Molecular Networking and Mass2Motif Analysis Prioritise Glycosylated Triterpenoid-Related Chemical Space in the Cytotoxic Fractions of Edible Sesbania grandiflora Flowers

Sesbania grandiflora flowers are widely consumed as vegetables in several South and Southeast Asian countries, but the chemical constituents associated with their reported cytotoxicity remain poorly defined. An ethanolic flower extract was separated into four solvent fractions, profiled by UHPLC-QTOF-MS/MS, and evaluated against A549 and HepG2 cells. Cytotoxicity was concentrated in the ethyl acetate and n-butanol fractions, with IC50 values ranging from 27.7 to 42.4 µg/mL. Among 3015 aligned features, 187 showed abundance patterns that tracked with the active fractions. Terpenoids accounted for 121 of these features, representing 64.7% of the activity-tracking set compared to 31.9% of the whole feature set, indicating a disproportionate representation of terpenoid-related chemistry. Eighteen molecular families were prioritised by family-level enrichment analysis, of which 13 were dominated by terpenoids. Complementary MS2LDA analysis identified nine enriched Mass2Motifs, including six triterpenoid-related motifs, two flavonoid-related motifs, and one structurally unresolved motif. A total of 334 features were supported by both family- and substructure-level analyses, with 80.5% classified as terpenoids. These enrichment analyses were exploratory and used for prioritisation rather than as confirmatory statistical tests. Chromatographic separation of the ethyl acetate fraction yielded nine NMR-confirmed constituents, including oleanolic acid, kaempferol, and 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl oleanolic acid (SGE20). SGE20 was the most active tested isolate, with IC50 values of 33.08 and 47.54 µM against A549 and HepG2 cells. The corresponding feature belonged to an enriched molecular family and carried enriched Mass2Motifs, thereby placing the most active tested isolate within the chemical region defined by the family- and substructure-level analyses. Overall, the combined biological and metabolomic analyses consistently prioritised glycosylated triterpenoid-related chemistry, together with a smaller flavonoid component, as the principal chemical region co-distributed with fraction-level MTT activity. Given the established dietary use of S. grandiflora flowers, these findings provide baseline chemical information for future studies examining how culinary processing and realistic dietary exposure affect the prioritised constituent families.