A.C. Paladini

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.

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.

New developments on the search for the endogenous ligand(s) of central benzodiazepine receptors.

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.

Acute stress induces an increase in rat cerebral cortex levels of n-butyl-β-carboline-3-carboxylate, an endogenous benzodiazepine binding inhibitor

The effect of an acute swimming stress in rats on the amount of n-butyl-?-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.

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.

Naturally occurring benzodiazepines in human milk.

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.

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.

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 5.10−10–10−8M; (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.

H-exchange behaviour and extent of reversible conformation changes in human, bovine, ovine, porcine and equine growth hormones.

Abstract The hydrogen exchange of highly purified preparations of human, bovine, ovine, porcine and equine growth hormones has been measured in various experimental conditions. All the hormones showed similar exchange behaviour. The kinetic classes of hydrogens in human, bovine, ovine and porcine growth hormones have been calculated. The extent of reversible conformation changes induced by pH variations or by urea was measured by the size of the new classes of very slow hydrogens appearing under these conditions. The greatest change was detected in human growth hormone. At physiological pH this protein was approx. 2–10 times more permeable to the solvent than all the other hormones tested.