Decreased forelimb ability in mice intracerebroventricularly injected with low dose 6-hydroxidopamine: A model on the dissociation of bradykinesia from hypokinesia.

Bradykinesia and hypokinesia represent well-known motor symptoms of Parkinson's disease (PD). While bradykinesia (slow execution of movements) is present in less affected PD patients and aggravates as the disease severity increases, hypokinesia (reduction of movement) seems to emerge prominently only in the more affected patients. Here we developed a model based on the central infusion of low dose (40µg) 6-hydroxydopamine (6-OHDA) in mice in an attempt to discriminate bradykinesia (accessed through forelimb inability) from hypokinesia (accessed through locomotor and exploratory activities). The potential beneficial effects of succinobucol against 6-OHDA-induced forelimb inability were also evaluated. One week after the beginning of treatment with succinobucol (i.p. injections, 10mg/kg/day), mice received a single i.c.v. infusion of 6-OHDA (40µg/site). One week after 6-OHDA infusion, general locomotor/exploratory activities (open field test), muscle strength (grid test), forelimb skill (single pellet task), as well as striatal biochemical parameters related to oxidative stress and cellular homeostasis (glutathione peroxidase, glutathione reductase and NADH dehydrogenases activities, lipid peroxidation and TH levels), were evaluated. 6-OHDA infusions did not change locomotor/exploratory activities and muscle strength, as well as the evaluated striatal biochemical parameters. However, 6-OHDA infusions caused significant reductions (50%) in the single pellet reaching task performance, which detects forelimb skill inability and can be used to experimentally identify bradykinesia. Succinobucol partially protected against 6-OHDA-induced forelimb inability. The decreased forelimb ability with no changes in locomotor/exploratory behavior indicates that our 6-OHDA-based protocol represents a useful tool to mechanistically study the dissociation of bradykinesia and hypokinesia in PD.

Fractal analysis of extra-embryonic vessels of chick embryos under the effect of glucosamine and chondroitin sulfates.

Like heparan sulfate proteoglycans, some monosaccharides and glycosaminoglycans, such as sulfated glucosamine (GS) and chondroitin (CS), integrate the vascular extracellular matrix and may influence vascular endothelial cell growth. To assess the effects of these substances on blood vessel formation, we used the chick yolk sac membrane (YSM) model and fractal geometry quantification, which provided an objective in vivo method for testing potential agents that promote vasculogenesis and angiogenesis. An image processing method was developed to evaluate YSM capillary vessels after they were implanted in a methylcellulose disk of GS or CS at a concentration between 0.001-0.1mg/disk (performed on 2-day old embryos). This method resulted in a binary image of the microvascular network (white vessels on a black background). Fractal box-counting (DBC) and information (DINF) dimensions were used to quantify the activity of GS and CS in vasculogenesis and angiogenesis. YSM treated with GS (0.001-0.1mg) and CS (0.03-0.1mg) showed an increase in fractal dimensions that corresponded to vitelline vessel growth compared to the control group (vehicle), with GS displaying higher fractal dimension values.

Southern Brazilian autumnal propolis shows anti-angiogenic activity: an in vitro and in vivo study.

The present study focuses on the effects of a hydro-alcoholic propolis extract collected in autumn (2010) in Santa Catarina State (Southern Brazil), on the angiogenesis, using in vitro and in vivo models. Cultures of human umbilical vein endothelial cells were used to assess the effects of propolis on viability, proliferation, and cell migration, as well as capillary tube formation. The propolis autumnal extracts significantly decreased the cell viability, based on CC50 values, which decreased (56%) from 297 to 130 µg/ml in 24 h and 72 h of treatment, respectively (cytotoxicity assay). The process of cell proliferation was decreased by 81.7 to 48.4% due to exposure (72 h) to 130-180 µg/ml of propolis extract, as compared with control (vehicle). In these same concentrations, the cell migration was also reduced by 39.6 to 12.6%, respectively (versus control). Furthermore, autumnal propolis extract (100-200 µg/ml) inhibited the tube-like structure formation (tubulogenesis) of endothelial cells on Matrigel™ (16.2-69.9% inhibition). The treatments performed in vivo with administration of 450 mg propolis.kg(-1) inhibited both angiogenesis and vasculogenesis by 82.3 and 66.5% in the chorioallantoic and yolk-sac membranes of chick embryos. Furthermore, by means of UV-vis-spectrophotometry, reverse phase-high performance liquid chromatography analysis and 1D and 2D-nuclear magnetic resonance experiments reveal higher contents of flavonoids and total phenolic compounds with predominance of the flavonol quercetin and the phenolic acids, e.g., gallic acid, protocatechuic acid and chlorogenic acid in the propolis hydro-alcoholic extract. Our findings related to the anti-proliferative, anti-migration, and anti-tubulogenic actions on human umbilical vein endothelial cell line agree with the inhibitory effects in the in vivo vessel formation exerted by propolis extract under study. The results also suggest that autumnal propolis extract might be potentially instrumental in providing alternative tools for angiogenic disease therapeutics.

Carotenoid and anthocyanin contents of grains of Brazilian maize landraces.

BACKGROUND: Carotenoid and anthocyanin contents of 26 maize landraces cultivated in southern Brazil were determined to evaluate their potential as natural colorants or functional food ingredients. RESULTS: The major carotenoids detected in the whole grain flour were zeaxanthin and lutein. Anthocyanins of landraces with purple starchy endosperm (Lingua de Papagaio and Mato Grosso Palha Roxa) were more extractable in methanol-HCl (1%, v/v), exhibiting 2.45 and 0.94 g kg(-1) of whole grains flour, respectively. In contrast, butanol-HCl (30%, v/v) was more effective for the extraction of anthocyanins from the purple-colored landraces Roxo 29 and Roxo 41; genotypes with pigments localized in the outer parts (pericarp) of grains (2.60 and 2.19 g kg(-1)). The Roxo 41 landrace showed the highest concentration of pigments, e.g. 11.72 10(-3) g kg(-1) of total carotenoids and 2.16 g kg(-1) of total anthocyanins. Similarly, the yellow-colored MPA 1 and the purple-colored Roxo 29 landraces showed prominent amounts of carotenoids (10.86 10(-3) g kg(-1)) and anthocyanins (2.60 g kg(-1)), respectively. CONCLUSION: Our findings suggest that the colored grains of maize landraces studied may hold promise for the development of grain-based functional foods or natural colorants regarding their carotenoid and anthocyanin contents and as genetic resource in breeding programs.

Metabolic fingerprint of Brazilian maize landraces silk (stigma/styles) using NMR spectroscopy and chemometric methods.

Aqueous extract from maize silks is used by traditional medicine for the treatment of several ailments, mainly related to the urinary system. This work focuses on the application of NMR spectroscopy and chemometric analysis for the determination of metabolic fingerprint and pattern recognition of silk extracts from seven maize landraces cultivated in southern Brazil. Principal component analysis (PCA) of the (1)H NMR data set showed clear discrimination among the maize varieties by PC1 and PC2, pointing out three distinct metabolic profiles. Target compounds analysis showed significant differences (p < 0.05) in the contents of protocatechuic acid, gallic acid, t-cinnamic acid, and anthocyanins, corroborating the discrimination of the genotypes in this study as revealed by PCA analysis. Thus the combination of (1)H NMR and PCA is a useful tool for the discrimination of maize silks in respect to their chemical composition, including rapid authentication of the raw material of current pharmacological interest.

Metabolomic analysis of Ocotea odorifera cell cultures: a model protocol for acquiring metabolite data.

Metabolomics constitutes a quantitative and qualitative survey of the whole metabolites of an organism as well as a tissue, reflecting the genome and proteome of a sample as analyzed. Advanced analytical spectroscopic and chromatographic techniques are used along with uni- or multivariate statistical data analysis, rapidly identifying up- or down-regulated metabolites in complex matrices. In this chapter, protocols for the analysis of target compounds (protocol I) and metabolomics (protocol II) of Ocotea odorifera cell cultures are described. In the first case, the target compound safrole, an aromatic ether used as a flavoring agent and also in the manufacture of insecticides, is analyzed in the organosolvent fraction of stable prototrophic cell lines of O. odorifera by gas chromatography-mass spectrometry. For metabolomics studies the protocol is designed to detect and quantify metabolites in the aqueous extract of O. odorifera cell lines by using high-resolution 1D- and 2D-nuclear magnetic resonance spectroscopy, followed by chemometric analysis of the 1H NMR spectra dataset. Protocol I has been successfully used, for example, in screening studies of cell lines able of producing safrole. Protocol II is suitable to detect the chemical features of a number of metabolite compounds in aqueous extracts of O. odorifera cell lines cultured under certain conditions, leading to new insights into metabolomics of that species.

Trans-resveratrol inhibits early blood vessel formation (vasculogenesis) without impairment of embryonic growth.

Resveratrol is a stilbene compound found in grapes and other sources. In this study we examined the effects of trans-resveratrol (4.38 - 438 microM/implant) in the vasculogenesis of yolk-sac membranes and its capacity to improve chick embryo growth. High concentrations of the stilbene (43.8 - 438 microM) significantly inhibited early vessel formation, decreasing the percentage vitelline vessels of 3.5-day embryos by 50% compared to the control. In addition, basic fibroblast growth factor-stimulated vasculogenesis (140% of vessels as compared to control) was partially reversed by t-resveratrol (35% of inhibition) and treatments with cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) as well a protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate) decreased the vessel number to 60%, 50%, and 44%, respectively. Treatments with t-resveratrol (4.38 - 43.8 microM/implant) significantly increased the body length of embryos incubated in vitro uncoupled from any impairment in the body shape or detectable embryotoxic effect. We suggest that the antivasculogenic activity and the enhancement in embryonic growth promoted by non acute treatments with t-resveratrol were, at least in part, due to PKC inhibition. We suggest that t-resveratrol can be usable not only as a reliable functional nutriment, but also is useful for the development of prophylactic and/or therapeutic agents for treatment of angiogenic-degenerative diseases.

A polysaccharide isolated from the brown seaweed Sargassum stenophyllum exerts antivasculogenic effects evidenced by modified morphogenesis.

A polysaccharide (Sarg) extracted from the brown marine alga Sargassum stenophyllum was studied for its antivasculogenic effects in both in vivo and in vitro assays, as well as for its capacity to modify embryonic morphogenetic processes endogenously regulated by bFGF, a well-known angiogenic stimulator. The antivasculogenic activity of Sarg (6-1500 microg/implant) was evaluated in a chick yolk sac membrane assay and the embryonic morphogenesis was measured as the percentage cephalic length. Sarg alone (96-1500 microg/implant) and co-administered with hydrocortisone (HC; 156 microg/implant) decreased the vitelline vessel number by 23-100% and 54-100% respectively. The polysaccharide potentiated the antivasculogenic effect of HC (42% inhibition). Basic fibroblast growth factor-stimulated vasculogenesis (141% of vessels as compared to control) was partially reversed by Sarg. The treatment with Sarg also decreased the percentage cephalic length of 3.5- and 4-day chick embryos (as cultured in vivo and in vitro, respectively), uncoupled from any impairment in the body shape or embryotoxic effect. Due to polyanionic characteristics of Sarg, which are similar to those seen in the heparin molecule, we suggest that this polysaccharide should modulate the activity of heparin-binding vascular growth factors (such as bFGF, which also acts as a morphogen) mimetically interfering with heparan sulfate proteoglycans during microvessel formation.

Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum.

The potential antiangiogenic and antitumoral properties of SargA, a polysaccharide extracted from the brown marine alga Sargassum stenophyllum, were studied in assays carried out in chick embryos and mice. Gelfoam plugs containing SargA (2-1500 microg/plug) implanted in vivo into fertilized 6-day-old chicken eggs induced dose-related antiangiogenic activity in the chorioallantoic membrane (CAM). By day 8, the highest dose of SargA alone decreased the vessel number in the CAM by 64%, but coadministered with hydrocortisone (156 microg/plug, which alone caused 30% inhibition) failed to potentiate its antiangiogenic effect. Combined with basic fibroblast growth factor (50 ng/plug), SargA (1500 microg/plug) abolished angiogenesis stimulated by this factor in both chick embryo CAM and in subcutaneous (s.c.) Gelfoam plugs implanted in the dorsal skin of Swiss mice (measured as plug hemoglobin content). Repeated s.c. injections of SargA (1.5 or 150 microg per animal per day for 3 days) close to B16F10 melanoma cell tumors in the dorsal skin of mice markedly decreased tumor growth in a dose-related fashion (by 40% and 80% at 2 weeks after the first injection, respectively), without evident signs of toxicity. SargA caused graded inhibitions of migration and viability of cultured B16F10 cells and also displayed antithrombotic activity in human plasma (5 mg/ml increased thrombin time 2.5-fold relative to saline). Thus, SargA exhibits pronounced antiangiogenic as well as antitumoral properties. Although the latter action of SargA might be related to the inhibition of angiogenesis, the polysaccharide also exerts cytotoxic effects on tumor cells. Because of its chemical characteristics and polyanionic constituents, we postulate that the polysaccharide SargA might modulate the activity of heparin-binding angiogenic growth factors.