Mario Baraldi

Pharmacological profile of apigenin, a flavonoid isolated from Matricaria chamomilla

Dried flowers of Matricaria chamomilla L. are largely used to provide sedative as well as spasmolytic effects. In the present study, we examined in particular the pharmacological property of a fraction isolated from a methanolic extract of M. chamomilla, which was identified by HPLC-MS-MS analysis as apigenin. By radioreceptor binding assays, we demonstrated the ability of the flavone to displace a specific radioligand, [(3)H]Ro 15-1788, from the central benzodiazepine binding site. Electrophysiological studies performed on cultured cerebellar granule cells showed that apigenin reduced GABA (gamma-aminobutyric acid)-activated Cl(-) currents in a dose-dependent fashion. The effect was blocked by co-application of Ro 15-1788, a specific benzodiazepine receptor antagonist. Accordingly, apigenin reduced the latency in the onset of picrotoxin-induced convulsions. Moreover, apigenin injected i.p. in rats reduced locomotor activity, but did not demonstrate anxiolytic, myorelaxant, or anticonvulsant activities. The present results seem to suggest that the inhibitory activity of apigenin on locomotor behaviour in rats cannot be ascribed to an interaction with GABA(A)-benzodiazepine receptor but to other neurotransmission systems, since it is not blocked by Ro 15-1788.

Behavioral characterisation of the flavonoids apigenin and chrysin.

The behavioral effects of acute administration of two flavonoids, apigenin and chrysin, contained in Matricaria chamomilla and in Passiflora incarnata, respectively, were studied in rats. The data demonstrate that in our experimental conditions, the two flavonoids were equally able to reduce locomotor activity when injected in rats at a minimal effective dose of 25 mg/kg. However, while chrysin exhibited a clear anxiolytic effect when injected at the dose of 1 mg/kg, apigenin failed to exert this activity. The sedative effect of these flavonoids cannot be ascribed to an interaction with GABA-benzodiazepine receptors, since it was not counteracted by the benzodiazepine antagonist Flumazenil. To the contrary, the anxiolytic effect of chrysin, which was blocked by the injection of Flumazenil, could be linked to an activation of the GABA(A) receptor unit.

Endogenous benzodiazepine receptor ligands in human and animal hepatic encephalopathy

Abstract: The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti‐benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti‐benzodiazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained ∼ 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased ∼ 10‐fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.

Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma

Increased number of peripheral benzodiazepine receptors (PBRs) have been found in some tumors outside the liver. The present study was to verify whether the PBR system is altered in hepatocellular carcinoma (HCC). The levels of endogenous benzodiazepine-like compounds (BZDs), measured by radioreceptor binding technique after HPLC purification and the endogenous ligand for PBRs, termed diazepam binding inhibitor (DBI), measured by radioimmunoassay utilizing a specific antibody for human DBI, were studied in the blood of 15 normal subjects, 12 liver cirrhosis and 10 patients with HCC. The levels of BZDs in serum were increased hundred fold in liver cirrhosis patients and slightly elevated in HCC patients. DBI was found to be increased in HCC patients. The binding recognition sites for PBRs (Bmax) were increased 4 to 7 fold in HCC tissue in comparison with that found in non-tumoral liver tissue (NTLT). On the contrary the concentrations of DBI were found to be significantly decreased in HCC tissue in comparison with the respective NTLT. These results seem to suggest an implication of PBRs and of their putative endogenous ligands in the metabolism of these neoplastic cells and possibly in their proliferation. The up-regulation of PBRs found in HCC tissue seems to indicate an increased functional activity of these receptors and opens up the possibility of new pharmacological and diagnostic approaches while the changes in the circulating endogenous ligands for the above receptors might be envisaged as early markers of tumorigenesis in liver cirrhosis.

Antiproliferative effects of Ceratonia siliqua L. on mouse hepatocellular carcinoma cell line

Extracts from pods and leaves of carob (Ceratonia siliqua L.) were tested for their ability to inhibit cell proliferation of mouse hepatocellular carcinoma cell line (T1). The two extracts showed a marked alteration of T1 cell proliferation in a dose-related fashion reaching the maximal effect at 1 mg/ml. Moreover, we demonstrated that leaf and pod extracts were able to induce apoptosis in T1 cell lines after 24-h treatment mediating a direct activation of the caspase 3 pathway. HPLC analysis revealed the presence of gallic acid, (-) epigallocatechin-3-gallate and (-) epicatechin-3-gallate in pod and leaf extracts, compounds well known to exert antiproliferative effects. Their concentration reached 6.28 mg/g in carob leaves and 1.36 mg/g in carob pods extract. The discovery that carob pod and leaf extracts contained antiproliferative agents could be of practical importance in the development of functional foods and/or chemopreventive drugs.

Influence of Eurycoma longifolia on the copulatory activity of sexually sluggish and impotent male rats.

AIM OF THE STUDY The root of Eurycoma longifolia Jack, native to South East Asia, has long been used as a male aphrodisiac remedy to treat sexual disorders. In the study we evaluated the influence of Eurycoma longifolia Jack on sexual behavior (including both motivation and copulatory performance) of sexually sluggish and impotent male rats. MATERIALS AND METHODS The root powder of the plant was orally administered to adult Sprague-Dawley male rats, classified as sexually sluggish or impotent taking in account their behavior in pre-experimental tests. Groups of 8 animals each were submitted to three different types of treatment: (1) acute at 3 dose levels (250, 500 and 1000 mg/kg); (2) subacute (daily for 6 days) at the dose of 500 mg/kg and (3) subchronic (daily for 12 days) at the same dose (500 mg/kg). Mount, intromission and ejaculation latencies and post-ejaculatory interval were recorded during the mating test in order to evaluate sexual performance. In addition the partner preference test was used to assess sexual motivation. Testosterone serum levels were measured in subacutely treated rats and compared with the values of controls receiving vehicle. RESULTS Concerning the copulatory activity of sexually sluggish rats, both acute (dosed at 500 and 1000 mg/kg) and subacute treatments with the root powder significantly reduced ejaculation latencies, increasing also the percentage of mounting and ejaculating animals; in addition the subacute administration reduced post-ejaculatory interval. In impotent rats both subacute and subchronic treatments increased the percentage of mounting and ejaculating rats. The motivational behavior of sluggish rats during the partner preference test was not affected by the treatments. Testosterone serum levels were increased in rats subacutely treated in comparison with controls. CONCLUSION Eurycoma longifolia root improved sexual performance but not motivation in sluggish rats after acute or subacute administration. The effect could be mainly ascribed to increased testosterone levels.

Antidepressants and opiates interactions: pharmacological and biochemical evidences.

Imipramine, chronically administered to rats (20 mg/Kg/day X 20) has a potent analgesic effect per se (hot plate test), increases morphine analgesia and intensifies morphine withdrawal syndrome precipitated by naloxone. Receptor binding studies performed with 3H-naloxone revealed that chronic administration of imipramine results in a marked increase of opiate binding sites in the brain. This increase persisted when the rats treated chronically with imipramine were rendered tolerant to morphine by s.c. implantation for 3 days of a pellet containing 100 mg of morphine. Since antidepressants exert their own analgesic effect, increase morphine analgesia and displace opiate receptor binding, it may be that by interacting with the opiate receptor complex imipramine induces supersensitivity in opiate recognition binding sites.

INCREASED EXPRESSION OF PERIPHERAL BENZODIAZEPINE RECEPTORS AND DIAZEPAM BINDING INHIBITOR IN HUMAN TUMORS SITED IN THE LIVER

The peripheral benzodiazepine receptor system triggers intracellular metabolic events and has been associated with cell proliferation. Its endogenous ligand, the diazepam binding inhibitor, contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. The present study was undertaken to verify whether this system is altered in tumors sited in the liver. Peripheral benzodiazepine receptors and diazepam binding inhibitor were studied using immunocytochemistry and in situ hybridization in 9 human tumors sited in the liver, in liver hyperplasia, cirrhotic nodular regeneration, intestinal adenocarcinoma and in surrounding non-tumoral tissue. Immunocytochemical staining and in situ hybridization demonstrated that peripheral benzodiazepine receptors and diazepam binding inhibitor were more prominently expressed in neoplastic cells than in non-tumoral tissue. They were present in the same cells, suggesting that diazepam binding inhibitor may act in an intracrine manner in these cells. Higher peripheral benzodiazepine receptors and diazepam binding inhibitor expression in tumor cells suggest an implication of this system in the metabolism of neoplastic cells. Furthermore the evaluation of peripheral benzodiazepine receptor and diazepam binding inhibitor expression might be useful in evaluating malignancy and in diagnostic approaches of tumors in liver tissue.

1,4-Benzodiazepines and gamma-aminobutyric acid: pharmacological and biochemical correlates.

The high affinity receptors or GABA present in brain interact with an endogenous thermostable inhibitor (GABA modulin) which allosterically modifies GABA binding sites. This is the type of GABA receptor that we term GABA2 receptor in comparison to GABA1 receptor which has low affinity for GABA and is not regulated by GABA modulin. The 1,4-benzodiazepines interact competitively with GABA modulin and thereby modify GABA2 receptor binding. In contrast the occupancy of GABA receptor increases the affinity of 1,4-benzodiazepine receptors for their specific agonist. The GABA modulin and both GABA receptors are located on the membranes of C6 and NB2a cells. The NB2a cell membranes also contain CL- ionophore, thus the complete receptor complex is present in the membranes of NB2a cell clone. It was proposed that the inability of clonazepam to displace 3H-diazepam from specific binding sites characterizes the nonneuronal 1,4-benzodiazepine receptor. This characterization was shown to relate to the properties of other membrane components rather than to the characteristics of the specific benzodiazepine receptors.

Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding.

3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior.