Flavonoid Composition and Bioactivities of Nymphaea ‘Blue Bird’: Analysis, Purification, and Evaluation

<i>Nymphaea</i> '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 <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, <i>Escherichia coli</i>, and <i>Candida albicans</i>). 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 <i>N</i>. 'Blue Bird' petals as a promising source of natural antioxidants and antimicrobials, providing a scientific basis for their future functional applications.