some spectra, peaks detected at m/z 113 (C₅H₅O₃⁻), m/z 101 (C₄H₅O₃⁻), m/z 71 (C₃H₃O₂⁻) and/or m/z 59 (C₂H₃O₂⁻) correspond to hexose fragments [13]. Acylation of glycosides with acetyl or malonyl moieties was recognized by additional mass and/or neutral loss of 42 amu (C₂H₂O) or 86 amu (C₃H₂O₃), respectively, while C-glycosides showed significant neutral losses of 90 amu and 120 amu resulting from 0,2 and 0,3-sugar ring cleavage [14].

3.1.1. Amino Acids and Amines

Several amino acids and amine derivatives were eluted early, as detected in chromatograms at Rt. 4–11 min. (Figure 1), and grouped in two major clusters, i.e., G and C, in negative and positive MNs, respectively (Figure 2 and Figure S1), from which 14 metabolites could be annotated in peaks 2–15 (Table 1). The identified metabolites included four amines and 10 derivatives of amino acids viz. lysine, iso/leucine, phenylalanine, and glutamine, in which decarboxylation (-CO₂, 44 amu), demethylation (-CH₂, 14 amu), deglycosilation, deamination (-NH₂, 17 amu) and dehydration (-H₂O, 18 amu) were the major fragmentation pathways matching the reported literature, GNPS library, and HMDB and MassBank databases. All identified metabolites were common between both fruits except for peaks 14 and 15 (Table 1), which were detected in L. siceraria at [M+H]⁺ 267.1337, C₁₃H₁₉N₂O₄⁺ and [M+H]⁺ 245.1859, C₁₂H₂₅N₂O₃⁺, identified as N-(methoxybenzyl)glutamine, which is previously reported in Cucurbitaceae [15] and Iso/Leucyl-Iso/Leucine dipeptide, respectively.

3.1.2. Organic Acids

6 Organic acids were identified in peaks 17–22 (Table 1) and grouped in cluster F in negative MN (Figure 2) based on decarboxylation and dehydration shared fragments. For example, homocitric acid in peak 18 (Table 1) was detected in L. siceraria for the first time at [M-H]⁻, C₇H₉O₇⁻ and fragmented into m/z 161, C₆H₉O₅⁻ (-COO, 44 amu), m/z 125, C₆H₅O₃⁻ [M-H-CO₂-2H₂O], and m/z 81, C₅H₅O⁻ [M-H-2CO₂-2H₂O].

3.1.3. Alkaloids

Aside from previous studies that reported the presence of alkaloids in cucumber at moderate levels [3,16,17], peak 16 at [M+H]⁺ 247.1446, C₁₄H₁₉N₂O₂⁺ appeared as a singleton in positive MN (Figure S1) at Rt. 11.4 min. (Table 1) and was identified as N,N,N-trimethyltryptophan betaine, known as lenticin or hypaphorin alkaloid, which is reported herein for the first time in cucumber fruit. Identification was based on the presence of diagnostic daughter ions at m/z 188, C₁₁H₁₀NO₂⁺, post the loss of N-trimethyl moiety, followed by decarboxylation at m/z 144 C₁₀H₁₀N⁺ or dehydration at m/z 170, C₁₁H₈NO⁺. The latter proceeded into ring cleavage and demethylation at m/z 146, C₉H₈NO⁺ and m/z 122 C₇H₈NO⁺, as explained in Figure S2, matching the reported literature [18] and HMDB spectrum (https://hmdb.ca/spectra/ms_ms/2947783, accessed on 11 January 2023). Lenticin is distributed in various vegetables and known to exhibit cardioprotective and neurological effects, and thus could be correlated with cucumbers’ cardioprotective reported effect for the first time using such a metabolomics approach [19]. It is believed that cucumber may encompass several other alkaloids, but a special extract targeting method with solvents of lower polarity is needed to enhance their detection.

3.1.4. Phenolics and Cinnamic Acid Derivatives

In total, 15 glycosylated and/or acylated derivatives of phenolic and cinnamic acids were eluted at Rt. 8–16 min. in peaks 23–38 (Table 1). MSn ions for cinnamic acid derivatives were evidenced at m/z 179, C₉H₇O₄⁻, m/z 161, C₉H₅O₃⁻ and m/z 135, C₈H₇O₂⁻ for caffeoyl, m/z 193, C₁₀H₉O₄⁻ and m/z 175, C₁₀H₇O₃⁻ for feruloyl, m/z 223, C₁₁H₁₁O₅⁻ and m/z 205, C₁₁H₉O₄⁻ for sinapoyl moieties (Table 1). While sinapic acid derivatives were detected in cucumber only, bottle gourd extract was exclusively enriched with conjugates of phenyl and caffeoyl or feruloyl glycosides (Figure S3–S5) that appeared in negative MN grouped in cluster J (Figure 2) and were identified in peaks 28, 29, 31–33 and 35–38 (Table 1). Such conjugates are reported herein for the first time in the genus Lagenaria based on the diagnostic peaks at m/z 123, C₇H₇O₂^– and m/z 105, C₇H₅O⁻ for hydroxymethylphenyl fragments or m/z 137, C₇H₅O₃^– and m/z 93, C₆H₅O⁻ for hydroxybenzoic acid fragments, or m/z 139, C₇H₇O₃⁻ and m/z 121, C₇H₅O₂⁻ for dihydroxymethoxybenzene fragments (Figure S3–S5). Hexosyl moieties in peaks 32, 37, and 38 (Table 1) were additionally acylated with malonic acid (C₃H₂O₃⁻, 86 amu), which is known to improve the biological effects of phenolics [20].

For example, peak 35 detected in bottle gourd at [M-H]⁻ 461.1449, C₂₃H₂₅O₁₀⁻ was identified as hydroxymethylphenyl-O-feruloyl-O-hexoside fragmented into m/z 337, C₁₆H₁₇O₈⁻ post losing hydroxymethylphenyl moiety, m/z 193, C₁₀H₉O₄⁻ for ferulic acid and its dehydrated and demethylated fragments at m/z 175, C₁₀H₇O₃⁻ and m/z 160, respectively, whereas ions at m/z 123, C₇H₇O₂⁻ and m/z 105, C₇H₅O⁻ were detected for hydroxymethlphenyl ion and its dehydrated form, respectively (Figures S3 and S4). The node of peak 35 in negative MN was directly connected to molecular ion [M-H]⁻ 547.1452 in peak 38 at C₂₆H₂₇O₁₃⁻ having an extra C₃H₂O₃⁻ (malonyl, 86 amu), and thus identified as hydroxymethylphenyl-O-feruloyl-O-malonylhexoside, which, in turn, was connected to [M-H]⁻ 709.1985, C₃₂H₃₇O₁₈⁻ having an additional C₆H₁₀O₅⁻ (hexosyl, 162 amu), and hence identified as hydroxymethylphenyl-O-hexosylferuloyl-O-malonylhexoside, aided by the necessary MSn ions and their predicted formulae to confirm their identification (Table 1). Little information is available on the bioactivities of such acylated conjugates and how they contribute to L. siceraria health effects; thus, further studies are required to explore their potential pharmacological effect.

3.1.5. Sesquiterpenes

The family Cucurbitaceae is known to accumulate di-/sesqui-/and triterpenes, to which various bioactivities are attributed [4,21] nevertheless, sesquiterpenes are the least reported in the literature. Cluster H in negative MN consists of five metabolites (Figure 2), from which peak 39 was detected in both fruit extracts and was identified as cymaroside A at [M-H]⁻ 443.1918, C₂₁H₃₁O₁₀⁻ (Table 1). Cynaroside A is sesquiterpene lactone-O-hexoside, abundant in artichoke, that undergoes deglycosilation at m/z 281, C₁₅H₂₁O₅⁻, decarboxylation at m/z 237, C₁₄H₂₁O₃, followed by sequential demethylation, dehydration and ring cleavage fragmentation processes to yield several diagnostic daughter ions, as explained in detail in Figure S6, in accordance with databases and the other literature [22]. Despite being previously detected/isolated from other green vegetables [23], this is the first report of its presence in F. Cucurbitaceae, and whether these fruits could serve as source of that sesquiterpene, similar to artichoke, should be considered. The other four metabolites clustered with cymaroside A sesquiterpene shared ions corresponding to lactone ring cleavage followed by sequential demethylation and dehydration at m/z 119, m/z 101, m/z 89, m/z 71, m/z 59 (Figure S6), but could not be completely identified.

3.1.6. Alkyl Glycosides

From cluster D in negative MN (Figure 2), four metabolites were detected exclusively in cucumber as formate adducts in peaks 44–47 that belong to the alkyl glycosides class (Table 1). Alkyl glycosides are formed of fatty alcohols glycosylated with sugars. In detail, peak 44, [M-H]⁻ detected at m/z 367.1608, C₁₅H₂₇O₁₀⁻ which was identified as butanol-O-pentosyl-hexoside and yielded fragment ions at m/z 235, C₁₀H₁₉O₆⁻ and m/z 73, post sequential loss of both sugars followed by demethylation at m/z 59, while MSn ions at m/z 161, 113, 101, 71 belonged to hexosyl fragments (Figure S7) in good accordance with the literature [24] and databases. Similarly, peaks 45 and 46 were identified as benzyl-O-pentosyl-hexoside and hexanol-O-pentosyl-hexoside, respectively. Such compounds were previously detected in several fruits as in apples, anise, cumin, and others [15,25,26,27]; however, according to our knowledge, this is the first report of their presence in Cucurbitaceae. The exact bioactivities of such class of compounds in these fruits have yet to be studied.

3.1.7. Flavonoids

Previous studies reported the presence of isoflavones and acylated/methoxylated apigenin and luteolin-O/C-glycosides in cucumber [28,29], while flavonoid-O/C-glycosides were reported in both species [3,4,30]. In this study, 24 flavonoids were detected and tentatively identified in both species, including flavones, flavonols, and isoflavones. C-glycosides were recognized by MSn ions corresponding to sugar ring cleavage, unlike O-glycosides, which showed intact release of the dehydrated sugar. Aglycones were differentiated based on their typical RDA fragments [31]. Identification was guided by GNPS-based networking, which allowed extrapolating peaks’ annotations to unknown compounds, as observed in clusters B and C, in positive and negative MNs, respectively (Figure S1 and Figure 2).

Acylated flavonoids were detected in peaks 52, 54, 61, 62, 66–68, and 71 (Table 1), in which cucumber was more abundant in aromatic, i.e., feruloyl (C₁₀H₈O₃, 176 amu) or coumaroyl (C₉H₆O₂, 146 amu), acylated flavones, whereas bottle gourd was enriched with flavone and flavonols acylated with aliphatic, i.e., malonyl (C₃H₂O₃, 86 amu) or acetyl (C₂H₂O, 42 amu), moieties. These differential metabolomics results could aid in annotating acetyl transferase enzyme by correlating metabolite data with gene expression data. For example, peak 54 was identified as iso/vitexin-O-couamroylhexoside, which was previously reported in cucumber [28]. The precursor ion detected at [M+H]⁺ 741.2041, C₃₆H₃₇O₁₇⁺ showed fragments at m/z 595, C₂₇H₃₁O₁₅⁺ [loss of coumaroyl, 146 amu], m/z 433 for iso/vitexin, C₂₁H₂₁O₁₀⁺ upon loss of O-hexosyl moiety, followed by MSn ions for C-hexosyl ring cleavage and aglycone RDA at m/z 415, m/z 397, m/z 313 and m/z 283 (Figure S8).

Methoxylated flavonoids were detected abundantly in bottle gourd fruit as in peaks 55, 58, 60, 62, 63, 64, 66–69, and 71 (Table 1), identified based on the neutral loss of one or more methyl (-CH₂, 14 amu) / methoxyl (-OCH₂, 30 amu) moieties. For example, peak 62 was identified as isorhamnetin-O-malonylhexoside at [M+H]⁺ 565.1186, C₂₅H₂₅O₁₅⁺ and was reported for the first time in both species (Figure S9). The precursor ion yielded fragments at m/z 479 [M+H-malonyl]⁺, m/z 317, C₁₆H₁₃O₇⁺ for isorhamnetin post deglycosylation followed by demethylation at m/z 303, C₁₅H₁₁O₇, dehydration at m/z 285, C₁₅H₉O₆⁺ and finally RDA fragments at m/z 229, m/z 151 and m/z 127 [32]. To our knowledge, this is the first report of di/methoxylated flavonoid-O-acylated hexoside in L. siceraria, as in peaks 64, 66–69, and 71 (Table 1).

Such a mixture of flavonoids promotes the various health effects of both species, since acylation and methoxylation are known to impart structural and functional modifications that improve flavonoids’ bioactivity by increasing their lipophilicity and intracellular bioavailability, thus exhibiting better receptor/ligand binding compared to their non-acylated/methoxylated analogues, as observed in cancer chemoprevention and anti-acetylcholine esterase activities [20,33,34,35].

3.1.8. Pterocarpans

Pterocarpans are derivatives of isoflavonoids de novo biosynthesized as a response to stress, mainly detected in legumes [36,37]. Herein, three pterocarpans were detected in cucumber grouped with flavonoids in negative MN cluster C (Figure 2) and identified in peaks 72–74 (Table 1). Identification was based on the diagnostic fragmentation pattern, showing sequential loss of two methyl groups (−2 × CH₂, 14 amu) followed by decarbonylation (-CO, 28 amu) confirmed by generated formulae [38]. For example, peak 72 in Table 1 showed the precursor ion at [M-H]⁻ 475.124, C₂₃H₂₃O₁₁⁻ that yielded MSn ions at m/z 313, C₁₇H₁₃O₆⁻ [M-H-C₆H₁₀O₅, hexosyl 162 amu], followed by demethylation at m/z 298 and m/z 283, C₁₅H₇O₆⁻, then loss of carbonyl at m/z 255, C₁₄H₇O₅⁻ and was thus identified as hedysarimpterocarpene A-O-hexoside (HPA-O-hexoside) in accordance with the reported literature [38] (Figure S10). Peak 72 node in cluster C was directly connected to [M-H]⁻ 517.1345, C₂₅H₂₅O₁₂⁻, having an additional 42 amu (-C₂H₂O⁻) and sharing the same fragments at m/z 313, m/z 298 and m/z 255, and was thus identified as hedysarimpterocarpene A-O-acetylhexoside (HPA-O-acetylhexoside). Similarly, peak 74 at [M-H]⁻ 313.0715, C₁₇H₁₃O₆⁻ was identified as hedysarimpterocarpene (HPA) (Table 1). This is the first report of pterocarpans in Cucurbitaceae; further studies are required to confirm their biosynthesis in plants other than legumes and present them as potential sources of pterocarpans.

3.1.9. Lignans

Three lignans belonging to the dibenzylbutanediol subclass were grouped in cluster A in positive MN as detected in bottle gourd for the first time at [M+H]⁺ 379.1747, [M+H]⁺ 363.1795 and [M+H]⁺ 347.1829 in peaks 75–77 (Table 1), identified as pentahydroxy-dimethoxylignan, known as carinol and secoisolariciresinol, previously reported in Cucurbitaceae [39], and trihydroxy-dimethoxylignan, respectively. Elemental composition of peaks 76, C₂₀H₂₇O₆⁺ and 77, C₂₀H₂₇O₅⁺ showed one and two hydroxyl moieties fewer than peak 75, C₂₀H₂₇O₇⁺, respectively. Identification was based on diagnostic fragments corresponding to sequential dehydration, demethylation, and demethoxylation prior to and/or after cleavage of the bis(benzylbutanediol) bonding (Figure S11) sharing MSn ions at m/z 137, C₈H₉O₂⁺, m/z 121, C₈H₉O⁺ m/z 107, C₇H₇O⁺ and m/z 93, C₇H₉⁺, in accordance with references and databases [40].

3.1.10. Saponins

Previously, cucumber was reported for the presence of triterpenoid saponins [41]. In this study, five saponins in peaks 78–82 were detected in cucumber fruit (Table 1). Peak 82 detected in positive ionization mode at [M+H]⁺ 943.5275, C₄₈H₇₉O₁₈⁺ was identified as soyasaponin I based on fragments at m/z 797, [M+H-rhm], m/z 635, C₃₆H₅₉O₉ [(M+H)-rhm-hex], 617, C₃₆H₅₇O₈ ⁺, m/z 599, C₃₆H₅₅O₇⁺, m/z 581, C₃₆H₅₃O₆⁺ and m/z 459, C₃₀H₅₁O₃⁺ for soyasapogenol B post loss of glucuronide moiety (C₆H₈O₆,176 amu) followed by dehydration, demethylation and RDA fragmentation of sapogenin at m/z 441, C₃₀H₄₉O₂⁺, m/z 423, C₃₀H₄₇O⁺, m/z 405, C₃₀H₄₅⁺, m/z 383, C₂₇H₄₃O⁺ and other fragments (Figure S12) matching references and databases [42]. On the other hand, all saponins were detected in negative ionization mode analysis as formate adducts (+46 amu, HCOOH), in which soyasaponin I showed few fragments indicative for the loss of hydroxymethyl, hydroxyl and ring A cleavage at m/z 397 and m/z 341, as predicted in HMDB spectra. In negative MN (Figure 2), cluster E consists of saponins correlated with soyasaponin I for their soyasapogenol B/E MSn ions at m/z 397 and m/z 341, in addition to other ions indicating terminal sugar loss or a fragment of hexose at m/z 113 (Table 1, Figure S13). From the aforementioned data, peaks 79 and 81 were annotated as soyasapogenol E-O-dihexosyl-O-glucuronide (soyasaponin Bd) and soyasapogenol B-O-rhamnosyl-O-pentosyl-O-hexosyl-O-glucuronide (melilotus saponin O1), respectively. The mixture of these saponins is reported in legumes [43], but this was the first time it was reported in cucumber. Other saponins maybe present in L. siceraria and C. sativus fruits that could be revealed upon using an extraction-targeting method instead of crude methanol extracts.

3.1.11. Fatty Acids/Amides

In total, 16 fatty acids and one fatty acyl amide were annotated in peaks 83–99, equally distributed in both species (Table 1) and grouped in the major clusters in both negative and positive MNs, A and E, respectively (Figure 2 and Figure S1). This included mono-, di-, tri-, and tetrahydroxylated fatty acids, as evidenced by the loss of water (H₂O,18 amu) and carboxylate (CO₂, 44 amu) moieties. Six saturated fatty acids were detected in peaks 83, 84, 86, 87, 98, and 99. For example, peak 86—detected in both species— was identified as azelaic acid at [M-H]⁻ 187.0975, C₉H₁₅O₄⁻ a dicarboxylic fatty acid, which showed fragment ions at m/z 169, C₉H₁₃O₃⁻ [M-H-H₂O], m/z 143, C₈H₁₅O₂⁻ [M-H-CO₂], m/z 125, C₈H₁₃O⁻ [M-H-H₂O-CO₂], m/z 97, C₆H₉O⁻, m/z 80, m/z 71, m/z 69 C₄H₅O⁻ and m/z 57, matching the literature [44]. Azelaic acid is incorporated into skin products for treatment of alopecia, as well as its reported cytotoxic and anti-inflammatory effects [44,45]. This could account for the popular usage of cucumber in skin preparations [3]. In contrast, 10 unsaturated fatty acids were detected in peaks 85, 88, and 90–97 (Table 1). Peak 85 was identified as octadecenamide at [M+H]⁺ 282.2796, C₁₈H₃₆NO⁺ showed fragment at m/z 265 C₁₈H₃₃O⁺ upon loss of ammonia (NH₃, 17 amu). The herein identified fatty acids matched reported literature on Cucurbitaceae seed oil contents [15].

3.1.12. Lysophospholipids

In negative MN (Figure 2), cluster B and cluster I grouped lysophospholipids and lysophosphatidic acids, respectively, from which peaks 100–107 were detected (Table 1). Metabolites detected at [M-H]⁻ 481.2203, C₂₁H₃₈O₁₀P⁻, [M-H]⁻ 497.2156, C₂₁H₃₈O₁₁P⁻, [M-H]⁻ 463.2100, C₂₁H₃₆O₉P⁻ and [M-H]⁻ 431.2202, C₂₁H₃₆O₇P⁻ are lysophophatidic acid derivatives, detected in cucumber only in peaks 100–102, and 104 (Table 1)

Overall, metabolome profiling of cucumber and bottle gourd based on GNPS-networking showed that both fruits share the abundance of amino acids, organic acids, flavones-C-glycosides, fatty acids and lysophospholipids in a comparable manner. By contrast, triterpenoid saponins, alkaloids, flavones-C-glycosides acylated with ferulic or coumaric acids, isoflavones alkyl glycosides and pterocarpans were exclusively detected in cucumber, and the latter two classes were reported for the first time in Cucurbitaceae. On the other hand, bottle gourd was further distinguished by the presence of dimethoxylated flavonoids acylated with malonic or acetic acids, lignans and conjugates of phenyl compounds with caffeic/ferulic acid-O-glycosides, all of which are reported for the first time in genus Lagenaria. Sesquiterpenes were more abundant in bottle gourd. except for cynaroside A, which was detected in both fruits for the first time as well. Successive extraction of fruits with solvents of different polarities is recommended to further reveal phytochemical classes that require targeting, such as alkaloids and saponins. The herein identified metabolites rationalized several of the reported pharmacological effects in both species, and further promoted them for new in vitro and in vivo medicinal research. Cucumber shared several phytochemicals that are characteristic to legumes (Fabaceae), e.g., soyasaponins, isoflavones, and pterocarpans, thus supporting the idea of an in-depth study of its biosynthetic pathways and gene expression data [37,43].

3.2. Aroma Profiling of L. siceraria and C. sativus Fruits Using SPME Coupled to GC-MS

SPME is well suited for the profiling of aroma for low-strength aroma food matrices, and also at low temperature for collection, compared to steam distillation, providing the true composition of volatile blends, [11] and this is the first time it has been reported in both species.

In this study, three independent replicates were analyzed under the same conditions via SPME-GC/MS and represented by total ion chromatograms (Figure 3A). 93 volatile constituents were detected in L. siceraria and C. sativus fruits and categorized into alcohols (11), aldehydes (16), ketones (14), fatty acids (3), oxides/ethers (7), S-containing compounds (1), monoterpenes (3), sesquiterpenes (5), esters (22), furans (1), and aliphatic (7) and aromatic (3) hydrocarbons (Table S1). Both species showed comparable aroma composition, as seen in the relative percentile of each class in Figure 3B.

In L. siceraria, alcohols and ethers were the major volatile classes, detected at 23.34% and 23.52%, respectively, whereas aldehydes were the most abundant in C. sativus (25.63%), followed by ethers (23.93 %) (Figure 3B, Table S1). Sesquiterpenes were detected in both fruits at low levels, i.e., 1.05% and 1.07% in bottle gourd and cucumber, respectively. Linalool is the major volatile constituent detected in both fruits at 18.76 % and 17.70 %, followed by safrole (11.4%, 10.5%), anethol (9.43%, 11.2%), and octanol (9.18%, 11.1%) in bottle gourd and cucumber, respectively.

Interestingly, hexenal, nonenal, and nonadienal (cucumber aldehyde) aldehydes that were reported to account for cucumber’s pleasant aroma [46] are herein detected in bottle gourd as well for the first time, at comparable levels except for 2,6-nonadienal (peak 19), which is more abundant in cucumber (Table S1).

3.3. Multivariate Data Analysis of Volatiles Dataset Acquired from SPME-GC/MS

Although notable differences in volatile constituents were observed visually between both fruits, multivariate data analyses were employed to classify both fruits in an untargeted manner using PCA and OPLS modelling (Figure S14). Samples segregation was observed along PC1 and PC2 to account for more than 80% of the total variance (Figure S14A,B). Supervised OPLS was further applied for its superiority in class separation [47] to obtain a model with good variance and prediction power (R² = 0.88, Q² = 0.82) (Figure S14C,D). The loading plot and S-loading plots identified metabolites mediating for samples separation as denoted by Mol. ion/Rt and labelled with their peak numbers in accordance with Table S1 (Figure S14B,D).

Examination of the PCA loading plot revealed that aldehydes were abundant in cucumber, represented by benzaldhyde (peak 14), octanal (peak 15), benzenacetaldehyde, (peak 17) and nonadienal (peak 19), in addition to anethol (peak 83), whereas bottle gourd was further enriched with ketones and esters, i.e., fenchone (peak 67) and methyl hexadecanoate (peak 58) (Table S1, Figure S14A,B).

Furthermore, OPLS score plot showed better discrimination of samples (Figure S14C). The S-loading plot derived from OPLS model (Figure S14D) confirmed aldehydes’ abundance in cucumber as discriminating metabolites, i.e., octanal (peak 15), benzenacetaldehyde (peak 17), and nonadienal (peak 19), versus anethol ether (peak 83) fenchone (peak 67), and methyl hexadecanoate (peak 58) in bottle gourd fruit.

The overall aroma of bottle gourd and cucumber is manifested by a mixture of characteristic volatiles. Linalool, that is, the major detected constituent in both fruits, is known to impart an orange oil-like aroma, whereas fenchone in bottle gourd possesses a pleasant camphor-like aroma and is incorporated in food as a perfumery flavor [48]. On the other hand, aldehydes in cucumber, i.e., nonadienal, nonenal, and hexenal are known to mediate for the pleasant characteristic aroma of fresh cucumber that is further enhanced upon chewing, while benzaldhyde has an almond-like odor, all of which are shared in bottle gourd as well at variable concentrations. [49,50,51]. In addition to their role in taste and odor, such volatiles are known to mediate for antimicrobial activity [51,52]. The study infers that bottle gourd possesses a consumer-pleasant aroma.

3.4. Unsupervised PCA Data Analysis of C. sativus and L. siceraria Primary Metabolites

To provide insight into C. sativus and L. siceraria fruits’ primary metabolome mediating for their nutritive value, GC–MS was employed, leading to the detection of 49 peaks. Chromatograms displayed a representative profile of C. sativus and L. siceraria fruits’ nutrient primary metabolites (Figure S15).

Metabolites were categorized into alcohols (6), amino acids (2), fatty acids/esters (12), nitrogenous compounds (1), organic acids (12), phenolic acids (1), steroids (2), sugars (9), sugar acids (1), and sugar alcohols (3), as shown in

Table 2. Primary metabolite analyzed using GC-MS of different C. sativus and L. siceraria with results expressed as relative percentile of the total peak area (n = 3).

Peak	Rt (min)	KI	Metabolite	M. formula	C. sativus	L. siceraria
Alcohols
1	5.31	987	Ethylene glycol, 2TMS derivative	C8H22O2Si2	1.39 ± 0.37	2.44 ± 1.19
2	5.60	1003	Propylene glycol, 2TMS derivative	C9H24O2Si2	0.11 ± 0.03	0.18 ± 0.08
4	6.62	1061	1,3 Propanediol di-TMS	C9H24O2Si2	0.21 ± 0.06	0.40 ± 0.17
7	8.23	1151	1,4-Butanediol, 2TMS derivative	C10H26O2Si2	0.12 ± 0.03	0.18 ± 0.10
11	9.89	1251	Triethylene glycol, 2TMS derivative	C12H30O4Si2	0.49 ± 0.14	0.89 ± 0.39
13	10.41	1285	Glycerol, 3TMS derivative	C12H32O3Si3	0.85 ± 0.20	0.86 ± 0.30
			Total alcohols		3.18 ± 0.82	4.95 ± 2.23
Amino acids
19	12.19	1401	Glycine, 3TMS derivative	C11H29NO2Si3	1.64 ± 0.27	2.41 ± 0.98
20	12.63	1434	β-Alanine, 3TMS derivative	C12H31NO2Si3	0.37 ± 0.08	0.69 ± 0.29
			Total amino acids		2.01 ± 0.35	3.10 ± 1.28
Fatty acids/esters
18	11.57	1360	Caprylic acid n-butyl ester	C12H24O4	5.70 ± 1.64	9.74 ± 4.49
33	18.77	1948	12-Methyltetradecanoic acid TMS ester	C18H38O2Si	0.38 ± 0.18	0.14 ± 0.09
35	19.76	2045	Palmitic Acid, TMS derivative	C19H40O2Si	3.18 ± 1.04	10.76 ± 10.50
37	20.72	2144	Margaric acid, TMS ester	C20H42O2Si	0.13 ± 0.03	0.35 ± 0.15
38	21.38	2214	Linoleic acid TMS ester	C21H40O2Si	0.40 ± 0.26	1.02 ± 0.94
39	21.42	2218	Oleic acid, TMS ester	C20H40O2Si	0.93 ± 0.38	3.34 ± 3.76
40	21.46	2223	α-Linolenic acid, TMS	C21H38O2Si	0.77 ± 0.56	2.53 ± 3.14
41	21.63	2242	Stearic acid, TMS derivative	C21H44O2Si	2.78 ± 0.71	6.51 ± 3.11
42	23.36	2440	Arachidic acid, TMS derivative	C23H48O2Si	0.04 ± 0.03	0.09 ± 0.03
43	24.62	2600	1-Monopalmitin 2TMS ether	C25H54O4Si2	0.35 ± 0.15	0.84 ± 0.84
46	26.08	2786	Glycerol monostearate, 2TMS derivative	C27H58O4Si2	0.26 ± 0.12	0.39 ± 0.27
47	26.30	2812	Sebacic acid, bis(2-ethylhexyl) ester	C26H50O4Si2	0.38 ± 0.11	0.66 ± 0.29
			Total fatty acids/esters		15.32 ± 5.19	36.38 ± 27.62
Nitrogenous compounds
10	9.80	1245	Urea, 2TMS derivative	C7H22N2OSi2	1.31 ± 0.48	4.10 ± 1.98
			Total nitrogenous compounds		1.31 ± 0.48	4.10 ± 1.98
Organic acids
3	6.44	1051	Methylmalonic acid (2TMS)	C10H22O4Si2	0.17 ± 0.06	0.28 ± 0.14
5	6.74	1068	Lactic acid, 2TMS derivative	C9H22O3Si2	1.43 ± 0.45	0.99 ± 0.38
6	7.01	1083	Glycolic acid, 2TMS derivative	C8H20O3Si2	0.22 ± 0.07	0.26 ± 0.09
9	9.74	1241	4-Hydroxybutanoic acid, 2TMS deriv.	C10H24O3Si2	1.77 ± 0.44	3.83 ± 1.81
12	9.94	1254	Benzoic acid, TBDMS derivative	C13H20O2Si	0.17 ± 0.05	0.21 ± 0.12
14	10.42	1286	Phosphoric acid, tris(trimethylsilyl) ester	C9H24O4PSi3	2.35 ± 0.85	2.22 ± 1.41
Peak	Rt (min)	KI	Metabolite	M. formula	C. sativus	L. siceraria
15	10.88	1315	L-Glutamic acid, N,N-di(3-methylbutyl)-, dimethyl ester	C17H33NO4	3.35 ± 0.93	5.92 ± 2.46
16	10.96	1320	Succinic acid (2TMS)	C10H22O4Si2	0.75 ± 0.21	1.21 ± 0.53
21	13.51	1500	Malic acid, 3TMS derivative	C13H30O5Si3	2.45 ± 0.35	1.00 ± 0.41
22	14.11	1545	3-Methylvaleric acid, TMS	C9H20O2Si	0.27 ± 0.09	0.47 ± 0.21
23	14.29	1559	Erythronic acid, tetrakis(trimethylsilyl) deriv.	C16H40O5Si4	0.06 ± 0.03	0.01 ± 0.01
25	17.19	1799	Azelaic acid, 2TMS derivative	C15H32O4Si2	0.26 ± 0.16	0.34 ± 0.13
			Total organic acids		13.24 ± 3.69	16.74 ± 7.70
Phenolic acids
45	25.62	2727	3,5-Dimethoxymandelic acid, di-TMS	C16H28O5Si2	0.11 ± 0.04	0.20 ± 0.08
			Total phenolic acids		0.11 ± 0.04	0.20 ± 0.08
Sterols
48	29.19	3180	Cholesterol, TMS derivative	C30H54OSi	0.37 ± 0.29	0.09 ± 0.06
49	31.11	3423	Lanost-7-en-3-ol, 9,11-epoxy-, acetate, (3β,11α)-	C32H52O3	0.08 ± 0.04	0.48 ± 0.50
			Total sterols		0.45 ± 0.33	0.57 ± 0.57
Sugars
26	17.55	1832	D-Fructopyranose, 5TMS derivative	C21H52O6Si5	9.72 ± 2.30	4.71 ± 2.82
27	17.65	1842	D-Fructose, 5TMS derivative	C21H52O6Si5	13.84 ± 1.53	5.59 ± 5.52
28	17.89	1865	Arabinofuranose, 4TMS derivative	C17H42O5Si4	0.88 ± 0.24	0.90 ± 0.50
29	17.89	1865	1-Deoxyglucose 4TMS	C18H44O5Si4	0.14 ± 0.02	0.16 ± 0.09
30	18.01	1877	D-Mannose, 5TMS derivative	C21H52O6Si5	0.04 ± 0.03	0.11 ± 0.03
31	18.39	1912	D-Fructose, 5TMS derivative isomer	C21H52O6Si5	3.01 ± 0.77	1.94 ± 1.22
32	18.44	1917	Galactopyranose, 5TMS derivative	C21H52O6Si5	14.24 ± 2.05	7.58 ± 5.59
34	19.33	2001	β-D-Glucopyranose, 5TMS derivative	C21H52O6Si5	19.36 ± 2.45	11.36 ± 8.18
44	25.34	2691	Sucrose, 8TMS derivative	C36H86O11Si8	1.55 ± 0.28	0.16 ± 0.05
			Total sugars		62.78 ± 9.68	32.51 ± 23.99
Sugar acids
17	11.29	1342	Glyceric acid, 3TMS derivative	C12H30O4Si3	0.17 ± 0.03	0.02 ± 0.02
			Total sugar acids		0.17 ± 0.03	0.02 ± 0.02
Sugar alcohols
8	8.33	1156	1,4-Anhydro-d-galactitol TMS derivative	C6H12O5	0.12 ± 0.02	0.24 ± 0.12
24	16.35	1729	Arabinitol, 5TMS derivative	C20H52O5Si5	0.14 ± 0.08	0.06 ± 0.01
36	20.49	2121	Myo-inositol, 6TMS derivative	C24H60O6Si6	1.18 ± 0.19	1.14 ± 0.47
			Total sugar alcohols		1.43 ± 0.29	1.44 ± 0.60

.

In C. sativus, sugars represented the most abundant class at ca. 63% of detected primary metabolites, mostly represented by monosaccharides at 61% and trace disaccharides at ca. 2%. Fructose, glucose, and galactose were the main identified monosaccharides, amounting to ca. 27, 19, and 14%, respectively, to account for the mild sweet taste of C. sativus (Table 2). The sugars in L. siceraria were detected at half that in C. sativus (ca. 33%), likewise represented by fructose, glucose, and galactose, though at lower levels than in C. sativus at 8–11%. Both samples encompassed trace levels of sugar acids and sugar alcohols (Table 2). Myo-inositol was the major detected sugar alcohol detected at 1%, which is in agreement with what was previously published for cucumber fruits [53].

12 Fatty acids/esters were identified in both fruit samples, i.e., cucumber and bottle gourd, amounting for ca. 15 and 36%, respectively (Table 2), mostly represented by saturated fatty acids detected at 13 and 29% in both fruit samples, respectively. The main identified fatty acids were palmitic, caprylic, and stearic acids (Table 2). At the cellular and tissue levels, palmitic acid performs a variety of important biological roles [54]. Caprylic acid is a medium-chain saturated fatty acid that exerts strong anti-atherosclerotic, anti-inflammatory, antifungal, and antibacterial effects [55,56]. Only three unsaturated fatty acids were identified, i.e., linoleic acid, oleic acid, and α-linolenic acid, amounting to 2 and 7% in cucumber and bottle gourd, respectively.

12 Organic acids were identified in both samples amounting to 13 and 17% in C. sativus and L. siceraria, respectively. Glutamic, malic, hydroxy butanoic, and lactic acids were the major identified organic acids in both fruits (Table 2). Organic acids are known to exert bactericidal activity acting as food acidulant preservatives [57].

Azelaic acid one of the organic acids identified, though at small levels, in both samples at 0.3%, and is well-reported for the treatment of a variety of skin conditions owing to its many skin-healing properties [58]. Whether L. siceraria has potential application in skin products comparable to that of C. sativus, with its well-reported anti-irritant, anti-acne, and skin-whitening properties has yet to be examined.

Amino acids were represented by two amino acids, viz. glycine and alanine, amounting to 2–3% of the total primary metabolites. Only one non-amino acid nitrogenous, i.e., urea, was identified in both fruits at 3–5% (Table 2).

3.5. Supervised Multivariate OPLS-DA Analysis of C. sativus and L. siceraria Primary Metabolites

(OPLS-DA) was further employed to confirm samples’ classification and primary metabolites’ marker determination obtained by GC-MS data-based PCA analysis. Both samples were modelled against each other (Figure S16C,D), showing clear separation as shown in the derived score plot, with R² = 0.99 (variance coverage) and a prediction goodness parameter Q² = 0.93 (Figure S16C). The corresponding S-plot (Figure S16D) showed that cucumber fruits were rich in sugars, i.e., glucose, galactose, and fructose, as listed in Table 2, found at a level twice that found in L. siceraria fruit, which accounts for its better recognition as fresh food, owing to its better taste. By contrast, the bottle gourd fruits were rich in fatty acids (Figure S16D), which confirmed the same markers obtained by PCA classification.

4. Conclusions

This study provided the first comparative metabolite profiling of cucumber and bottle gourd fruits via HR-UPLC/MS/MS based on GNPS-networking in both modes, allowing for annotation of 107 metabolites, i.e., amino acids (14), organic acids (6), phenolic/cinnamic acid derivatives (15), alkaloids (1), flavonoids (24), pterocarpans (3), alkyl glycosides (4), sesquiterpenes (5), saponins (5), lignans (3), fatty acids/amides (16), and lysophospholipids (8). Overall, both fruits share an abundance of amino acids, organic acids, flavones-C-glycosides, fatty acids and lysophospholipids in a comparable manner. However, triterpenoid saponins, alkaloids, flavones-C-glycosides acylated with ferulic or p-coumaric acids, alkyl glycosides, and pterocarpans were exclusively detected in cucumber, and the latter two classes were reported in this study for the first time to be present in Cucurbitaceae. On the other hand, bottle gourd was further distinguished by the presence of dimethoxylated flavonoids acylated with malonic or acetic acids, lignans, and conjugates of phenyl compounds with caffeic/ferulic acid-O-glycosides, all of which were reported for the first time in genus Lagenaria. Sesquiterpenes were more abundant in bottle gourd, except for cynaroside A, which was detected in both fruits for the first time. The herein-identified metabolites rationalized several of the reported pharmacological effects of both species and further promoted them for new in vitro and in vivo medicinal research. Several of the herein-detected phytochemicals require further bioactivity studies to study their potential health effects, e.g., pterocarpans, alkyl glycosides, and phenyl-cinnamic acids conjugates. Aroma profiling via SPME-GC/MS analysis detected 93 volatiles at comparable levels in both species, responsible for the pleasant aroma of bottle gourd, despite its enrichment with ketones and esters. 49 Silylated primary metabolites were detected in both species via GC/MS analysis at comparable levels, of which bottle gourd was further enriched with fatty acids, and cucumber with sugars.