Luteolin as a major anti-fatigue compound in coix seed seedling flavonoids: oxidative stress alleviation and intestinal microbiota modulation.

Our previous studies have identified an active coix seed seedling extract (CSSE) with anti-fatigue potential, yet its anti-fatigue material basis remains to be fully elucidated. Here, a secondary-purified flavonoid of CSSE (PPCSSE) was prepared and characterized. The major anti-fatigue compound of PPCSSE was identified by liquid chromatography-tandem mass spectrometry, network pharmacology and molecular docking. Results showed that the flavonoid purity of PPCSSE reached 75.05 ± 0.11%. Four candidate compounds in PPCSSE (luteolin, 4-coumaric acid, caffeic acid and isoferulic acid) were identified as its potential anti-fatigue compounds targeting the PI3K-AKT pathway, among which luteolin exhibited the strongest binding affinity for the pathway's core targets. In vitro, luteolin alleviated H2O2-induced oxidative damage in C2C12 myoblasts, restored cell viability and enhanced ATPase activity. In vivo, luteolin also markedly enhanced exercise endurance and energy metabolism, dramatically mitigated fatigue-induced injury and effectively reversed intestinal microbiota imbalance in mice, which was related to the activation of the PI3K-AKT/mTOR pathway. These findings demonstrated that luteolin served as the major anti-fatigue active component in PPCSSE, providing a theoretical basis for the development of functional anti-fatigue products.