Flavonoid compounds isolated from Tibetan herbs, binding to GABA_A receptor with anxiolytic property

Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABA_A receptors (GABA_ARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABA_ARs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABA_AR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation.

The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABA_ARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABA_ARs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids.

Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABA_ARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, K_i = 0.59 μM), a flavone that binds to recombinant α1-3/5 subunit-containing GABA_ARs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABA_ARs.

This present study indicated that the flavones, especially DMS, are novel GABA_AR ligands and therapeutic potential candidates for anxiety.