Assessment of the Acute Toxicity and Anxiolytic-Like Effect of α-Humulene in Adult Zebrafish (Danio rerio)

The sesquiterpene α-humulene (alpha-humulene, humulene, or HUM) is recognized for its anti-inflammatory actions. However, its pharmacological potential in the central nervous system (CNS) remains largely unexplored. In this study, we evaluated the acute toxicity and behavioral responses of adult zebrafish (Danio rerio) to intraperitoneal (IP) injection of HUM. The behavioral parameters examined were number of line crossings (open-field test) and time spent in the light zone of the tank (light/dark test). Then were performed experiments with standard antagonists to investigate the possible mechanism of action. Humulene exhibited low acute toxicity (LD₅₀ > 40 mg kg^-1, IP) and produced a maximal anxiolytic-like effect at an intermediate dose of 20 mg kg^-1 IP. The pharmacological dissection revealed that this effect was independent of the benzodiazepine (B_ZD) allosteric binding site on the gamma-aminobutyric acid (GABA) type A receptor (GABA_AR), but it was completely reversed by granisetron (G_RAN), a serotonin (5-HT) type 3 receptor (5-HT₃R) antagonist. Further in silico analysis revealed a low-affinity binding and an allosteric interaction of α-humulene, respectively, with GABA_AR and 5-HT₃R channels. Together, these results suggest a noncanonical mechanism involving both GABAergic and serotonergic systems.