B-ring methoxy substitution modulates flavokawain membrane interactions and cytotoxicity

Flavokawains are naturally occurring chalcone derivatives from Piper methysticum with promising pharmacological properties. This study investigated the biological and physicochemical characteristics of five flavokawain derivatives (A1-A5) differing in B-ring methoxy substitution patterns to elucidate structure-activity relationships. All derivatives exhibited low hemolytic activity toward human erythrocytes, indicating minimal cytotoxicity to red blood cells. Membrane interaction studies using fluorescent probes suggest preferential partitioning toward the hydrophobic core, inducing concentration-dependent rigidification, with effects most pronounced for mono-methoxy derivatives (A2, A3) and diminished for di-methoxy compounds (A4, A5). Dynamic light scattering analyses showed that A1-A3 did not markedly change liposome size or PDI, whereas A4 and A5 increased vesicle size and polydispersity, suggesting stronger membrane perturbation and/or possible aggregation-related effects. Human serum albumin (HSA) binding assays showed that all derivatives formed stable complexes, with binding affinity strongly dependent on the B-ring methoxy substitution pattern. Cytotoxicity assays using canine B-cell leukemia (CLB70) and lymphoma (CLBL-1) cell lines revealed concentration-dependent effects, with flavokawain B (A1) and 2-methoxy-flavokawain B (A2) displaying the strongest cytotoxic activity (IC₅₀ ≈ 8-16 μM). However, these compounds also showed limited selectivity toward malignant versus non-malignant canine cell lines under the tested conditions. Overall, the results identify B-ring substitution as a key determinant of membrane interactions, albumin binding, and cellular response, and provide a basis for further structural optimization of flavokawain derivatives with improved selectivity.