Integrated Approach Reveals Fermented Moringa oleifera Leaves Extracts’ Impact on Mouse Sleep

Sleep disturbances are linked to metabolic and neurological dysregulation. Moringa oleifera leaves, rich in bioactive compounds, may improve sleep via gut–brain axis modulation. This study investigated the sleep-enhancing effects of fermented Moringa oleifera leaf extract (FM) in mice using metabolomics, gut microbiota analysis, network pharmacology, and molecular docking. A 1:1 combination of Lactobacillus plantarum GDMCC 1.2685 and L. swissii GDMCC 1.791 optimally fermented FM, increasing GABA by 1.67-fold and total amino acids to 46,058.20 ± 845.53 μg/g. FM shortened sleep latency, increased sleep duration, and elevated brain GABA while reducing glutamate (Glu) and Glu/GABA ratios. Hypothalamic metabolomics identified seven sleep-related metabolites, implicating glycerophospholipid, tryptophan, and purine metabolism pathways. FM also reduced Mycobacterium anisopliae (a gut bacterium associated with insomnia) and increased the Firmicutes/Bacteroidetes ratio. Network pharmacology revealed that FM’s effects were mediated via GABA, Glu, and serotonin (5-HT) pathways. These findings demonstrate that FM improves sleep by modulating hypothalamic neurotransmitters and gut microbiota, exerting sedative-hypnotic effects through amino acid, purine, and energy metabolism.