Claudia Wolfman

Possible anxiolytic effects of chrysin, a central benzodiazepine receptor ligand isolated from Passiflora Coerulea

The pharmacological effects of 5,7-dihydroxyflavone (chrysin), a naturally occurring monoflavonoid that displaces [3H]flunitrazepam binding to the central benzodiazepine (BDZ) receptors, were examined in mice. In the elevated plus-maze test of anxiety, diazepam (DZ, 0.3-0.6 mg/kg) or chrysin (1 mg/kg) induced increases in the number of entries into the open arms and in the time spent on the open arms, consistent with an anxiolytic action of both compounds. The effects of chrysin on the elevated plus-maze was abolished by pretreatment with the specific BDZ receptor antagonist Ro 15-1788 (3 mg/kg). In the holeboard, diazepam (1 mg/kg) and chrysin (3 mg/kg) increased the time spent head-dipping. In contrast, high doses of DZ (6 mg/kg) but not of chrysin produced a decrease in the number of head dips and in the time spent head-dipping. In the horizontal wire test, diazepam (6 mg/kg) had a myorelaxant action. In contrast, chrysin (0.6-30 mg/kg) produced no effects in this test. These data suggest that chrysin possesses anxiolytic actions without inducing sedation and muscle relaxation. We postulate that this natural monoflavonoid is a partial agonist of the central BDZ receptors.

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.

Chrysin (5,7-di-OH-flavone), a naturally-occurring ligand for benzodiazepine receptors, with anticonvulsant properties.

Chrysin (5,7-di-OH-flavone) was identified in Passiflora coerulea L., a plant used as a sedative in folkloric medicine. Chrysin was found to be a ligand for the benzodiazepine receptors, both central (Ki = 3 microM, competitive mechanism) and peripheral (Ki = 13 microM, mixed-type mechanism). Administered to mice by the intracerebroventricular route, chrysin was able to prevent the expression of tonic-clonic seizures induced by pentylenetertrazol. Ro 15-1788, a central benzodiazepine receptor antagonist, abolished this effect. In addition, all of the treated mice lose the normal righting reflex which suggests a myorelaxant action of the flavonoid. The presence in P. coerulea of benzodiazepine-like compounds was also confirmed.

Benzodiazepine receptors in rat cerebral cortex and hippocampus undergo rapid and reversible changes after acute stress

Rats were submitted to forced swimming and were killed 15 min after initiation of the stress and at 1 h, 1 day and 4 days thereafter. Immediately after the stress there was a decrease of 30% in the density of [3H]flunitrazepam binding sites in the cerebral cortex and of 27% in the hippocampal formation, with no changes in all the other brain areas studied. These changes in the number of benzodiazepine receptors were also corroborated by the binding of [3H]ethyl-beta-carboline carboxylate. For both ligands there were no changes in affinity. These effects were selective for the benzodiazepine receptors and no changes in alpha 1, alpha 2 and beta adrenoceptors and in dopaminergic receptors were observed. One hour after the stress, the number of benzodiazepine receptors had recovered in the cerebral cortex (8% above the control) and had increased greatly in the hippocampal formation (+53%). One day after the stress, the [3H]flunitrazepam binding in the cerebral cortex reached the normal level but it was still slightly elevated (+16%) in the hippocampus. These results are discussed in relation to some contradictory findings in the literature and to the fact that the hippocampal formation is related to neural mechanisms underlying behavior and neuroendocrine regulation.

Proconvulsant and anxiogenic effects of n-butyl β carboline-3-carboxylate, an endogenous benzodiazepine binding inhibitor from brain

The discovery of n-butyl beta carboline-3-carboxylate (beta CCB) as an endogenous substance of brain capable of interacting with the central benzodiazepine receptor, and the fact that this beta carboline increases in the cerebral cortex of rats undergoing acute stress, led us to study the pharmacological properties of beta CCB in mice. Using 3-mercaptopropionic acid in subconvulsant doses, it was found that this beta carboline, although not being a convulsant, has a proconvulsant action, as indicated by the number of mice undergoing convulsions and the reduction in latency. This proconvulsant effect was observed both with IP or ICV injections and was blocked by the benzodiazepine receptor antagonist RO 15-1788. In an open-field test the injection of 0.3 mg/kg of diazepam increased the number of squares crossed, while beta CCB had the opposite effect, reducing the squares crossed in a dose dependent manner between 1 and 30 mg/kg. This drug also increased the time of freezing and decreased the number of rearings. These changes were partially counteracted by the injection of 3.6 mg/kg of RO 15-1788. In the plus-maze test, 10 mg/kg chlordiazepoxide increased the number of entries and the time spent in the open arms, while the beta carboline produced the opposite effect. The conclusion reached is that beta CCB has both proconvulsant and anxiogenic actions, behaving as an inverse agonist for the central benzodiazepine receptor.

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.

Benzodiazepine-like molecules, as well as other ligands for the brain benzodiazepine receptors, are relatively common constituents of plants.

The presence of benzodiazepine (BZD)-like molecules as well as of other substances with affinity for the brain BZD-receptors was explored in eight non-flowering plants known to contain biflavonoids, three flowering plants used as sedatives in folkloric medicine and one plant extensively used in Argentina, Uruguay, Brazil and Paraguay as a tea substitute. All the plants examined contained substances which bound to the central BZD-receptors and the majority of them also had BZD-like compounds detected by their specific interaction with a monoclonal antibody against BZDs. In various cases this last type of compound was present in amounts which exceeded trace levels (0.5-1.0 ng/g). The biological or clinical significance for humans of all these substances should be explored.

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.

Post-training down-regulation of memory consolidation by a GABA-A mechanism in the amygdala modulated by endogenous benzodiazepines

In rats, amygdala benzodiazepine-like immunoreactivity decreases by 29% immediately after the animals step down from the platform of an inhibitory avoidance apparatus and decreases by a further 45% immediately after they receive a training footshock. The decrease is attributable to a release of diazepam or diazepam-like molecules. The immediate post-training intraamygdala injection of the central benzodiazepine antagonist flumazenil (10 nmole/amygdala) causes memory facilitation, and that of the GABA-A agonist muscimol (0.005 to 0.5 nmole) causes retrograde amnesia. Pretraining ip flumazenil administration (2.0 and 5.0 mg/kg) attenuates the effect of post-training muscimol by a factor of at least 100. The higher dose of pretraining flumazenil also causes memory facilitation. The data suggest that post-training consolidation is down-regulated by a GABA-A mechanism in the amygdala modulated by endogenous benzodiazepines released during training and at the time of consolidation.

Inhibitory Avoidance Training Induces Rapid and Selective Changes in3[H]AMPA Receptor Binding in the Rat Hippocampal Formation

The AMPA receptor has been shown to participate in the synaptic mechanisms involved in certain forms of learning and memory. We have previously demonstrated that the posttraining infusion of 6-cyano-7-nitroquinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor blocker, into the dorsal hippocampus of rats, causes retrograde amnesia of an inhibitory avoidance training. Here, we report on the effect of this learning task on 3[H]AMPA binding to frozen rat brain sections. By using a quantitative autoradiographic analysis, we were able to demonstrate that the binding of 3[H]-AMPA was increased by 40-80% in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampal formation of rats trained in a step-down inhibitory avoidance paradigm, compared to naive, shocked, and free exploration controls. This effect was evident between 30 and 180 min after training, and it was mainly due to an increase in the density, but not in the affinity of binding sites. No alterations in 3[H]AMPA binding were observed either in those animals that received only the footshock (shocked group) or in animals that were submitted to 1 min of free exploration of the training box (free exploration group). In the rest of the brain regions, including the frontal cortex, entorhinal cortex, striatum, amygdala, cerebellum, and thalamus, the 3[H]AMPA binding remained unchanged. In addition, the binding of 3[H]muscimol and 3[H]-flunitrazepam to the GABAA/benzodiazepine receptor complex was unaltered in all the experimental groups. In conclusion, rats submitted to a one-trial inhibitory avoidance training showed a rapid, selective, and specific increase in 3[H]AMPA binding in the hippocampal formation. The present findings support the hypothesis that hippocampal AMPA receptors are involved in the neural mechanisms underlying certain forms of learning and memory.