Glabridin Hypnosis in Zebrafish Larvae Is Associated With Effects on Multiple Anesthetic Target Receptors

Abstract

BACKGROUND: R-Glabridin is a major flavonoid of licorice (Glycyrrhiza glabra) root and known to modulate GABAA receptors, which are targets of many clinical hypnotics. However, R-glabridin hypnotic activity has not been reported in animals. METHODS: Inverted photomotor responses (IPMRs) were used to assess the hypnotic effects of natural R-glabridin and synthetic R/S-glabridin in wild-type zebrafish larvae and transgenic larvae lacking functional GABAA receptor β3 subunits (β30/0). Two-electrode voltage-clamp electrophysiology in Xenopus oocytes heterologously expressing ion channels quantified the effects of R-glabridin on wild-type and mutated human α1β3γ2L GABAA receptors, NR1B/NR2A N-methyl-D-aspatate (NMDA) receptors, and α4β2 neuronal nicotinic (nnACh) receptors. RESULTS: IPMRs in wild-type zebrafish larvae identified R/S-glabridin as an inhibitor (IC50 = 7.5 µM; 95% confidence interval [CI], 5.9–9.3 µM) that was about half as potent as R-glabridin (IC50 = 4.4. µM; 95% CI, 3.6–5.4 µM). In β30/0 zebrafish larvae, R-glabridin inhibited IPMRs with IC50 = 7.5 µM (95% CI, 5.6–10.0 µM). Electrophysiologic studies revealed that R-glabridin directly activated and positively modulated α1β3γ2L GABAA receptors. Modulation was significantly reduced by α1L232W and β3N265M mutations in the β+/α- transmembrane intersubunit sites where etomidate binds, but not by 5 other point mutations in 4 other transmembrane modulator binding sites. NMDA and nnACh receptors were inhibited by R-glabridin. DISCUSSION/CONCLUSIONS: Our findings in zebrafish larvae indicate that IPMR inhibition by R-glabridin is more potent than S-glabridin and that β3-containing GABAA receptors contribute significantly to this behavioral effect. Molecular studies show that R-glabridin modulates at least 3 known anesthetic-sensitive ion channels, suggesting that it is a multimodal hypnotic.