Profiling Class-Wide Bioactivities of Flavonoids While Minimizing Compound-Specific Effects Using an Equimolar Mixture Strategy

Flavonoids share a C₆-C₃-C₆ core yet vary in side-chain decorations. Here, we tested whether an equimolar cross-subclass mixture could serve as a "class reference" by capturing class-wide bioactivities and diluting outlier effects. Twenty flavonoids across five subclasses were blended and tested alongside five single-flavonoid representatives─luteolin, quercetin, naringenin, EGCG, and genistein. In rat PC12 pheochromocytoma cells, the flavonoid mixture enhanced NGF-induced neuronal differentiation and activated reporters of neurofilament, cAMP, NF-κB, and antioxidant response elements, paralleling the effects of the representatives and indicating conserved neurotrophic activity. All flavonoidic samples were nontoxic at 5 μM but became toxic at 50 μM, except naringenin. Notably, luteolin-induced mitochondrial depolarization and cytotoxicity were buffered as the proportion of other flavonoids increased. The mixture bound amyloid-β_1-42 ∼10× more weakly than EGCG, evidencing dilution of a specific interaction. This proof-of-concept study offers tools and a framework to map flavonoids' functions in nutritional and pharmacological contexts.