| Summary: | <b> </b>To elucidate the mechanism of anti-ferroptosis and examine structural optimization in natural phenolics, cellular and chemical assays were performed with 2′-hydroxy chalcone butein and dihydroflavone (<i>S</i>)-butin. C11-BODIPY staining and flow cytometric assays suggest that butein more effectively inhibits ferroptosis in erastin-treated bone marrow-derived mesenchymal stem cells than (<i>S</i>)-butin. Butein also exhibited higher antioxidant percentages than (<i>S</i>)-butin in five antioxidant assays: linoleic acid emulsion assay, Fe<sup>3+</sup>-reducing antioxidant power assay, Cu<sup>2+</sup>-reducing antioxidant power assay, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO<sup>•</sup>)-trapping assay, and α,α-diphenyl-β-picrylhydrazyl radical (DPPH<sup>•</sup>)-trapping assay. Their reaction products with DPPH<sup>•</sup> were further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Butein and (<i>S</i>)-butin produced a butein 5,5-dimer (<i>m/z</i> 542, 271, 253, 225, 135, and 91) and a (<i>S</i>)-butin 5′,5′-dimer (<i>m/z</i> 542, 389, 269, 253, and 151), respectively. Interestingly, butein forms a cross dimer with (<i>S</i>)-butin (<i>m/z</i> 542, 523, 433, 419, 415, 406, and 375). Therefore, we conclude that butein and (S)-butin exert anti-ferroptotic action via an antioxidant pathway (especially the hydrogen atom transfer pathway). Following this pathway, butein and (S)-butin yield both self-dimers and cross dimers. Butein displays superior antioxidant or anti-ferroptosis action to (S)-butin. This can be attributed the decrease in π-π conjugation in butein due to saturation of its α,β-double bond and loss of its 2′-hydroxy group upon biocatalytical isomerization.
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