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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.

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.

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 presence of benzodiazepine-like molecules was detected radioimmunologically in the plasma and milk of 12 women and in the plasma of 9 men. All subjects were non-users of benzodiazepines. The concentration of these biological materials expressed as diazepam equivalents per mL amounted to 2.54 +/- 0.74 ng in male plasma; to 2.20 +/- 0.35 ng in female plasma and to 1.91 +/- 0.54 ng in milk. Further investigation of the active compounds in milk permitted the unequivocal identification of diazepam, both free and bound to a presumably protein carrier and, at least, three more benzodiazepine-like molecules. Their origin either from dietary sources or as a result of endogenous biosynthesis is still unclear.

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.

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.

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 μM) 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.

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 μCi 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 [ 3 H] tryptophan, a peak of radioactivity containing material that inhibited the binding of [ 3 H] flunitrazepam to both the BDZ receptor and the anti-BDZ inonoclonal 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 [ 3 H] phenylalanine, [ 14 C] glycine, [ 14 C] methionine or [ 14 C] tyrosine alone or in different combinations were micro-injected 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.

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 5.10−10–10−8 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 andN-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.

A substance having benzodiazepine-binding inhibitory activity has been extracted from 18 kg of gray matter of bovine cerebral cortex and purified to homogeneity. This substance inhibits competitively [3H]flunitrazepam and ethyl beta-[3H]carboline-3-carboxylate binding with high affinity (Ki, 3 nM), but it is inactive upon 3H-labeled Ro 5-4864, [3H]quinuclidinyl benzylate, [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol, and upon high-affinity [3H]muscimol binding. This inhibitor has been identified as n-butyl beta-carboline-3-carboxylate (beta-CCB) by fast atom bombardment mass spectroscopy (Mr, 268) and electron bombardment fragmentography, ultraviolet and fluorescence spectra, coelution in HPLC with standard beta-CCB, and by the exact correspondence in Ki with beta-CCB on the displacement of [3H]flunitrazepam binding. The possible artificial formation of beta-CCB has been discarded by a series of control experiments including addition of tryptophan to the starting homogenate, extraction from liver, isolation and purification by an alternative procedure avoiding organic solvents, and by the impossibility of making beta-CCB from beta-carboline-3-carboxylic acid or its methyl ester in the conditions of our extraction and purification procedures.

The effect of an acute swimming stress in rats on the amount of n -butyl-β-carboline-3-carboxylate, an endogenous benzodiazepine receptor binding inhibitor, was investigated. In 15 min this substance increased two fold in the cerebral cortex of the stressed rat and this increase was blocked by the previous injection of diazepam; however, no changes were observed in the cerebellum with stress. These results are discussed in relation to previous findings that, after the acute stress, [ 3 H]flunitrazepam binding decreases in cerebral cortex and hippocampus, but not in cerebellum. A possible relationship between this benzodiazepine receptor binding inhibitor and the state of “anxiety” produced by stress is postulated.

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.

This review describes three new research developments that have occurred since 1983, in relation to the possible identification of endogenous ligand(s) for the benzodiazepine central receptor (BZD-R). The polypeptides diazepam binding inhibitor (DBI) and the ODN of Guidotti and Costa, as well as the endozepines of Shoyab and Todaro are considered in their affinities and pharmacological actions. The work of the De Blas group on the presence of benzodiazepines in brain, confirmed by us and other groups, is commented and the discovery, in our own laboratory, of n-butyl-?-carboline-3-carboxylate as a possible putative ligand, having high affinity for the BZD-R and showing proconvulsant and "anxiogenic" properties, is described. In the concluding remarks, the possibility that two or more endogenous ligands with opposing activity could regulate the BZD-GABA receptor complex is postulated.

original Fil: Caputto, Ranwel. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Trucco, Raúl E.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Cardini, Carlos E.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Paladini, Alejandro C.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Blanco y negro 2 páginas en pdf LFL-PI-O-ART. Artículos científicos Unidad documental simple AR-HYL-2018