Experimental study on Satureja montana as a treatment for premature ejaculation.

AIM OF THE STUDY: Satureja montana (winter savory) is a medicinal plant traditionally used to treat different disorders including male sexual dysfunction. In this study we evaluated the effect of Satureja montana hydroalcoholic extract on copulatory behavior of sexually potent male rats. MATERIALS AND METHODS: The extract was orally administered acutely or repetitively for 8 consecutive days at the doses of 25 and 50 mg/kg. The main parameters of sexual behavior, mount (ML), intromission (IL), ejaculation (EL) latencies and post-ejaculatory interval (PEI), were evaluated in animals submitted to mating test and multiple ejaculations test. Testosterone serum levels were measured in rats acutely treated with Satureja montana extract dosed at 50 mg/kg. In addition the open field test was conducted to evaluate the locomotor behavior. RESULTS: When acutely administered at both dosages, the extract was able to significantly increase EL and decrease intromission frequency (IF) in comparison with controls. The significant increase in EL was found also when the extract was subacutely administered, daily for 8 consecutive days, at the dose of 25 mg/kg. In the multiple ejaculations test, EL values of treated rats were significantly increased during the 1st and 2nd sequence in comparison with controls; in addition only rats treated with the extract were able to reach the 4th ejaculation within 30 min. Testosterone serum level measured in rats acutely treated with Satureja montana at the dose of 50 mg/kg was significantly increased in rats in comparison with controls. Finally, the locomotor activity recorded in the open field test was not affected by the acute administration of the plant extract. CONCLUSIONS: These data suggest that Satureja montana could be considered as a natural remedy for the treatment of premature ejaculation delaying ejaculation latency without exerting any negative effect on the other parameters of sexual behavior and without exerting a sedative effect. In addition the increased serum level of testosterone confirms the positive influence of Satureja montana on male sexual function.

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.

Experimental evidence of the anaphrodisiac activity of Humulus lupulus L. in naïve male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In the folk medicine Humulus lupulus L. (hops) is mainly recommended as a mild sedative with antispasmodic and digestive properties. It is also reputed to exert an anaphrodisiac effect but it is still lacking the experimental evidence of this activity. AIM OF THE STUDY: To evaluate the influence of Humulus lupulus extract on sexual behavior of both naïve and sexually potent male rats; thereafter to investigate the role of 8-prenylnarigenin (8-PN) in the effect displayed by the hop extract. MATERIALS AND METHODS: Sprague-Dawley male rats both naïve and sexually potent were acutely administered with the hop extract dosed at 5, 10, 25 and 50 mg/kg. In addition the extract was administered daily for 10 consecutive days at the dose of 0.25 mg/kg/day in sexually potent animals. The pure compound 8-PN was acutely administered in naïve rats at the dosages of 5, 12.5 and 25 microg/kg. All the animals were screened for their sexual behavior manifestation during the mating test. RESULTS: In naïve rats the acute administration of Humulus lupulus extract at the doses of 25 and 50 mg/kg significantly reduced the percentage of mounting and ejaculating animals, in comparison to vehicle controls. The other parameters recorded during the mating test were not affected by the hop extract. In sexually potent rats nor the acute neither the repeated administration of the extract modified their copulatory behavior. The pure compound 8-PN failed to influence male sexual behavior of naïve rats. CONCLUSION: Humulus lupulus extract exerted an anaphrodisiac effect only in naïve rats by inhibiting their mounting and ejaculating behavior. The presence of 8-PN in the extract could be only partially involved in the observed anaphrodisiac effect.

Natural endogenous ligands for benzodiazepine receptors in hepatic encephalopathy.

Benzodiazepines of natural origin (NBZDs) have been found in human blood and brains as well as in medicinal plants and foods. In plasma and brain tissue there are i.e. diazepam and nordiazepam equal to commercial drugs but there are also other benzodiazepine-like compounds termed "endozepines", which act as agonists at the benzodiazepine receptors of central type (CBR). A synthetic pathway for the production of NBZDs has not yet been found, but it has been suggested that micro-organisms may synthesize molecules with benzodiazepine-like structures. Hence NBZDs could be of both endogenous and exogenous source and be considered as natural anxyolitic and sedative. Interestingly there are also natural compounds, such as the polypeptide Diazepam Binding Inhibitor (DBI) acting as an "inversive agonist" implicated in fair and panic disorders. It has been suggested that NBZDs may play a role in the pathogenesis of hepatic encephalopathy (HE). Multidirectional studies evaluated NBZDs levels (1) in the blood of normal subjects, of cirrhotic with or without HE and in commercial benzodiazepine consumers; (2) in the blood of cirrhotic treated or not with a non-absorbable antibiotic; (3) in several constituents of our diet. In conclusion, NBZDs increase sometime in cirrhotics with or without HE but they reach concentrations not higher than those found in commercial benzodiazepines consumers. Hence NBZDs must be considered as occasional precipitating factor of HE and benzodiazepine antagonists only symptomatic drugs. The finding that NBZDs may be in part synthesized by intestinal bacterial flora and in part constituent of our diet underlines the importance to feed cirrhotic patients with selected food.

Evidence that the beta-acids fraction of hops reduces central GABAergic neurotransmission.

Humulus lupulus (hops) is traditionally used as a tranquilizing herbal remedy. Here, we investigated the in vivo and in vitro effect of hop beta-acids on central nervous system function. Oral administration of beta-acids (5-10mg/kg) in rats produced an increased exploratory activity in the open field, a reduction in the pentobarbital hypnotic activity and a worsening of picrotoxin-induced seizures. When dosed at 10mg/kg, beta-acids increased, in the elevated plus maze, open arm entries reducing in parallel those in closed arms. In the forced swimming test, we observed a reduction in the immobility time that could suggest an antidepressant-like activity. Electrophysiological studies performed on cerebellar granule cells in culture showed that the beta-acids fraction decreased GABA-evoked current in a dose-dependent way. The effect was not inhibited by the benzodiazepine antagonist Ro 15-1788. Benzodiazepine receptors involvement was also excluded by [(3)H]-Ro 15-1788 binding assay. In conclusion, the behavioral effects of beta-acids fraction could be explained by a reduction in the GABAergic activity although we cannot rule out the involvement of other neurotransmitter systems.

New insight in the neuropharmacological activity of Humulus lupulus L.

The purpose of the present study was to investigate the effects of Humulus lupulus CO2 extract and its fraction containing alpha-acids on the central nervous system of rats. Both tested substances were able to prolong pentobarbital sleeping time, without affecting the latency to the loss of the righting reflex. This effect was dose-dependent, starting from a minimal dose of 10 mg/kg. Neither the extract nor its alpha-acid fraction affected the locomotor activity in the open field test or exerted an anxiolytic effect in rats submitted to the elevated plus-maze test. Interestingly both compounds reduced the immobility time during the behavioral despair test when administered three times (24, 5 and 1 h) before the test. In conclusion this report shows that Humulus lupulus CO2 extract exerts: (a) a pentobarbital sleep-enhancing property without influencing the motor behavior of rats; (b) an antidepressant activity. The same effects were elicited by the administration of the Humulus lupulus fraction containing alpha-acids, which can be considered as the major responsible for the enhanced pentobarbital effect and for the antidepressant property.

Activity of single components of Ferula hermonis on male rat sexual behavior.

The influence of the single components of Ferula hermonis extract on sexual behavior was studied in male rats. Sexually potent and sluggish/impotent animals were orally treated acutely (2.5 mg/kg) and subchronically (0.25 mg/kg/day for 10 days) with ferutinin, teferdin and teferin. Ferutinin alone acutely administered in potent rats was able to reduce mount and intromission latencies, while in sluggish/impotent animals, it induced the same effects and additionally shortened the ejaculation latency, as teferdin did. Both substances increased testosterone levels in rats. Unlike teferdin, ferutinin subchronically administered in potent rats negatively affected appetitive and consummatory sexual behavior, reducing also testosterone serum levels. In conclusion, if repetitively administered, ferutinin was able to stimulate sexual behavior after acute ingestion, but exerted a negative influence on the sexual capacity of potent male rats, whereas teferdin only improved copulatory performance of sluggish/impotent animals.

Opposite effect of acute and subchronic treatments with Ferula hermonis on copulatory behavior of male rats.

Sexually potent and sluggish/impotent male rats were orally treated with an extract of Ferula hermonis (30 and 60 mg/kg). The acute administration stimulated sexual motivation in potent rats and improved copulatory performance in sluggish/impotent rats. This last effect was elicited only by the higher dose, which, in parallel, increased serum testosterone levels in rats. On the contrary, when the extract was subchronically administered (10 days) a marked reduction in the percentage of rats achieving ejaculation was detected, together with a general impairment of the copulatory pattern. Furthermore, the repeated administration of the extract (6 mg/kg/day for 10 days) resulted in a significant reduction of testosterone levels in comparison with controls. The present results discourage a repeated assumption of F. hermonis, while suggesting its acute administration to improve the performance in sexual dysfunctions.

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.

Extraction and purification from Ceratonia siliqua of compounds acting on central and peripheral benzodiazepine receptors.

The presence of molecules with high affinity for central and peripheral benzodiazepine receptors was determined in the pod and leaves of Ceratonia siliqua (carob). The amount of the substances able to selectively bind the central benzodiazepine receptor recovered from carob pods and leaves was respectively 12.17 and 18.7 ng diazepam equivalent/g. The amount of compounds active on peripheral benzodiazepine receptor in both pods and leaves was higher in comparison with the central one, being 49.83 and 40.00 PK 11195 equivalent/g, respectively. In particular the compounds acting on peripheral benzodiazepine receptors were found to be extremely concentrated in the young leaves (2572.57 ng PK 11195 equivalent/g). The presence of substances with central benzodiazepine activity in carob extracts seems of great importance in view of the possibility to use carob extract as potential natural products with anxiolytic-sedative effects. Moreover, the prevalence in leaves of substances acting on peripheral benzodiazepine receptor suggests the possible utilisation of leave extracts as chemopreventive agents.

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.

Synthesis and anticonvulsant activity of novel and potent 6,7-methylenedioxyphthalazin-1(2H)-ones.

In this paper, we describe the synthesis of a series of novel substituted 4-aryl-6,7-methylenedioxyphthalazin-1(2H)-ones. The anticonvulsant activity of these compounds against audiogenic seizures was evaluated in DBA/2 mice after intraperitoneal (ip) injection. Most of these derivatives are more active than 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive AMPA receptor antagonist. As deduced by the rotarod test, all the compounds exhibit a toxicity lower than that of 1. Within the series of derivatives submitted to investigation, 4-(4-aminophenyl)-2-butylcarbamoyl-6,7-methylenedioxyphthalazin -1(2H)-one (21) proved to be the most active compound and is 11-fold more potent than 1 (i.e., ED50 3.25 micromol/kg for 21 versus ED50 35.8 micromol/kg for 1). When compared to 1, compound 21 as well as its analogue 4-(4-aminophenyl)-6,7-methylenedioxyphthalazin-1(2H)-one (16) show a longer lasting anticonvulsant activity. Compound 21 also effectively suppresses seizures induced in Swiss mice by maximal electroshock (MES) and pentylenetetrazole (PTZ). Furthermore, it antagonizes in vivo seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), 2-amino-3-(3-hydroxy-5-tert-butyl-isoxazol-4-yl)propionic acid (ATPA), and kainate (KA), and its anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 16 and 21 to antagonize KA-evoked currents in primary cultures of granule neurons was tested. They behaved as antagonists, but they proved to be less effective than 1 and 1-(4-aminophenyl)-3,4-dihydro-4-methyl-3-N-methylcarbamoyl-7,8-met hylenedioxy-5H-2,3-benzodiazepine (2, GYKI 53655) to reduce the KA-evoked currents.

Endogenous benzodiazepines.

The existence of endogenous benzodiazepines such as diazepam and nordiazepam has been provided in human blood and brains as well as in medicinal plants and foods. It must be stressed, however, that in plasma and brain tissue there are also other benzodiazepine-like compounds termed 'endozepines' which are not halogenated. A synthetic pathway for the production of benzodiazepine-like compounds and endozepines has not yet been found, hence it may be surmised that these compounds could be of exogenous source. Changes in the level of endogenous circulating benzodiazepines due to food or drug ingestion could be responsible for pathological conditions. Clinical experiments were designed in order to study the levels of the endogenous benzodiazepines in vegetables and in the blood of control subjects and of cirrhotic patients. These patients accumulate benzodiazepines because of decreased liver metabolization capacity and impaired renal secretion, reaching plasma concentrations similar to those recorded in commercial benzodiazepine consumers.

Evidence that total extract of Hypericum perforatum affects exploratory behavior and exerts anxiolytic effects in rats.

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.

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.

Synthesis and anticonvulsant activity of novel and potent 2,3-benzodiazepine AMPA/kainate receptor antagonists.

We have previously shown that 1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) in analogy to the structurally related 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive AMPA/kainate receptor antagonist. We now report the synthesis of 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (4a-h) and 1-aryl-3, 5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-thiones (5a-c). The activity of all compounds, intraperitoneally (ip) injected, was evaluated against audiogenic seizures in DBA/2 mice and against seizures induced by maximal electroshock (MES) and pentylenetetrazole (PTZ) in Swiss mice. Some of the new compounds 4 and 5 showed remarkable anticonvulsant activity, and their toxicity, as evidenced by the rotarod test, is lower than that of 1. The time course of anticonvulsant activity of derivatives 4b and 5b,c was studied and compared to that of 1 and 3b,c. Compounds 4a,b and 5a-c antagonize seizures induced by AMPA and kainate (KA) and their anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 3c, 4b, and 5c to antagonize KA-evoked currents in primary cultures of granule neurons was tested and compared with that of the parent compounds 1 and 1-(4-aminophenyl)-3, 4-dihydro-4-methyl-3-methylcarbamoyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine (2, GYKI 53655).

Opioid peptides of the pituitary and hypothalamus: changes in pregnant and lactating rats.

Immunoreactive (Ir) beta-endorphin concentrations were determined in plasma, anterior pituitary (AP), neurointermediate pituitary lobe (NIL) and mediobasal hypothalamus (MBH) of pregnant (12-14 and 18-20 days) and fertile control rats, during labour and lactation. Immunoreactive Met-enkephalin concentrations were also evaluated in the MBH. Concentrations of Ir beta-endorphin in plasma, AP and NIL of rats during early and late pregnancy were significantly higher than in controls, the plasma and AP contents showing an increasing pattern in the second half of gestation. During labour, Ir beta-endorphin concentrations in plasma and AP reached the highest values, whereas those in NIl remained unchanged. Lactating rats showed Ir beta-endorphin concentrations in NIL and plasma in a range similar to that found in pregnant rats, resulting in concentrations in the AP not significantly different from those of nonpregnant controls. Immunoreactive beta-endorphin and Ir Met-enkephalin concentrations in MBH of pregnant rats were almost twice as high as in controls, rising markedly during labour; during lactation levels were in the same range as in non-pregnant controls. These results indicate that pregnancy and labour are characterized by high plasma, pituitary and hypothalamic concentrations of Ir-beta-endorphin as well as by high hypothalamic Ir Met-enkephalin levels, and that Ir beta-endorphin concentrations vary differently during pregnancy, lactation and labour in the two pituitary lobes, supporting the existence of different control mechanisms in the AP and NIL.

Opiate receptors and beta-endorphin levels in brain areas of dogs with portal-systemic encephalopathy.

Clinical observation has indicated a supersensitivity to morphine in patients with hepatic encephalopathy. With the aim of clarifying the issue, radioreceptor binding studies of opiate receptors were performed in frontal cortex and hypothalamus of 6 dogs with mild portal-systemic encephalopathy induced by chronic treatment with dimethylnitrosamine followed by porto-caval shunt end-to-side. beta-Endorphin assays were performed in the same areas with radioimmunoassay. Opiate receptors labeled with [3H]naloxone in both areas showed a significant increase in the receptor densities (Bmax) without changes in the dissociation constant (KD). In parallel beta-endorphin levels showed a decline during the development of encephalopathy in both areas. The increased densities of opiate receptors in the mild stage of encephalopathy may explain the supersensitivity to morphine in patients with liver diseases.