Calycosin

Glycitein (≥95%; CAS 40957-83-3), prunetin (≥95%; CAS 552-59-0), biochanin A (≥99%; CAS 491-80-5), formononetin (≥99%; CAS 485-72-3), and glycitin (≥95%; CAS 40246-10-4) were from Extrasynthese (Lyon, France). Daidzien (≥98%; CAS 486-66-8), genistein (≥98%, CAS 446-72-0), daidzin (≥95%; CAS 552-66-9), and genistin (≥95%; CAS 529-59-9) were from Sigma-Aldrich (St. Louis, MO, USA). Calycosin (99%; CAS 25389-94-0) was from Phytolab (Vestenbergsgreuth, Germany), and S-equol (97%; CAS 531-95-3) was from Toronto Research Chemicals (Toronto, Canada). The FLuc positive control was resveratrol (>99%; CAS 501-36-0; Sigma-Aldrich, St. Louis, MO, USA), and the RLuc positive control was H-89 (99.34%; CAS 130964-39-5; MedChemExpress, Monmouth Junction, NJ, USA). The isoflavonoids were dissolved in cell-grade dimethylsulfoxide (DMSO; 99.9%; CAS 67-68-5; Sigma-Aldrich, St. Louis, MO, USA) and serially diluted to prepare 100× stock solutions. All of the isoflavonoids were screened in the in vitro FLuc and RLuc inhibition assays at 1, 10, and 100 µM final concentrations. Dose–response curves were generated for the active isoflavonoids and for the positive controls to determine the IC₅₀ values.