Effect and mechanism of wedelolactone as antioxidant-coumestan onOH-treated mesenchymal stem cells

The antioxidant properties of coumestans have been investigated previously using inappropriate methods, which has resulted in misleading and inaccurate mechanisms being reported to account for their antioxidant behavior. In this study, the phenolic coumestan wedelolactone increased the viability of OH -treated mesenchymal stem cells (MSCs) in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl assay. Mechanistic analysis in vitro suggested that wedelolactone could scavenge various radicals, including OH , O 2 - , 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl ( DPPH ) radicals, as well as reducing Cu2+ and chelating Fe2+. Configuration analysis based on a ball and stick model indicated that the 3′,4′-catechol moiety of wedelolactone was acting as an Fe2+-chelating site. The main product of the reaction between wedelolactone and DPPH was analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry, which gave a mass ion with an m/z value of 709. This peak yielded several fragments with m/z values of 663, 543, 501 and 483. Quantitative evaluation revealed that the antioxidant activity of wedelolactone was 1.62 times higher than that of Trolox. Based on these results, we concluded that (i) wedelolactone can efficiently protect MSCs against OH -induced damage and that this protective effect provides preliminary evidence for the application of wedelolactone in transplantation of MSCs (especially for osteoporosis); (ii) the main antioxidant mechanism of wedelolactone involves direct radical-scavenging via a single electron transfer (SET) → radical adduct formation (RAF) pathway, whereas the Fe2+-chelating activity of wedelolactone represents a minor pathway; and (iii) the direct radical-scavenging and Fe2+-chelating pathways can both be attributed to the catechol moiety rather than the coumestan skeleton.