Proceedings of the British Pharmacological Society at http://www.pA2online.org/abstracts/Vol10Issue3abst006P.pdf

P689

Saffron extract and trans-crocetin inhibits excitotoxicity by inhibition of postsynaptically located glutamate receptors in rat brain neurons

Karen Nieber 1, Frauke Berger 1, Andreas Hensel 2. 1University of Leipzig, Institute of Pharmacy, 04103 Leipzig, Germany, 2University of Münster, Institute of Pharmaceutical Biology and Phytochemisty, 48149 Münster, Germany

Excessive glutamate levels have been detected following brain trauma or cerebral ischemia, resulting in unregulated stimulation of NMDA receptors. These conditions are thought to elicit a cascade of excitation-mediated neuronal damage. NMDA receptor antagonists are well established in the treatment of these disorders, but adverse CNS effects limit their use. A now approach is saffron. Saffron, the dried stigmata of Crocus sativus L., has been used for centuries as an herbal remedy for various central and peripheral diseases such as depression and gastrointestinal mal function. Recent studies have revealed neuroprotective effect of the herbal drug and some of the secondary lead compounds. A new approach has resulted for radioligand binding studies indicating that the hydro- ethanolic (80 vol.-%) Crocus sativus L extract (CSE) and isolated trans-crocetin show an affinity to the phencyclidine binding site of the NMDA receptor (Lechtenbegr et al., 2008) Since no detailed pharmacological mechanisms are available to explain the binding experiments, this study was due to elucidate neuroprotective properties and to examine the cellular targets of CSE and trans-crocetinon neuronal cells. Rat neuroblastom cell line B104 possessed functional ionotopic glutamate receptors. In MTT and LDH assays the treatment of the cells with glutamate (0.1-20 mM) for 24 h decreased metabolic activity and increased cell death, respectively, both in a concentration-dependent manner. When cells were treated with glutamate (10 mM) in the presence of CSE (10-500 µg/ml) or trans- cocetin (1-50 µM), a concentration-dependent inhibition of the glutamate-induced excitotoxicity was found. To evaluate the underlying mechanisms membrane potential, input resistance and postsynaptic potentials (PSPs) were recorded using intracellular placed microelectrodes in pyramidal cells of the rat cingulate cortex. CSE (100 µg/ml) and trans-cocetin (10 µM) inhibited the PSPs by approximately 27%. To investigate the influence of CSE and trans-crocetin on the NMDA and the non-NMDA component of the PSPs, the components were isolated by application of specific antagonists. Glutamate added into the organ bath induced a membrane depolarization. CSE (10-200 µg/ml) decreased the glutamate-induced membrane depolarization and inhibited the evoked PSPs. Additionally, CSE (100 µg/ml) inhibited the isolated NMDA and non-NMDA component of the PSPs and decreased the NMDA (20 µM) and kainate (1 µM) induced depolarization, whereas the AMPA (1 µM) induced depolarization was not affected. Trans-crocetin (1-50 µM) showed inhibitory effects on the glutamate-induced membrane depolarization and evoked PSPs comparable to CSE. Trans- crocetin (10 µM) decreased the NMDA (20 µM) induced membrane depolarisation, but compared to CSE trans-crocetin did not inhibit the isolated non-NMDA component of the PSPs. Our study indicates differences between CSE and trans-crocetin in regulation the glutamatergic neurotransmission in the rat cingulated cortex. The apparent differences between CSE and trans-cocetin may be explained by participation of other current unidentified constituents in the multi-component mixture CES. In conclusion, this is, to our knowledge, the first report to provide evidence of the inhibition of postsynaptically located NMDA and kainite receptors by a hydro-ethanolic Crocus sativus L extract, which is partly mediated by trans-cocetin. These mechanisms contribute to the neuroprotective effect of saffron.