The anxiolytic-like effects of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) in mice.

The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts of Aloysia polystachya (Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0 mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0 mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolic extract also significantly increased the percentage of both entries (1.0 and 100.0 mg/kg) and the time spent (10.0 and 100.0 mg/kg) into the open arms of the elevated plus maze (EPM). Nevertheless, the binding of (3)H-flunitrazepam ((3)H-FNZ) to the benzodiazepine binding site (BDZ-bs), in washed crude synaptosomal membranes from rat cerebral cortex, was not affected by the semi-purified components from Aloysia polystachya. These results indicate an anxiolytic-like profile of action for the extract of Aloysia polystachya without sedative side effect, being this activity probably mediated by other mechanism than BDZ-bs modulation at the GABA(A) receptors.

Molecular modeling and QSAR analysis of the interaction of flavone derivatives with the benzodiazepine binding site of the GABA(A) receptor complex.

A large number of structurally different classes of ligands, many of them sharing the main characteristics of the benzodiazepine (BDZ) nucleus, are active in the modulation of anxiety, sedation, convulsion, myorelaxation, hypnotic and amnesic states in mammals. These compounds have high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Since 1989 onwards our laboratories established that some natural flavonoids were ligands for the BDZ-bs which exhibit medium to high affinity in vitro and anxiolytic activity in vivo. Further research resulted in the production of synthetic flavonoid derivatives with increased biochemical and pharmacological activities. The currently accepted receptor/pharmacophore model of the BDZ-bs (Zhang, W.; Koeler, K. F.; Zhang, P.; Cook, J. M. Drug Des. Dev. 1995, 12, 193) accounts for the general requirements that should be met by this receptor for ligand recognition. In this paper we present a model pharmacophore which defines the characteristics for a ligand to be able to interact and bind to a flavone site, in the GABA(A) receptor. closely related to the BDZ-bs. A model of a flavone binding site has already been described (Dekermendjian, K.; Kahnberg, P.; Witt, M. R.; Sterner, O.; Nielsen, M.; Liljerfors, T. J. Med. Chem. 1999, 42, 4343). However, this alternative model is based only on graphic superposition techniques using as template a non-BDZ agonist. In this investigation all the natural and synthetic flavonoids found to be ligands for the BDZ-bs have been compared with the classical BDZ diazepam. A QSAR regression analysis of the parameters that describe the interaction demonstrates the relevance of the electronic effects for the ligand binding, and shows that they are associated with the negatively charged oxygen atom of the carbonyl group of the flavonoids and with the nature of the substituent in position 3'.

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone: new additions to the 6,3'-disubstituted flavone family of high-affinity ligands of the brain benzodiazepine binding site with agonistic properties.

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.

6-Chloro-3'-nitroflavone is a potent ligand for the benzodiazepine binding site of the GABA(A) receptor devoid of intrinsic activity.

6-Chloro-3'-nitroflavone integrates a list of nearly 70 flavone derivatives synthesized in our laboratories. The effects of 6-chloro-3'-nitroflavone on the benzodiazepine binding sites (BDZ-BSs) of the GABA(A) receptor were examined in vitro and in vivo. 6-Chloro-3'-nitroflavone inhibited the [3H]flunitrazepam ([3H]FNZ) binding to rat cerebral cortex membranes with a Ki of 6.68 nM and the addition of GABA to extensively washed membranes did not modify its affinity for the BDZ-BSs (GABA-shift = 1.16+/-0.12). The binding assays performed in rat striatal and cerebellar brain membranes showed that this compound has similar affinity to different populations of BDZ-BSs. Electrophysiological experiments revealed that 6-chloro-3'-nitroflavone did not affect GABA(A)-receptors (GABA(A)-Rs) responses recorded in Xenopus oocytes expressing alpha1beta2gamma2s subunits, but blocked the potentiation exerted by diazepam (DZ) on GABA-activated chloride currents. In vivo experiments showed that 6-chloro-3'-nitroflavone did not possess anxiolytic, anticonvulsant, sedative, myorelaxant actions in mice or amnestic effects in rats; however, 6-chloro-3'-nitroflavone antagonized diazepam-induced antianxiety action, anticonvulsion, short-term, and long-term amnesia and motor incoordination. These biochemical, electrophysiological, and pharmacological results suggest that 6-chloro-3'-nitroflavone behaves as an antagonist of the BDZ-BSs.

6-Methyl-3'-bromoflavone, a high-affinity ligand for the benzodiazepine binding site of the GABA(A) receptor with some antagonistic properties.

6-Methyl-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the GABA(A) receptor (BDZ-bs) with Ki values between 10 and 50 nM in different brain regions. The GABA ratio of 1.03 for [(3)H]flunitrazepam binding to cerebral cortex, 0.76 for cerebellum, 0.7 for hippocampus, 0.7 for striatum, and 0.8 for spinal cord indicated an antagonistic or weak inverse agonistic profile of 6-methyl-3'-bromoflavone on BDZ-bs. Unlike classical benzodiazepines, it had no anticonvulsant, anxiolytic, myorelaxant, sedative, amnestic or motor incoordination effects. However, it antagonized the muscle relaxant, the sedative effect, and the changes in locomotor activity induced by diazepam. Taken together, these findings suggest that 6-methyl-3'-bromoflavone has an antagonistic profile on the BDZ-bs.

Flavonoids and the central nervous system: from forgotten factors to potent anxiolytic compounds.

The list of activities of plant flavonoids did not include effects on the central nervous system (CNS) up to 1990, when our laboratory described the existence of natural anxiolytic flavonoids. The first of these was chrysin (5,7-dihydroxyflavone), followed by apigenin (5,7,4'-trihydroxyflavone) and flavone itself. Semisynthetic derivatives of flavone obtained by introducing halogens, nitro groups or both in its molecule, give rise to high affinity ligands for the benzodiazepine receptor, active in-vivo; 6,3'-dinitroflavone, for example, is an anxiolytic drug 30 times more potent than diazepam. The data collected in this paper make clear that some natural flavonoids are CNS-active molecules and that the chemical modification of the flavone nucleus dramatically increases their anxiolytic potency.

Pharmacological characterization of 6-bromo-3'-nitroflavone, a synthetic flavonoid with high affinity for the benzodiazepine receptors.

6-Bromo-3'-nitroflavone is a synthetic flavone derivative that selectively recognizes benzodiazepine receptors and has potent anxiolytic-like effects. Here, we describe in detail its pharmacological characterization. When i.p. injected in mice, 6-bromo-3'-nitroflavone (0.01-0.3 mg/kg) had an anxiolytic-like effect in the elevated plus-maze test. This effect was blocked by the specific benzodiazepine receptor antagonist, flumazenil. In addition, it exhibited anxiolytic-like actions when given orally (1 mg/kg). 6-Bromo-3'-nitroflavone did not exhibit myorelaxant effects (up to 30 mg/kg, i.p.). Unlike diazepam, this flavonoid produced no anterograde amnesia in a one-trial inhibitory avoidance learning. On the other hand, 6-bromo-3'-nitroflavone possessed mild anticonvulsant activity (0.1 mg/kg, i.p.) and provoked sedative-depressant actions only at doses 100-1000 times higher than those producing anxiolytic-like effects. 6-Bromo-3'-nitroflavone (0.1-1 mM) produced a lower potentiation of gamma-amino-butyric acid (GABA)-stimulated 36Cl- influx (126-138%) in comparison to diazepam (0.1 mM: 166%) in cerebral cortical membrane vesicles. Taken together, these findings suggest that 6-bromo-3'-nitroflavone has anxiolytic-like action possibly behaving as a partial agonist of the benzodiazepine receptors.

Detection of benzodiazepine receptor ligands in small libraries of flavone derivatives synthesized by solution phase combinatorial chemistry.

Solution phase combinatorial synthesis of flavone derivatives and evaluation of their affinity for the central benzodiazepine receptors is described. The libraries preparation is simple and provides a convenient method for rapid compound generation and screening. Thirty one new compounds were obtained of which the most promising, as high affinity benzodiazepine receptor ligands, were 6-bromo-3'-fluoroflavone; 6,3'-dichloroflavone; 6-bromo-3'-chloroflavone and 6-chloro-3'-bromoflavone.

Neuroactive flavonoids: new ligands for the Benzodiazepine receptors.

Flavonoids isolated from plants used as tranquilizers in folkloric medicine have a selective affinity, for central benzodiazepine receptors (BDZ-Rs) and some of them possess a pharmacological profile compatible with a partial agonist action. Synthetic derivatives of the common flavone nucleus, give rise to high affinity ligands when electronegative groups are introduced in carbons 6 and/or 3'. Representative compounds such as 6,3'-dinitroflavone and, 6-bromo-3'-nitroflavone exhibit a high affinity for the BDZ-Rs (Ki = 1.5 to 30 nM) and have anxiolytic effects not associated with myorelaxant, sedative or amnesic actions. These compounds or similar ones, could lead to improved therapeutic drugs in the treatment of anxiety.

Overview--flavonoids: a new family of benzodiazepine receptor ligands.

Benzodiazepines (BDZs) are the most widely prescribed class of psychoactive drugs in current therapeutic use, despite the important unwanted side-effects that they produce such as sedation, myorelaxation, ataxia, amnesia, ethanol and barbiturate potentiation and tolerance. Searching for safer BDZ-receptor (BDZ-R) ligands we have recently demonstrated the existence of a new family of ligands which have a flavonoid structure. First isolated from plants used as tranquilizers in folkloric medicine, some natural flavonoids have shown to possess a selective and relatively mild affinity for BDZ-Rs and a pharmacological profile compatible with a partial agonistic action. In a logical extension of this discovery various synthetic derivatives of those compounds, such as 6,3'-dinitroflavone were found to have a very potent anxiolytic effect not associated with myorelaxant, amnestic or sedative actions. This dinitro compound, in particular, exhibits a high affinity for the BDZ-Rs (Ki = 12-30 nM). Due to their selective pharmacological profile and low intrinsic efficacy at the BDZ-Rs, flavonoid derivatives, such as those described, could represent an improved therapeutic tool in the treatment of anxiety. In addition, several flavone derivatives may provide important leads for the development of potent and selective BDZ-Rs ligands.

Sedative and hypnotic properties of Salvia guaranitica St. Hil. and of its active principle, Cirsiliol.

Salvia guaranitica St. Hil. is a traditional medicinal plant used in Latin America as sedative. We have recently demonstrated the presence of cirsiliol in its extracts and found that this flavonoid is a competitive low affinity benzodiazepine receptor ligand (Marder et al., 1996). This report describes the pharmacological properties of Salvia guaranitica extracts and of its active principle, cirsiliol. A partially purified fraction of this plant, administered intraperitoneally in mice (in a dose equivalent to 3 g of the fresh plant), exhibited sedative and hypnotic effects as measured in the hole board and in the pentobarbital-induced sleep tests, respectively. On the other hand, this fraction had no anxiolytic or myorelaxant effects. In the pentobarbital-induced sleep test, cirsiliol (2-10mg/kg, i. p.) exhibited a dose-dependent hypnotic action. In contrast, it did not produce myorelaxant (up to 30mg/kg) or anticonvulsant (up to 10mg/kg) effects. Cirsiliol was found to be more potent in displacing (3)H Zolpidem binding (K(i) = 20 LiM) than (3)H flunitrazepam binding (K(i) = 200 µM) to benzodiazepine receptors from rat cerebral cortex. It is concluded that Salvia guaranitica extracts and its active principle cirsiliol, possess sedative and hypnotic properties; cirsiliol produces these effects probably acting on the so-called type I benzodiazepine receptor.

Anxioselective properties of 6,3'-dinitroflavone, a high-affinity benzodiazepine receptor ligand.

6,3'-Dintroflavone is a synthetic flavone derivative with high affinity for central benzodiazepine receptors that has anxiolytic effects. Here, we describe its biochemical and pharmacological characterization. 6,3'-Dinitroflavone inhibited differentially [3H]flunitrazepam binding to central benzodiazepine receptors in several brain regions, showing a lower Ki value in the cerebellum (central benzodiazepine receptor type I-enriched area), and a higher Ki value in the spinal cord and in the dentate gyrus (central benzodiazepine receptor type II-enriched area). When i.p. injected in mice, 6,3'-dinitroflavone had a potent anxiolytic effect in the elevated plus maze test. This effect was blocked by the specific central benzodiazepine receptor antagonist, Ro 15-1788. 6,3'-Dinitroflavone did not exhibit anticonvulsant or myorelaxant effects in mice or amnestic effects in rats. Moreover, it abolished the myorelaxant effect of diazepam. On the other hand, 6,3'-dinitroflavone possessed a mild sedative action only at doses 100-300-fold greater than the anxiolytic one. Based on these findings, we suggest that 6,3'-dinitroflavone has a benzodiazepine partial agonist profile, with low selectivity for central benzodiazepine receptor types I and II.

6-Bromoflavone, a high affinity ligand for the central benzodiazepine receptors is a member of a family of active flavonoids.

6-Bromoflavone, obtained by bromination of flavanone, binds to central benzodiazepine receptors with a Ki=70 nM and has a clear anxiolytic activity in mice, at 0.5 mg/kg i.p. A survey of the structure/affinity relationship for those receptors in a series of natural and synthetic flavonoids is presented.

Cirsiliol and caffeic acid ethyl ester, isolated from Salvia guaranitica, are competitive ligands for the central benzodiazepine receptors.

Ligands for the central benzodiazepine receptors have been detected in the crude ethanolic extract of Salvia guaranitica. The compounds responsible for the activity were isolated by bioassay-directed fractionation and have been characterised as cirsiliol (5, 3', 4'-trihydroxy 6,7-dimethoxyflavone) and caffeic acid ethyl ester.

Apigenin, a component of Matricaria recutita flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects.

The dried flower heads of Matricaria recutita L. (Asteraceae) are used in folk medicine to prepare a spasmolytic and sedative tea. Our fractionation of the aqueous extract of this plant led to the detection of several fractions with significant affinity for the central benzodiazepine receptor and to the isolation and identification of 5,7,4'-trihydroxyflavone (apigenin) in one of them. Apigenin competitively inhibited the binding of flunitrazepam with a Ki of 4 microM and had no effect on muscarinic receptors, alpha 1-adrenoceptors, and on the binding of muscimol to GABAA receptors. Apigenin had a clear anxiolytic activity in mice in the elevated plusmaze without evidencing sedation or muscle relaxant effects at doses similar to those used for classical benzodiazepines and no anticonvulsant action was detected. However, a 10-fold increase in dosage produced a mild sedative effect since a 26% reduction in ambulatory locomotor activity and a 35% decrement in hole-board parameters were evident. The results reported in this paper demonstrate that apigenin is a ligand for the central benzodiazepine receptors exerting anxiolytic and slight sedative effects but not being anticonvulsant or myorelaxant.

Isolation of pharmacologically active benzodiazepine receptor ligands from Tilia tomentosa (Tiliaceae).

Tilia species are traditional medicinal plants widely used in Latin America as sedatives and tranquilizers. For this purpose, the infusion of their inflorescences is used to prepare a tea. In this study extracts of inflorescences from Tilia tomentosa Moench, one of the species found in the market, were purified using a benzodiazepine (BZD) binding assay to detect BZD receptor ligands in the different fractions. One of the ligands was identified as kaempferol, but it had low affinity (Ki = 93 microM) for this receptor, and did not produce sedative or anxiolytic effects in mice. On the other hand, a complex fraction, containing as yet unidentified constituents, but probably of a flavonoid nature, when administered intraperitoneally in mice, had a clear anxiolytic effect in both the elevated plus-maze and holeboard tests, two well validated pharmacological tests to measure anxiolytic and sedative compounds. This active fraction had no effect on total and ambulatory locomotor activity. In conclusion, our results demonstrate the occurrence of active principle(s) in, at least, one species of Tilia that may explain its ethnopharmacological use as an anxiolytic.

In vivo formation of benzodiazepine-like molecules in mammalian brain.

The possible cerebral formation of benzodiazepine (BDZ)-like molecules was evaluated in rats bilaterally microinjected into either the lateral ventricles (ICV) or the hippocampus (IH), with 10-70 microCi of different radiolabeled amino acids. After 3, or 24 h, the rats were sacrificed and BDZ-like molecules from total brain or hippocampus were purified by reversed phase HPLC. At 24 h. but not at 3 h of the ICV or IH microinjections with [3H] tryptophan, a peak of radioactivity containing material that inhibited the binding of [3H] flunitrazepam to both the BDZ receptor and the anti -BDZ monoclonal antibody MAb 21-7F9 was obtained. This active labeled fraction eluting just before diazepam also bound directly and specifically to the BDZ receptor and to MAb 21-7F9. No peak of radioactivity containing BDZ-like material was obtained when [3H] phenylalanine, [14C] glycine, [14C] methionine or [14C] tyrosine alone or in different combinations were microinjected into the hippocampus. The present results, together with our previous findings on the in vitro production of BDZ-like molecules in rat brain homogenates or slices (11), strongly suggest that the mammalian brain is capable of synthesizing low molecular weight substances possessing BDZ-like activity.

Benzodiazepines in the brain. Their origin and possible biological roles.

Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5 x 10(-10)-10(-8) M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam and N-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.

Production of benzodiazepine-like compounds in bovine rumen.

The presence of benzodiazepine-like molecules was detected radioimmunologically in bovine rumen contents and in incubates of ruminal contents with homogenates of several common grasses. A similar production was found "in vivo" in samples obtained from a grazing cow with a rumen cannula.

The hydration of a zwitterion, glycine, as a function of pH.

1. The hydration numbers of glycine (concentrations 1--3 M), as a function of concentration, were determined by surface tension measurements, using octan-1-ol as a 'reference' substance. 2. The hydration number of glycine at the isoelectric point decreased from 17.7 to 10.7 upon increasing the concentration from 1 to 3 M. 3. The changes in hydration of glycine as a function of the pH are due to the difference between hydrations of the ionized functional groups (NH+3, COO-) and the added ions (Me+ A-).