Flavonoids from Leptospermum scoparium with affinity to the benzodiazepine receptor characterized by structure activity relationships and in vivo studies of a plant extract.

The New Zealand Myrtacea Leptospermum scoparium Forst. contains lipophilic flavonoids which interact specifically with benzodiazepine receptors. For an indepth characterization of their binding behavior, structure activity relationships were delineated which are in accord with results obtained by quantum-chemical and spectroscopic methods. Inhibition experiments have been performed by a radio receptor assay with [3H]flunitrazepam and IC50-values of 2.1 microM for 5,7-dimethoxyflavone (1), 45 microM for 5,7-dimethoxy-6-methylflavone (2), 3.3 microM for 5-hydroxy-7-methoxy-6-methylflavone (3) and 40 microM for 5-hydroxy-7-methoxy-6,8-dimethylflavone (4) have been measured. Flavanones 5 to 8, however, at concentrations < or = 0.1 mM, did not show a 50% inhibition of the binding radiolignand. The agonistic profile of the flavones was determined indirectly by TBPS-shift experiments which revealed a negative cooperation with the TBPS/picrotoxinin-binding site. To characterize the biologically active conformations, energy minima were calculated using the semiempirical method AM1. The steric arrangement of the substituents for all global minima calculated were in accord with homonuclear NOE-experiments. A correlation of the geometry of the lowest energy conformers with corresponding IC50-values reveals an increase of the affinity towards the benzodiazepine receptor, when the substituents at the flavones are coplanar to the aromatic system and R3 represents a sterically demanding methylgroup. Analyses of the global minima of 5,7-Dimethoxyflavone and diazepam showed one conformer each, in which the methoxy substituent in R3 and the N-methyl on the one hand and the corresponding carbonyl oxygens as well as the unsubstituted phenyl rings on the other were nearly superimposable. The flavanones lacking the double bond between C-2 and C-3 have angular structures, whereby the loss of affinity to the receptor can be explained. From the locomotion study with rats, an in vivo sedating, possibly even anxiolytic effect of the dry extract of the tincture prepared from Leptospermum scoparium by use of 70% ethanol, could be concluded. At doses of 50 mg and 250 mg of the dry extract per kg of body weight, an unequivocal but not linear dose-activity relationship in respect to the moving activities of the animals was determined. Upon an application of 500 mg of this extract per kg body weight, by contrast, only a negligible reduction of the moving activity was found in relation to a control group. We suppose that at higher doses, activating compounds of the extract come to the fore pharmacologically neutralizing the primarily sedating effect.