In vitro Study of the Interaction of Tilia europeae L. Aqueous Extract with GABA A Receptors in Rat Brain C. Cavadas  , C. A. Fontes Ribeiro†, M. S. Santos‡, A. P. Cunha§, T. Macedo†, M. M. Caramona and M. D. Cotrim Laboratory of Pharmacology, Faculty of Pharmacy, †Department of Pharmacology, IBILI, Faculty of Medicine, §Laboratory of Pharmacognosy, Faculty of Pharmacy, ‡ Center for Neurosciences of Coimbra, Department of Zoology, University of Coimbra, 3000 Coimbra, Portugal The interaction of GABA A receptor-complex in rat brain was investigated in vitro with aqueous extracts obtained from the inflorescences of Tilia europeae L., using the [ 3 H]muscimol and [ 3 H]flunitrazepam binding techniques to synaptic membranes and the uptake of 36 Cl - to synaptoneurosomes from cortices. The extract inhibited [ 3 H]muscimol binding, stimulated 36 Cl - uptake by synaptoneurosomes and displaced at high concentrations, the [ 3 H]flunitrazepam bound to synaptic membranes. When analysed by HPLC, the aqueous extract of Tilia europeae L. contained several amino acids, including GABA (about 100 M). This GABA content can justify the displacement of [ 3 H]muscimol produced by the extract but it did not increase the binding of [ 3 H]flunitrazepam, as expected. Probably the extract contains other benzodiazepine-like substances which displace the [ 3 H]fluni- trazepam binding and counteract the expected GABA-induced increase in [ 3 H]flunitrazepam binding. © 1997 by John Wiley & Sons, Ltd. Keywords: Tilia europeae; amino acid content; GABA A receptor; rat brain. INTRODUCTION Aqueous extracts of inflorescences of Tilia species are used in folklore medicine as tranquilizers or mild sedative substances (Costa, 1986). However, their mechanism of action in the central nervous system is not known. It is well known that sedation in the central nervous system is mainly mediated by the GABA/benzodiazepine/chloride ionophore receptor complex (GABA A receptor-complex) (Biggio et al., 1989). The GABA A receptors, which can be alloster- ically modulated by benzodiazepines or barbiturates, mediate an inhibitory effect by increasing Cl - influx, which induces membrane hyperpolarization and neuronal inhibi- tion (Cooper et al., 1991; Sieghart, 1992). The aim of our study was therefore to study the interaction of the aqueous extract of Tilia europeae inflorescences with the GABA A receptor-complex in rat brain. The affinity of the extract for the competitive and the benzodiazepines modulatory sites was studied, respectively, by the evaluation of the binding of [ 3 H]muscimol, a GABA A receptor specific agonist, and [ 3 H]flunitrazepam to synaptic membranes. Since the measurement of 36 Cl - uptake by synaptoneur- osomes has been used extensively to evaluate drug action at the GABA A receptor complex (Allan and Harris, 1986), the effects of Tilia extracts on 36 Cl - uptake by synaptoneur- osomes of rat cortices were also studied. Synaptoneurosomes are vesicular structures containing both pre- and post-synaptic membranes which form closed vesicles that are able to maintain a membrane potential (Hollingsworth et al., 1985; DeLorey and Brown, 1992). MATERIALS AND METHODS Materials. [Methylamine- 3 H]muscimol (specific activity 7.3 Ci/mmol), [N-methyl- 3 H]flunitrazepam (specific activ- ity 1.85 Ci/mmol), 35 Cl - (12.8 mCi/g) were obtained from Amersham International, Buckinghamshire, UK; HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), gamma-aminobutyric acid (GABA), picrotoxin, rutin, caffeic acid, 2,5-difeniloxazol (PPO), p-bis[2(5- feniloxazoil)]-benzeno (POPOP) from Sigma Chemical Co, St Louis, MO, USA; Muscimol from Research Bio- chemicals Incorporated, Natick, MA, USA; Triton X-100 from Merck, Germany. Extract preparation. Tilia europeae L. inflorescences were obtained at the Botanic Garden of Coimbra and identified by the Botanic Institute of the University of Coimbra. The inflorescences were dried at room temperature and then kept at - 20°C. To prepare the aqueous extract, the dried material was crushed in a mechanical mill and passed through a 20 mesh sieve. The resulting powder (3 g) was resuspended in 100 mL of boiled distilled water; after cooling, the infusion was filtered under vacuum. The content of hydrocinnamic acids (caffeic acid as standard) and flavonoids (rutin as standard) was determined as described by Lamaison et al. (1991) and Lamaison and Carnat (1990), respectively. The extracts used in the experiments contained 0.84 ± 0.05 mg of caffeic acid per mL and 0.11 ± 0.03 mg of rutin per mL of extract. Preparation of crude synaptic membranes. Crude synaptic membranes were isolated from rat brain cortices as described previously (Cavadas et al., 1995). Briefly, rats were decapitated and the cortices rapidly removed and  Author to whom correspondence should be addressed. CCC 0951–418X/97/010017–05 © 1997 by John Wiley & Sons, Ltd. Accepted (revised) 31 May 1996 PHYTOTHERAPY RESEARCH, VOL. 11, 17–21 (1997)