Petals of Crocus sativus L. as a potential source of the antioxidants crocin and kaempferol.

Saffron from the province of L'Aquila, in the Abruzzo region of Italy, is highly prized and has been awarded a formal recognition by the European Union with EU Protected Designation of Origin (PDO) status. Despite this, the saffron regions are abandoned by the younger generations because the traditional cultivation of saffron (Crocus sativus L.) is labour intensive and yields only one crop of valuable saffron stamens per year. Petals of the saffron Crocus have had additional uses in traditional medicine and may add value to the crops for local farmers. This is especially important because the plant only flowers between October and November, and farmers will need to make the best use of the flowers harvested in this period. Recently, the petals of C. sativus L., which are considered a waste material in the production of saffron spice, were identified as a potential source of natural antioxidants. The antioxidants crocin and kaempferol were purified by flash column chromatography, and identified by thin layer chromatography (TLC), HPLC-DAD, infrared (IR), and nuclear magnetic resonance ((1)H &(13)C NMR) spectroscopy. The antioxidant activity was determined with the ABTS and DPPH tests. The antioxidant activities are mainly attributed to carotenoid and flavonoid compounds, notably glycosides of crocin and kaempferol. We found in dried petals 0.6% (w/w) and 12.6 (w/w) of crocin and kaempferol, respectively. Petals of C. sativus L. have commercial potential as a source for kaempferol and crocetin glycosides, natural compounds with antioxidant activity that are considered to be the active ingredients in saffron-based herbal medicine.

Ultrastructure of isolated mouse ovarian follicles cultured in vitro.

BACKGROUND: In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles. METHODS: The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH. RESULTS: The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). CONCLUSIONS: It is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.

Development of the microcirculation of the secondary ossification center in rat humeral head.

This work investigated the origin and development of microcirculation in the rat humeral head and the expression of vascular endothelial growth factor (VEGF) as a factor supporting the vascular growth and the development of the secondary ossification centers. Sixty rats aging 1, 3-4, 6-8, 11, and 21 days, 5 weeks, and 4 and 8 months were used. Samples of humeral head were collected for histology and immunohistochemistry for VEGF. Some animals were perfused with Mercox resin in order to obtain vascular corrosion casts (vcc) observed by scanning electron microscopy (SEM). No cartilage canals were present at birth. At 6 days postnatal, blood vessels coming from the perichondrium and the region near the capsule attachment invaded the cartilage; at 11 days postnatal, signs of calcification were present and within the third week some bone trabeculae were formed. Just before the vascular invasion of the epiphysis, a positive reaction for VEGF was localized in chondrocytes of the epiphyseal cartilage close to the capsule insertion. During the development and expansion of the secondary ossification center, VEGF expression was higher in chondrocytes but decreased when epiphysis was diffusely ossified. VEGF was expressed also by mesenchymal cells present in and around the fibrous tissue where the secondary ossification center will develop. SEM vcc confirmed that vessels penetrating into the epiphysis arose merely from the periosteal and the capsular networks, and vascular connections with the diaphyseal circulation were not evident. These observations demonstrated that VEGF production by chondrocytes begun some days after birth, supported the rapid vascular growth from the surrounding soft tissues, and was chronologically related to the development of the secondary ossification center in rat proximal humerus. Finally, the possible role of VEGF as mediator of angiogenesis and, at least indirectly, as a trigger factor also in the ossification and the bone remodeling of the secondary ossification centers has been discussed.