5,7,3′,4′,5′‐Pentamethoxyflavone, a Flavonoid Monomer Extracted From Murraya paniculata (L.) Jack, Alleviates Anxiety Through the A2AR/Gephyrin/GABRA2 Pathway

The sedative and hypnotic properties of 5,7,3′,4′,5′‐pentamethoxyflavone (PMF), a monomer extracted from the leaves of Murraya paniculata (L.) Jack, have been reported. However, the role of PMFs in the development of anxiety remains uncertain. An anxiety model was developed using chronic unpredictable mild stimulation (CUMS). Kunming mice were randomly allocated to the following groups: control, CUMS, PMF (50 mg/kg), PMF (100 mg/kg), and diazepam (3 mg/kg). The anxiolytic effects of PMFs were evaluated using elevated plus maze (EPM) test and open field test (OFT). Enzyme‐linked immunosorbent assay (ELISA) kits were used to analyze the serum levels of corticosterone (CORT), 5‐hydroxytryptamine (5‐HT), gamma‐aminobutyric acid (GABA), and cyclic adenosine monophosphate (cAMP) in the hippocampus. High‐throughput‐16S rRNA sequencing was performed to investigate its effect on the composition of the gut microbiota. Subsequently, western blotting was performed to assess the expression of GABAergic synaptic‐associated proteins. PMF effectively mitigated CUMS‐induced anxiety‐like behavior. Further examination revealed that PMF treatment ameliorated dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis and increased 5‐HT and GABA levels in the hippocampus. Notably, the ability of PMF to maintain the stability of GABAergic synapses by enhancing the species composition of the gut microbiota and acting on the adenosine a2a receptor (A2AR)/gephyrin/gamma‐aminobutyric acid A receptor alpha 2 (GABRA2) pathway revealed a previously unrecognized mechanism for the anxiolytic effect of PMF. These findings suggest that PMF enhances the expression of A2AR, preserves GABAergic synaptic stability, and reduces anxiety by modulating the microbiota composition. Thus, it holds promise as an anxiolytic agent.