Manuela Zavatti

Pharmacognostic and pharmacological profile of Humulus lupulus L

The present review describes the morphological, phytochemical and ethnopharmacological aspects of Humulus lupulus L. (Cannabinaceae) and summarizes the most interesting findings obtained in the preclinical and clinical research related to the plant. The female inflorescences of Humulus lupulus (hops), well-known as bittering agent in brewing industry, have long been used in traditional medicine mainly to treat sleep disturbances. However the sedative activity is still under investigation in order to recognize the active principles responsible for the neuropharmacological effects observed in laboratory animals, and their mechanism of action. Here we report the data from our experiments as well as those obtained from other researchers, focusing on the variability of the results. Other traditional applications of hops as stomachic, antibacterial and antifungal remedy have been supported by in vivo and/or in vitro investigations. In recent years some prenylated chalcones present in hops have received much attention for their biological effects: in particular, xanthohumol has been shown to exert cancer chemopreventive activity in in vitro experiments, while 8-prenylnaringenin has been characterized as one of the most potent phytoestrogens isolated until now. Nevertheless much additional work is needed to open up new biomedical application of these compounds.

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.

Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization

IntroductionThe main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells and amniotic fluid stem cells, combined with collagen scaffold to reconstruct critical-size cranial bone defects in an animal model.MethodsWe performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks, cranial tissue samples were taken for histological and immunofluorescence analysis.ResultsWe observed a new bone formation in all of the samples but the most relevant differences in defect correction were shown by stem cell–collagen samples 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold.ConclusionThese data confirmed the strong potential of bioengineered constructs of stem cell–collagen scaffold for correcting large cranial defects in an animal model and highlighting the role of stem cells in neovascularization during skeletal defect reconstruction.

Influence of ferutinin on bone metabolism in ovariectomized rats. II: Role in recovering osteoporosis

The aim of the present investigation, which represents an extension of a previous study, was to investigate the effect of ferutinin in recovering severe osteoporosis due to estrogen deficiency after rat ovariectomy and to compare phytoestrogen effects with those of estrogens commonly used in hormone replacement therapy (HRT) by women with postmenopausal osteoporosis. The animal model used was the Sprague–Dawley ovariectomized rat. Ferutinin was orally administered (2 mg kg−1 per day) for 30 or 60 days starting from 2 months after ovariectomy (i.e. when osteoporosis was clearly evident) and its effects were compared with those of estradiol benzoate (1.5 μg per rat twice a week, subcutaneously injected) vs. vehicle‐treated ovariectomized (OVX) and sham‐operated (SHAM) rats. Histomorphometric analyses were performed on trabecular bone of lumbar vertebrae (4th and 5th) and distal femoral epiphysis, as well as on cortical bone of femoral diaphysis. Bone histomorphometric analyses showed that ferutinin seems to display the same effects on bone mass recorded with estradiol benzoate, thus suggesting that it could enhance the recovery of bone loss due to severe estrogen deficiency in OVX rats. On this basis, the authors propose listing ferutinin among the substances representing a potential alternative for the treatment of postmenopausal osteoporosis, which occurs as a result of estrogen deficiency.

In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp

AIM To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.

Leptin increases growth of primary ossification centers in fetal mice

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg−1) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new‐born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross‐sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.

Improved sexual behavior in male rats treated with a Chinese herbal extract: hormonal and neuronal implications

AIM To investigate the influence of an extract obtained from five Chinese medicinal plants on sexual behavior of adult male rats. METHODS The extract was administered at doses of 30, 60 and 120 mg/kg by oral gavage, acutely (one time, 45 min before mating test) or subchronically (daily for 10 days) in sexually potent and sexually sluggish/impotent rats. Sexual behavior, serum levels of luteinizing hormone (LH) and testosterone (T) were evaluated in treated rats and compared with controls receiving vehicle. The effect of the extract on central dopaminergic neurotransmission was assessed in the nucleus accumbens using a microdialysis technique. RESULTS In sexually potent rats, both acute and subchronic treatment with the extract dosed at 30 and 60 mg/kg reduced mount latency and intromission latency. In sluggish/impotent rats, the acutely administered extract at the dose of 60 mg/kg shortened ejaculation latency, whereas subchronically administered at the doses of 30 and 60 mg/kg, reduced mount, intromission and ejaculation latencies, increasing also the percentage of mounting and ejaculating rats. The extract dosed at 60 mg/kg significantly increased LH and T following acute and subchronic administration and increased 3,4-dihydroxyphenylacetic acid levels in the nucleus accumbens, 30 min after the acute administration. CONCLUSION The improvement in both appetitive and consummatory components of sexual behavior observed in male rats treated with the extract could be ascribed to increased serum T level in parallel with the activation of the central dopaminergic system.

Role of ferutinin in the impairment of female sexual function induced by Ferula hermonis.

In the present study, we evaluated the effects of single components of Ferula hermonis extract on female rat sexual behaviour. Ovariectomized rats hormonally primed with estradiol benzoate (1.5 or 10 microg/rat s.c.) and progesterone (500 microg/rat s.c.) were acutely treated by oral gavage with ferutinin, teferin and teferdin (2.5 mg/kg). Thereafter they were tested for: a) partner preference, b) receptivity, c) proceptivity, d) paced mating behaviour. In the partner preference test, the choice of the female for a sexually active male was not influenced by the different treatments. Similarly, during the paced mating test, the contact-return latencies as well as the percentage of exits from the male compartment were not different in control and treated rats. Therefore none of the three compounds showed the capacity to alter sexual motivation. On the other hand, ferutinin, but not teferin and teferdin, significantly inhibited female receptivity. These results suggest a primary role of ferutinin in the impairment of sexual behaviour elicited by F. hermonis extract in hormone primed-female rats.

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. 17β-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.

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. Ferutinins 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.