Characterization of the Composition of Bioactive Fractions from Flowers That Protect against HO-Induced Oxidative Damage through the PI3K/AKT/Nrf2 Pathway.

flowers (DOF) have previously been established as a promising source of natural antioxidants, and it is ideally suited for processing to prepare functional foods and food additives. The precise extraction processes employed, however, can alter the composition and antioxidant properties of the resultant products, and the characteristic compounds associated with the active fractions prepared from DOF or their mechanisms of action have yet to be reported. To clarify the molecular mechanisms through which these active fractions function for the first time, chromatography was used to separate DOF extracts, yielding five fractions (Fr. (a-e)). Analyses of the antioxidant activity for these different fractions revealed that Fr. (d) presented with the most robust bioactivity. Levels of total flavonoids were then measured, revealing that antioxidant activity levels were positively correlated with total flavonoid content. Fr. (d) was found to contain 20 flavonoids in HPLC-Triple-TOF-MS/MS analyses. At the cellular level, Fr. (d) was found to induce increases in the levels of protective antioxidant factors (SOD and GSH-Px) while reducing the levels of reactive oxygen species (ROS), damage-associated factors (MDA, NO, TNF-α, IL-1β, and IL-6), and inducible nitric oxide synthase (iNOS) expression in C2C12 cells that had been stimulated with HO. These data thus provided support for Fr. (d) prevention of oxidative stress and inflammation. Network pharmacology analyses further suggested that Fr. (d) can help protect against oxidative stress through its effects on PI3K/Akt-related signaling activity. Fr. (d) was subsequently found to upregulate PI3K/Akt pathway-related proteins, nuclear transcription factor 2 (Nrf2), and heme oxygenase 1 (HO-1) in addition to suppressing Kelch-like epoxide-related protein 1 (Keap1) expression. In summary, Fr. (d) was found to suppress PI3K/Akt/Nrf2 pathway activation, ultimately alleviating inflammation and oxidative stress as predicted with a network pharmacology approach. Future studies aimed at clarifying the composition and mechanistic activity of DOF Fr. (d) will likely help establish it as a functional food capable of promoting health and longevity.