Insomnia, a prevalent health challenge, often requires pharmacological interventions to improve sleep onset, maintenance, and quality. Benzodiazepines and Z-drugs, like other positive modulators, enhance the inhibitory effects of gamma-aminobutyric acid (GABA) by stabilizing the open conformation of the GABAA receptor (GABAAR) chloride ion channels, facilitating the transition to sleep. However, prolonged use raises concerns, including dependence and cognitive issues. Among herbal alternatives, Humulus lupulus (hops) is gaining attention due to its role as a natural relaxant, sleep aid, and brewing component. Neuroactive phytochemicals in hops may modulate GABAARs differently from benzodiazepines. This research uncovers these hop constituents and potential therapeutic mechanisms.

The α-acid humulone and hop prenylflavonoids (PFs), including xanthohumol/isoxanthohumol, 6/8-prenylnaringenin, enhanced GABA-induced displacement of [3H]EBOB, a GABAAR function marker, in the low micromolar range. These potent effects were flumazenil-insensitive and α6β3δ subtype-selective. Molecular docking at the α1β2γ2 isoform identified the extracellular α+/β− interface as the PF binding site. An additional 6-prenylnaringenin site was recognized at the extracellular α+/γ2− interface, aligning with its inhibition of [3H]flunitrazepam and [3H]Ro 15-4513 binding. Given humulone’s prominence and relatively high potency, its activity was confirmed electrophysiologically, where it enhanced GABA-evoked currents in the sedation-mediating α1β3γ2 subtype. In mice, humulone reduced locomotor activity, shortened sleep onset induced by pentobarbital, and prolonged sleep duration induced by either pentobarbital or ethanol. Moreover, [3H]EBOB binding assays showed synergies between humulone and ethanol, and additive interactions with PFs, suggesting enhanced alcohol intoxication in hop-rich beers.

In summary, we revealed positive modulators of GABAARs that act independently of the classical benzodiazepine site. 6-prenylnaringenin also acts as a silent modulator with the potential to block benzodiazepine responses. Humulone plays a pivotal role in the sedative and sleep-promoting properties of hops. These findings offer novel mechanistic insights into hop neuroactive constituents and potential avenues for sleep aid optimization.