Flavonoid Composition and Bioactivities of Nymphaea 'Blue Bird': Analysis, Purification, and Evaluation.

Nymphaea 'Blue Bird', a tropical water lily prized for its ornamental appeal, has been less explored as a source of bioactive flavonoids. This study developed an efficient extraction and purification protocol for flavonoids from this plant and compared their distribution and bioactivities across different tissues. Supercritical CO2 fluid extraction (SFE) proved optimal, yielding 2.56% under conditions of 24.3 MPa, 39 °C, 91 min, and a CO2 flow rate of 16 L/min. Subsequent purification with HPD500 macroporous resin enhanced flavonoid purity from 3.05% to 11.46%. Among the tissues analyzed, petals contained the highest levels of total flavonoids (6.43 mg/g) and total phenolics (45.71 mg/g), and exhibited the most potent antioxidant (as shown by the lowest EC50 values for ABTS+ and DPPH scavenging) and broad-spectrum antibacterial activities (indicated by the lowest MIC and MBC against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans). Antibacterial efficacy was generally superior against Gram-positive bacteria. Widely targeted metabolomics identified 560 metabolites, predominantly flavonols and flavonoids. Principal component and cluster analyses revealed tissue-specific metabolite profiles. KEGG enrichment analysis underscored the significance of the flavonoid biosynthetic pathway, and key differential metabolites-such as luteolin, myricetin, taxifolin, and quercetin-were strongly correlated with the observed bioactivities. These results highlight N. 'Blue Bird' petals as a promising source of natural antioxidants and antimicrobials, providing a scientific basis for their future functional applications.