Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells.

AIMS: Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects. MAIN METHODS: hAFSCs were cultured in a standard medium or in an osteoblastic medium for 14 or 21days and ferutinin was added at 10-8M. Immunofluorescence techniques and Western-blot 21analysis were used to study estrogen receptors and signaling pathways. KEY FINDINGS: In both undifferentiated and differentiated hAFSCs we identified ERα and GPR30 with a nuclear or cytoplasmatic localization, respectively. The presence of ferutinin in the osteoblastic medium leads to an increase in ERα expression. To dissect the role of estrogen receptors, MPP and G15 were used to selectively block ERα and GPR30, respectively. Notably, ferutinin enhanced osteoblastic differentiation in cells challenged with G15. Ferutinin was able to increase ERK and Akt phosphorylations with a different timing activation. These phosphorylations were antagonized by PD0325901, a MEK inhibitor, and wortmannin, a PI3K inhibitor. Both MPP and G15 inhibited the ferutinin-induced MEK/ERK and PI3K/Akt pathway activations. In the osteoblastic condition, PD0325901, but not wortmannin, reduced the expression of OPN and RUNX-2, whereas ferutinin abrogated the down-modulation triggered by PD0325901. SIGNIFICANCE: PI3K/Akt pathways seems to mediate the enhancement of hAFSCs osteoblastic differentiation triggered by ferutinin through ERα.

Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells.

INTRODUCTION: Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. METHODS: After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. RESULTS: Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. DISCUSSION: AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

Enrichment in c-Kit+ enhances mesodermal and neural differentiation of human chorionic placental cells.

OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.

Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells.

AIMS: The phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs). MAIN METHODS: AFSCs and DPSCs were cultured in a differentiation medium for 14 or 21days with or without the addition of Ferutinin at a concentration ranging from 10(-11) to 10(-4)M. 17ß-Estradiol was used as a positive drug at 10(-8)M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed. KEY FINDINGS: MTT assay revealed that Ferutinin, at concentrations of 10(-8) and 10(-9)M, enhanced proliferation of both AFSCs and DPSCs after 72h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14days of culture in both populations. SIGNIFICANCE: The enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.

Effects of the cannabinoid antagonist SR 141716 on sexual and motor behaviour in receptive female rats.

1. The aim of the present study was to investigate the effect of the cannabinoid antagonist/inverse agonist SR 141716 (SR) on the receptive behaviour and sexual motivation of female rats. 2. Partner preference, receptivity and proceptivity were evaluated in ovariectomized female rats primed with oestrogen and progesterone and administered SR (1 or 2.5 mg/kg, i.p.) 20 min prior to testing. 3. In the partner preference test, a reduced interest in both stimulus animals (a sexually active male and an ovariectomized hormone-primed female) was detected in rats treated with SR at both doses, but no effect on preference score was observed. In the receptivity test, pronounced reductions in lordosis quotient, lordosis rating and in the percentage of receptive females were found in SR-treated rats compared with control rats. Proceptive behaviours were not significantly affected by either dose of SR. 4. In addition, we explored the behavioural effects induced by SR in female rats using the open field test. Only at the higher dose (i.e. 2.5 mg/kg) did SR markedly increased grooming and scratching behaviour. 5. The results demonstrate the ability of SR to reduce female sexual receptivity, but not sexual motivation. The reduction does not seem strictly related to the motor alterations induced by the cannabinoid antagonist.

Influence of Griffonia simplicifolia on male sexual behavior in rats: behavioral and neurochemical study.

The seeds of Griffonia simplicifolia Baill. are rich in 5-HTP (5-hydroxytryptophan), a direct precursor of the neurotransmitter serotonin. In the present study we investigated the influence of the plant extract on male sexual behavior. The seed extract was orally administered to Sprague-Dawley male rats at three dose levels (25, 50 and 100 mg/kg) both acutely and subchronically (daily for 9 days). Mating test with receptive female rats was performed 60 min after the acute treatment or the last dose when repetitively administered. Mount, intromission and ejaculation latencies and post-ejaculatory interval were recorded. Food intake and body weight were measured over the 9-day period of treatment. Microdialysis technique was used to detect the extracellular levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in rat brain following the acute administration of the extract dosed at 100mg/kg. The acute treatment significantly increased mount latency (at any dosage), intromission and ejaculation latencies (at 100 mg/kg) and post-ejaculatory interval (at 50 and 100 mg/kg). On the contrary the subchronic treatment failed to exert a significant influence on copulatory behavior. The daily administration of the extract dosed at 50 and 100 mg/kg for 9 days significantly reduced food intake and body weight. Finally in the microdialysis experiments we found a dramatic increase in 5-HT and its metabolite 5-HIAA.

Anxiolytic-like effect of Griffonia simplicifolia Baill. seed extract in rats.

The seeds of Griffonia simplicifolia Baill., a tropical shrub native to West Africa, are rich in 5-hydroxy-l-tryptophan (5-HTP), a direct precursor in the synthesis of serotonin (5-HT). In spite of the modern therapeutic application of Griffonia simplicifolia seed extract in mood disorders, no scientific evidence has been provided till now. For this reason the aim of our study was to investigate the effect of Griffonia simplicifolia seed extract on anxiety behavior. Griffonia simplicifolia seed extract, dosed at 1, 5, 10 and 25 mg/kg, was orally administered in rats which were submitted to the dark-light test and open field test, 60 min after the treatment. In the dark-light test, the administration of the extract at the doses of 10 and 25 mg/kg was able to significantly increase the time spent in the light compartment (P<0.05). In the open field test, the extract dosed at 5, 10 and 25 mg/kg induced an anti-tigmotactic effect, as indicated by a significant increase of time spent in the central area of the open field (P<0.01). In conclusion these findings indicate that Griffonia simplicifolia seed extract exerts anxiolytic-like effect in rats and suggest its potential usefulness for the treatment of anxiety in humans.

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.

Griffonia simplicifolia negatively affects sexual behavior in female rats.

At present Griffonia simplicifolia is used in food supplement aimed to treat mood disorders as well as to reduce food intake and body weight. The plant has gained increasing interest for its high content in 5-hydroxy-L-tryptophan (5-HTP) particularly in the seed. The present study was designed to evaluate the influence of a seed extract of the plant, dosed at 25, 50 and 100 mg/kg, on the sexual behavior of ovariectomized hormone-primed rats after acute and subchronic treatment. The single administration of G. simplicifolia significantly reduced lordosis response and increased rejection behavior in female rats treated with the highest dose while it did not influence proceptive behaviors. On the other hand the subchronic administration of the extract significantly reduced proceptivity but not receptivity, and increased rejection behavior. All the tested dosages were able to markedly decrease food intake and body weight after a 9-day treatment. Taken together the present results, possibly ascribed to increased levels of 5-hydroxytryptamine (5-HT) in the brain, suggest a cautious administration of the plant extract owing to its negative influence on female sexual behavior.

Evidence that isopropylthioxanthone (ITX) is devoid of anxiolytic and sedative effect.

Isopropylthioxanthone (ITX) is a well-known photo-initiator in ultraviolet light-cured inks frequently used in milk packaging materials, yoghurt, ready-to-feed infant formula, and other drinks. Traces of ITX have been found in milk and, as a consequence, there was considerable interest in studying the biological activity of this molecule and its potential hazard for the human health. Although the ITX genotoxic effects have been excluded by the European Food Safety Authority (EFSA), the US Environmental Protection Agency (USEPA) is still examining its possible toxic potential depending on a dose-effect ratio. Little is known about the ITX activity on the function of the central nervous system and cerebral neurotransmitters. Using behavioural, biochemical, and electrophysiological tests, the authors have found that: (1) ITX did not exert an in vivo anxiolytic or sedative effect when administered orally to rats; (2) ITX did not affect the binding characteristics of central and peripheral benzodiazepine receptors studied in vitro; and (3) ITX did not influence the ability of gamma-Aminobutyric acid (GABA) to increase the chloride channel permeability studied by patch clamp technique in a single neuron of cultured cerebellar granule cells.

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.

The phytoestrogen ferutinin improves sexual behavior in ovariectomized rats.

The present study was designed to examine the effect of ferutinin chronic administration on sexual behavior of ovariectomized non-estrogen-primed rats. Starting from 3 weeks after ovariectomy, female rats were orally treated with ferutinin at the doses of 0.2 and 0.5 mg/kg, daily for 4 weeks. Ferutinin's effect was compared with that of estradiol benzoate, subcutaneously injected at the dose of 1.5 microg/rat twice a week. Animals were tested for sexual motivation, receptivity and proceptivity after 1, 2 and 3 weeks of treatment and for paced mating behavior after 4 weeks of treatment. Before each experimental test, they received progesterone injection (500 microg/rat). Both dosages of ferutinin significantly increased the receptive behavior in a time-dependent manner, as well as estradiol benzoate did. Also proceptive behaviors increased in ferutinin-treated animals in comparison with control ones. During the partner preference test ferutinin was able to induce a significant preference for a sexually active male over a sexually receptive female. Moreover, ferutinin restored a normal paced mating behavior, which had been suppressed by ovariectomy. These results show that ferutinin exerts an estrogenic activity in ovariectomized non-estrogen-primed female rats.

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.