Natural Synergistic Anti‐Fatigue Formula Development via Zebrafish Model and Combination Index Method

ABSTRACT Many plant and fungal extracts (PFEs) show anti‐fatigue properties through various pathways, but synergistic formulas and mechanisms are unclear. We screened six PFEs using a sodium sulfite (Na 2 SO 3 )‐induced exercise fatigue zebrafish model and developed a synergistic anti‐fatigue formula based on the combination index (CI) method. Red ginseng (RG), Lycium ruthenicum (LR) and Ganoderma lucidum (GL) extracts were selected as formulation materials based on their potential synergistic possibilities. The formula demonstrated a strong synergistic effect on recovery rate with a CI of 0.04 when RG, LR, and GL were mixed at concentrations of 3.6, 4.0, and 14 μg/mL, respectively. Ginsenosides Re, LR anthocyanins and GL polysaccharides are the main anti‐fatigue components. The contents of ATP and SOD were increased by 39.1% and 53.9%, while lactic acid and malondialdehyde (MDA) contents were decreased by 31.3% and 15.6%. Zebrafish swimming ability was restored by 97.6% relative to the control group, demonstrating a fatigue recovery effect comparable to that of Red Bull. The formula exerts anti‐fatigue effects by regulating muscle contraction, energy metabolism and oxidative stress at the relative rRNA expression. These findings highlight the promising potential of rationally designed PFEs combinations for functional food.