The dietary flavonoids apigenin and ()-epigallocatechin gallate enhance the positive modulation by diazepam of the activation by GABA of recombinant GABA A receptors Erica L. Campbell a , Mary Chebib b , Graham A.R. Johnston a, * a Department of Pharmacology, The University of Sydney, Sydney 2006, NSW, Australia b Faculty of Pharmacy, The University of Sydney, Sydney 2006, NSW, Australia Received 4 June 2004; accepted 8 July 2004 Abstract The dietary flavonoids apigenin, genistein and ()-epigallocatechin gallate (EGCG) inhibited the activation by GABA (40 mM) of recombinant human a1b2g2L GABA A receptors expressed in Xenopus laevis oocytes with IC 50 values of 8, 30 and 15 mM, respectively. Apigenin and genistein also acted as GABA antagonists at flumazenil-insensitive a1b2 GABA A receptors, indicating that they were not acting as negative modulators through flumazenil-sensitive benzodiazepine sites on GABA A receptors. In addition to these GABA A antagonist effects, a novel second order modulatory action was found for apigenin and EGCG on the first order enhancement of GABA responses by diazepam. Apigenin (1 mM) and EGCG (0.1 mM) enhanced the modulatory action of diazepam (3 mM) on the activation by GABA (5 mM) of recombinant human a1b2g2L GABA A receptors by up to 22% and 52%, respectively. This was not found with genistein, nor was it observed with enhancement by allopregnanolone or pentobarbitone. # 2004 Elsevier Inc. All rights reserved. Keywords: Apigenin; ()-Epigallocatechin gallate; Genistein; GABA A receptors; Diazepam; Flavonoids; Modulation; Herbal medicine 1. Introduction Flavonoids were first linked to GABA A receptors when three isoflavans isolated from bovine urine were shown to inhibit diazepam binding to brain membranes [1]. They have a range of activities on GABA A receptors [2], acting as positive and negative modulators of GABA A receptor function. While many of these actions are sensitive to the benzodiazepine antagonist flumazenil consistent with the ability of flavonoids to inhibit benzodiazepine binding, flumazenil-insensitive actions have also been described indicating that flavonoids can act at sites other than clas- sical benzodiazepine sites on GABA A receptors. For exam- ple, the positive modulatory effects of 6-methylflavone [3] and negative modulatory effects of amentoflavone [4] on recombinant a1b2g2L GABA A receptors expressed in oocytes are insensitive to flumazenil. Furthermore, flavo- noids are known to inhibit the action of GABA on fluma- zenil-insensitive r1 GABA C receptors [5]. The present study describes a novel action of two flavonoids, apigenin and ()-epigallocatechin gallate (EGCG), that enhance the positive modulation of the activation by GABA of recom- binant a1b2g2L GABA A receptors by diazepam. Apigenin (5,7,4 0 -trihydroxyflavone, Fig. 1) is a common flavonoid found in a range of plants, including chamomile. The traditional use of chamomile tea as a treatment for insomnia and anxiety led to investigations of its active constituents. Apigenin was proposed to be a benzodiaze- pine partial agonist and produced potent anxiolysis (and mild sedation at high doses) without any other benzodia- zepine-like effects [6]. However, Avallone et al. [7] found that apigenin fitted the profile of a benzodiazepine inverse agonist and was sedative and mildly pro-convulsant, but not anxiolytic. Dekermendjian et al. [8] reported that apigenin fitted the profile of a benzodiazepine antagonist. Thus, apigenin appears to have a variety of effects at the benzodiazepine site of the GABA A receptor, but the nature of these effects is unclear. (+)-Catechin and ()-epicatechin (Fig. 2) are the most common flavans in nature, appearing in many common www.elsevier.com/locate/biochempharm Biochemical Pharmacology 68 (2004) 1631–1638 Abbreviation: EGCG, ()-epigallocatechin gallate. * Corresponding author. Tel.: +61 2 9351 6117; fax: +61 2 9351 2891. E-mail address: grahamj@mail.usyd.edu.au (Graham A.R. Johnston). 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.07.022