Functional Robustness of Flavonoidic Mixtures Across Different Subclasses: Identification of Redundant Individual Members Through Compositional Perturbation

Flavonoids commonly occur not in isolation but as compositionally variable mixtures whose structurally diverse members often share antioxidant and other biological properties. This functional overlap may render flavonoid mixtures that are intrinsically robust to compositional variation, a possibility we investigated here using our previously established equimolar mixture of 20 flavonoids (MIX20). First, each flavonoid was individually depleted, and equivalence to MIX20 was assessed using two one-sided tests (TOSTs) with prespecified 80–125% bounds. Most leave-one-out mixtures were statistically equivalent to MIX20 across cell-free antioxidant assays, cellular oxidative damage, and antioxidant response element reporter activation, indicating substantial functional redundancy. Second, we generated 20 biased mixtures, each containing one flavonoid at 50% of the total flavonoid concentration. Their retained bioactivities across assays supported resilience to substantial compositional changes. The generality and boundaries of these findings were further examined using flavonoid subsets derived from MIX20 and an independent eight-flavonoid panel, with functional verification extended to α-glucosidase inhibition, mitochondrial membrane potential, cytotoxicity, and paraquat-induced oxidative stress in Caenorhabditis elegans. These results establish a systematic framework for assessing compositional robustness in the tested flavonoid mixtures and may provide a basis for future evaluation of other phytochemical systems.