ABSTRACT
Abstract Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several studies related to its phytoestrogenic activity. The aim of this study was to establish germination and cell cultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured and wild plants harvested in two different seasons. Murashige Skoog medium supplemented with naphthalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultures were analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cultivation. The chemical analysis was performed by ultra performance liquid chromatography, using the linearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrations of isoflavones detected in wild plants were different in the two harvest periods and contrasted in content when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentration of isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promote reduction of clumps and ended up altering the characteristics of secondary metabolites production in some cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g-1), and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g-1) at 60 days. The methods used for this research were effective, and the red clover plants of the analyzed variety can be cultivated in vitro aiming the commercial productivity by having contents greater than or equal to the wild plants in the periods studied, even without the use of elicitors during the cultivation.
ABSTRACT
To elucidate the histological findings of the anlage of the mandibular condyle during very early developmental stages, we analyzed sagittal and frontal plane serial sections of mouse fetuses for which the gestational period was precisely determined. An aggregate of mesenchymal cells around the buccal nerve (peripheral cell aggregate) could be seen at 12.0 days post-conception (dpc). Another cell aggregate (core cell aggregate), which almost coincided with the outline of the condylar head, was detected on the inside of the dome-shaped peripheral cell aggregate at 12.75 dpc. The cells of the peripheral cell aggregate were gradually flattened in accordance with cell differentiation, and formed a fibrous sheath covering the condylar head by 15.0 dpc. The cells of the central region of the core cell aggregate differentiated into hypertrophic chondrocytes by 14.5 dpc, whereas the cells of the fringe of the core cell aggregate differentiated into osteogenic cells to form the bone collar by 15.0 dpc. The continuity of the anlage of the condyle with that of the mandibular ramus was first recognized at 13.0 dpc. As the anlage of the mandibular condyle was observed histologically during very early developmental stages, further research is necessary to characterize the development of this anlage in greater detail.