Multiplatform metabolomics and chemometrics reveals anticancer metabolites in Clinacanthus nutans against nasopharyngeal carcinoma

Plant metabolites are important natural sources of anti-cancer agents. This study aimed to identify potential metabolites and lipids from Clinacanthus nutans leaves (CNL) as natural anti-nasopharyngeal carcinoma (anti-NPC) agents. Three different CNL extracts, namely ethanol (E-CNL), ethanol/water (1:1, v/v) (EW-CNL), and water (W-CNL), were used for analysis. Following methyl tert-butyl ether/methanol/H 2 O liquid–liquid separation, each CNL sample was simultaneously separated into an upper-phase nonpolar lipidome and lower-phase polar metabolites. Using liquid chromatography–mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS), anti-NPC activity, metabolomics, lipidomics, and chemometric-profile analysis of the CNL extracts were investigated and compared. Results showed that E-CNL had the highest anti-NPC potential with an IC 50 value of 145.08, 163.62, and 133.43 μg/mL on NPC cell lines HK-1, C666–1, and CNE2, respectively. Through metabolomics and lipidomics analyses, 20 metabolites and 20 lipids were identified as the most influential compounds based on LC-MS/MS profiling, whereas 11 metabolites were identified by GC-MS profiling. The chemical composition of the crude extracts was significantly different. The key parameters influencing the ultrasonic-assisted extraction of bioactive E-CNL extract were systematically optimized using the Box-Behnken design (BBD) combined with response surface methodology (RSM). This optimization process resulted in a maximum extraction yield of 8.903 %. This study holds considerable significance for drug discovery and development, and offers the potential to identify novel anti-NPC agents derived from plant sources. • Fifty-one top vip compounds were identified in the CNL extracts by GC-MS and LC-MS/MS. • E-CNL was discriminated from EW-CNL and W-CNL based on the GC-MS and LC-MS/MS profiling. • E-CNL exhibited strong anti-NPC on cell lines HK-1, C666–1, and CNE2. • Chemometrics revealed Kuwanon C, Linoleate, Stigmasterol, and γ -Sitosterol as bioactive compounds.