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

ETHNOPHARMACOLOGICAL RELEVANCE: 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 GABAA receptors (GABAARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABAARs 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 GABAAR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation. MATERIALS AND METHODS: 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 GABAARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABAARs 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. RESULTS: 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 GABAARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, Ki = 0.59 µM), a flavone that binds to recombinant α1-3/5 subunit-containing GABAARs, 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 GABAARs. CONCLUSIONS: This present study indicated that the flavones, especially DMS, are novel GABAAR ligands and therapeutic potential candidates for anxiety.

Flavonoids isolated from Tibetan medicines, binding to GABAA receptor and the anticonvulsant activity.

BACKGROUND: Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders. PURPOSE: The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAA) receptor complex, and to search for anticonvulsant compounds without undesirable effects such as myorelaxation and sedation. STUDY DESIGN AND METHODS: The affinities of these flavonoids for the BZ-S of GABAA receptors were determined by [3H]flunitrazepam binding to mouse cerebellum membranes in vitro. And the anticonvulsant, myorelaxant and sedative effects were determined by pentylenetetrazol (PTZ)-induced seizure and electrogenic seizure protection, rotarod test and locomotor activity test, respectively. RESULTS: Fifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAA receptors. Furthermore, 2',4',5,7-tetrahydroxy-5',6-dimethoxyflavone (DMF, IC50 value of 0.10 µM), a flavone isolated from ACN, presented high anticonvulsant activity against chemical-induced seizures and electrogenic seizures, without myorelaxation and sedation. CONCLUSION: This study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.

Dihydromyricetin as a novel anti-alcohol intoxication medication.

Alcohol use disorders (AUDs) constitute the most common form of substance abuse. The development of AUDs involves repeated alcohol use leading to tolerance, alcohol withdrawal syndrome, and physical and psychological dependence, with loss of ability to control excessive drinking. Currently there is no effective therapeutic agent for AUDs without major side effects. Dihydromyricetin (DHM; 1 mg/kg, i.p. injection), a flavonoid component of herbal medicines, counteracted acute alcohol (EtOH) intoxication, and also withdrawal signs in rats including tolerance, increased anxiety, and seizure susceptibility; DHM greatly reduced EtOH consumption in an intermittent voluntary EtOH intake paradigm in rats. GABA(A) receptors (GABA(A)Rs) are major targets of acute and chronic EtOH actions on the brain. At the cellular levels, DHM (1 µM) antagonized both acute EtOH-induced potentiation of GABA(A)Rs and EtOH exposure/withdrawal-induced GABA(A)R plasticity, including alterations in responsiveness of extrasynaptic and postsynaptic GABA(A)Rs to acute EtOH and, most importantly, increases in GABA(A)R α4 subunit expression in hippocampus and cultured neurons. DHM anti-alcohol effects on both behavior and CNS neurons were antagonized by flumazenil (10 mg/kg in vivo; 10 µM in vitro), the benzodiazepine (BZ) antagonist. DHM competitively inhibited BZ-site [(3)H]flunitrazepam binding (IC(50), 4.36 µM), suggesting DHM interaction with EtOH involves the BZ sites on GABA(A)Rs. In summary, we determined DHM anti-alcoholic effects on animal models and determined a major molecular target and cellular mechanism of DHM for counteracting alcohol intoxication and dependence. We demonstrated pharmacological properties of DHM consistent with those expected to underlie successful medical treatment of AUDs; therefore DHM is a therapeutic candidate.