Mechanisms involved in the endothelium-dependent vasodilatory effect of an ethyl acetate fraction of Cyathea phalerata Mart. in isolated rats' aorta rings.

The species Cyathea phalerata Mart. is a tree fern, commonly known as "xaxim", which is found in tropical and subtropical areas of Brazil. The present study investigated the mechanisms related with the vasorelaxant effects of an Ethyl Acetate Fraction (EAF) obtained from C. phalerata in rats' thoracic aorta rings. In pre-contracted vessels, EAF (0.1-1000 µg/mL) caused a concentration-dependent relaxation. The endothelium denudation, the nitric oxide (NO) synthase and guanylyl cyclase inhibitor reduced the vasodilation, indicating the participation of NO/cGMP pathway in its effect. The relaxation of EAF was abolished in the absence of extracellular Ca2+ and was significantly decreased in the presence of Ca2+ entry blocker, suggesting that Ca2+ influx plays an important role in EAF effect and probably in eNOS activity. However, the PI3K/Akt pathway is not responsible for eNOS phosphorylation/activation. The vasodilator effect of EAF was partially inhibited by KCl 40 mM and almost totally abolished with L-NOARG + KCl 40 mM, indicating also the role of hyperpolarization in its effect. Calcium activated K+ channels are not involved in the EAF-induced hyperpolarization. The COX inhibitor, indomethacin, slightly reduced the vasodilation induced by EAF. In addition, EAF did not alter the relaxant effects of NO-donor, indicating that the relaxant activity cannot be attributed to free radical-scavenging properties. In conclusion, the present study showed that the EAF, causes an endothelium-dependent vasorelaxant effect in aorta that mainly involves the NO-cGMP pathway, hyperpolarization and prostanoids. The vasorelaxant activity of EAF can be attributed to the occurrence of polyphenol compounds.

Phytochemical Composition, Antioxidant Activity, and the Effect of the Aqueous Extract of Coffee (Coffea arabica L.) Bean Residual Press Cake on the Skin Wound Healing.

The world coffee consumption has been growing for its appreciated taste and its beneficial effects on health. The residual biomass of coffee, originated in the food industry after oil extraction from coffee beans, called coffee beans residual press cake, has attracted interest as a source of compounds with antioxidant activity. This study investigated the chemical composition of aqueous extracts of coffee beans residual press cake (AE), their antioxidant activity, and the effect of topical application on the skin wound healing, in animal model, of hydrogels containing the AE, chlorogenic acid (CGA), allantoin (positive control), and carbopol (negative control). The treatments' performance was compared by measuring the reduction of the wound area, with superior result (p < 0.05) for the green coffee AE (78.20%) with respect to roasted coffee AE (53.71%), allantoin (70.83%), and carbopol (23.56%). CGA hydrogels reduced significantly the wound area size on the inflammatory phase, which may be associated with the well known antioxidant and anti-inflammatory actions of that compound. The topic use of the coffee AE studied improved the skin wound healing and points to an interesting biotechnological application of the coffee bean residual press cake.

In vivo potential hypoglycemic and in vitro vasorelaxant effects of Cecropia glazioviistandardized extracts

ABSTRACTThe aim of this study is to investigate the effect of Cecropia glaziovii Snethl, Urticaceae, extracts on the oral glucose tolerance curve, on glycemia in alloxan-induced diabetic rats and vasorelaxant effect after the extraction process, and to standardize the extractive solutions. The effects of the process variables and their interactions were calculated in relation to dry residue, pH, total phenolic results and chemical marker content. Furthermore, the effect of the extracts (400 mg/kg), chlorogenic (2 or 15 mg/kg) and caffeic acids (2 mg/kg) were investigated on the oral glucose tolerance curve and on glycemia in alloxan-induced diabetic rats. Oral administration of ethanol extracts 4d20 and 8d20 significantly improved glucose tolerance in the hyperglycemic rats. Chlorogenic and caffeic acids, as well as the association of the compounds were able to significantly reduce glycemia after oral gavage treatments. On the other hand, the aqueous extracts did not alter the glycemia. The aqueous extracts (8020 and 9030) and only the higher dose of chlorogenic acid presented a significant effect on serum glucose lowering in diabetic rats. Additionally, the IC50 reveals that the ethanol extracts presented more potent vasodilator effects than the aqueous extracts in aortic rings. This study shows that C. glazioviistandardized extracts exhibits antihyperglycemic action, is able to improve glucose tolerance and has a potent vascular relaxing effect. These results are probably linked to concentrations of the main phenolic compounds of the extracts.

Diphenyl diselenide protects endothelial cells against oxidized low density lipoprotein-induced injury: Involvement of mitochondrial function.

Elevated levels of oxidized low density lipoprotein (oxLDL) are considered to be one of the major risk factors for atherosclerosis and cardiovascular morbidity. The early stages of atherosclerosis are initiated by the accumulation of oxLDL and the induction of toxic effects on endothelial cells, resulting in endothelial dysfunction. The aim of this study was to investigate how diphenyl diselenide (DD), an organoselenium compound, protect vascular endothelial cells against the toxic effects of oxLDL in vitro. Our data showed that the treatment of bovine endothelial aortic cells (BAEC) with DD (0.1-1 µM) for 24 h protected from oxLDL-induced reactive species (RS) production and reduced glutathione (GSH) depletion. Moreover, DD (1 µM) per se improved the maximal mitochondrial respiratory capacity and prevented oxLDL-induced mitochondrial damage. In addition, DD could prevent apoptosis induced by oxLDL in BAEC. Results from this study may provide insight into a possible molecular mechanism underlying DD suppression of oxLDL-mediated vascular endothelial dysfunction.

Diphenyl diselenide differently modulates cardiovascular redox responses in young adult and middle-aged low-density lipoprotein receptor knockout hypercholesterolemic mice.

OBJECTIVES: The present work aimed to investigate the effect of (PhSe)2 on cardiovascular age-related oxidative stress in hypercholesterolemic mice. METHODS: To this end, LDL receptor knockout (LDLr(-/-) ) mice, 3 months (young adult) and 12 months (middle-aged) old, were orally treated with (PhSe)2 . KEY FINDINGS: Hypercholesterolemia, regardless of age, impaired the mitochondrial antioxidant defence in the cardiac tissue, which was characterized by a decline in mitochondrial aortic glutathione (GSH) levels and increased reactive oxygen species production in the heart. (PhSe)2 treatment improved GSH levels, thioredoxin reductase (TRxR) and GSH reductase (GR) activity, and decreased malondialdehyde levels in the heart of young adult LDLr(-/-) mice. Moreover, (PhSe)2 increased GPx activity in both age groups, and GR activity in the aorta of middle-aged LDLr(-/-) mice. CONCLUSIONS: Therefore, (PhSe)2 enhances the antioxidant defences in the cardiovascular system of LDLr(-/-) mice, which could explain its success as an anti-atherogenic compound.

Diphenyl diselenide prevents cortico-cerebral mitochondrial dysfunction and oxidative stress induced by hypercholesterolemia in LDL receptor knockout mice.

Recent studies have indicated a causal link between high dietary cholesterol intake and brain oxidative stress. In particular, we have previously shown a positive correlation between elevated plasma cholesterol levels, cortico-cerebral oxidative stress and mitochondrial dysfunction in low density lipoprotein receptor knockout (LDLr(-/-)) mice, a mouse model of familial hypercholesterolemia. Here we show that the organoselenium compound diphenyl diselenide (PhSe)2 (1 mg/kg; o.g., once a day for 30 days) significantly blunted the cortico-cerebral oxidative stress and mitochondrial dysfunction induced by a hypercholesterolemic diet in LDLr(-/-) mice. (PhSe)2 effectively prevented the inhibition of complex I and II activities, significantly increased the reduced glutathione (GSH) content and reduced lipoperoxidation in the cerebral cortex of hypercholesterolemic LDLr(-/-) mice. Overall, (PhSe)2 may be a promising molecule to protect against hypercholesterolemia-induced effects on the central nervous system, in addition to its already demonstrated antiatherogenic effects.

Cardioprotective effects of a proanthocyanidin-rich fraction from Croton celtidifolius Baill: focus on atherosclerosis.

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies have pointed to proanthocyanidins as promising molecules that could prevent the development of several coronary syndromes by inhibiting the atherogenic process. The present study was designed to investigate the antiatherogenic effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius Baill (Euphorbiaceae) barks. In isolated human LDL, PRF caused a concentration-dependent inhibition of Cu2+-induced oxidative modifications, evidenced by the increasing of the lag phase of lipid peroxidation and decreasing in the oxidation rate (Vmax), moreover, the protein moieties from LDL were protected against Cu2+-induced oxidation. In human umbilical vein endothelial cells (HUVECs), PRF reduced the ROS production stimulated by oxidized LDL. Herein, we demonstrate that oral treatment with PRF improved endothelium-dependent vasorelaxation in hypercholesterolemic LDL receptor knockout mice (LDLr-/-), however, the fraction did not modify plasma lipids and atherosclerotic lesion size in this experimental model. Finally, our results showed for the first time that PRF prevent isolated LDL oxidation, decrease oxidative stress in endothelial cells and improve endothelial function in mice.

Anti-tumour effects of elatol, a marine derivative compound obtained from red algae Laurencia microcladia.

OBJECTIVES: This paper aims to evaluate the anti-tumour properties of elatol, a compound (sesquiterpene) isolated from algae Laurencia microcladia. METHODS: In-vitro and in-vivo anti-tumour properties of elatol were investigated using: MTT assays to assess the cytotoxic effects; flow cytometry analysis to examine the cell cycle and apoptosis; Western blot analysis for determination of the expression of cell cycle and apoptosis proteins; and study of in-vivo tumour growth in mice (C57Bl6 mice bearing B16F10 cells). KEY FINDINGS: Elatol exhibited a cytotoxic effect, at least in part, by inducing cell cycle arrest in the G(1) and the sub-G(1) phases, leading cells to apoptosis. Western blot analysis demonstrated that elatol reduced the expression of cyclin-D1, cyclin-E, cyclin-dependent kinase (cdk)2 and cdk4. A decrease in bcl-xl and an increase in bak, caspase-9 and p53 expression was also observed. In the in-vivo experiment, treatment with elatol was able to reduce tumour growth in C57Bl6 mice. CONCLUSIONS: Elatol promotes a delay in the cell cycle, probably in the G(1)/S transition, activating the apoptotic process and this could be responsible, at least in part, for the in-vivo effects observed. Taken together, the in-vitro and in-vivo experiments suggested that elatol has anti-tumour properties. Further studies should be conducted to clarify the mechanism of action.

High performance liquid chromatography determination of cucurbitacins in the roots of Wilbrandia ebracteata Cogn. / Determinação de cucurbitacinas em raízes de Wilbrandia ebracteata Cogn. por cromatografia líquida de alta eficiência

Roots of Wilbrandia ebracteata Cogn., Cucurbitaceae, used in folk medicine for treatment of rheumatic disease, are rich in cucurbitacins. Dihydrocucurbitacin B is the most abundant cucurbitacin while cucurbitacin B is a minor component. A reverse-phase HPLC system was developed for simultaneous quantitative assay of these cucurbitacins in the roots. The optimised experimental conditions were acetonitrile/H(2)0 40:60, flow-rate 1.2 mL/min., detection at 230 nm and isocratic elution. A variety of sample preparation modes were tested and the extraction with dichloromethane under reflux gave better results. The validation process included linearity, accuracy, repeatability and intermediate precision. The calibration curve of dihydrocucurbitacin B was linear from 40.00 to 400 μg/mL, the recovery was 95.5±3.01 percent, the intermediate precision was found to be 1.64 percent and the repeatability varied between 1.30 to 2.05 percent. The calibration curve of cucurbitacin B was linear from 4.00 to 240 μg/mL, intermediate precision was found to be 2.29 percent and repeatability varied between 1.03 to 2.95 percent. Analysis of the same specimen of W. ebracteata every year from 2002 to 2005 revealed a great rise on the cucurbitacin B concentration after the root was attacked by an herbivore.

Raízes de Wilbrandia ebracteata Cogn. (Cucurbitaceae), tradicionalmente empregada no tratamento de doenças reumáticas, contém cucurbitacinas, sendo di-hidrocucurbitacina B a mais abundante, enquanto cucurbitacina B está presente em menor quantidade. Foi desenvolvido um método para determinação quantitativa destas cucurbitacinas. Os parâmetros selecionados foram: eluente isocrático acetonitrila/H(2)0 40:60, fluxo 1,2 mL/min. e detecção em 230 nm. Diversas formas de preparo da amostra foram testadas, sendo que extração com diclorometano sob refluxo forneceu o melhor resultado. O processo de validação incluiu: linearidade, exatidão, repetibilidade e precisão intermediária. A curva de calibração para a di-hidrocucurbitacina B foi linear de 40.00 to 400 μg/mL, a recuperação foi 95,5±3.01 por cento, a precisão intermediária, 1,64 por cento e a repetibilidade variou entre 1,30 a 2,05 por cento. A curva de calibração da cucurbitacina B foi linear de 4,00 to 240 μg/mL, a recuperação encontrada foi igual a 96,6±2.45 por cento, a precisão intermediária, 2,29 por cento e a repetibilidade variou entre 1,03 a 2,95 por cento. Análise do mesmo espécime de W. ebracteata uma vez por ano de 2002 a 2005 revelou grande aumento no teor de cucurbitacina B após a raiz ter sido atacada por herbívoro.

Evaluation of the antitumoral effect of dihydrocucurbitacin-B in both in vitro and in vivo models.

AIMS: We evaluated both in vitro and in vivo antitumoral properties of an isolated compound from Wilbrandia ebracteata, dihydrocucurbitacin-B (DHCB), using B16F10 cells (murine melanoma). MATERIALS AND METHODS: We made use of MTT and (3)H-Thymidine assays to investigate the cell viability and cell proliferation, flow cytometry analysis to monitor cell cycle and apoptosis, western blot analysis to evaluate the expression of cell cycle proteins, imunofluorescence analysis and in vivo tumor growth and metastasis. RESULTS: Dihydrocucurbitacin-B significantly reduced cell proliferation without important effects on cells viability. DHCB lead cells to accumulate in G2/M phases accompanied by the appearance of polyploid cells, confirmed by fluorescence assays that demonstrated a remarkable alteration in the cell cytoskeleton and formation of binuclear cells. Annexin-V-FITC incorporation demonstrated that DHCB did not induce apoptosis. About 10 microg/mL DHCB was found to decrease cyclin-A, and especially in cyclin-B1. The in vivo experiments showed that DHCB treatment (once a day up to 12 days; p.o.) was able to reduce the tumor growth and lung metastasis up to 83.5 and 50.3%, respectively. CONCLUSIONS: Dihydrocucurbitacin-B reduces cell proliferation due to a decrease in the expression of cyclins, mainly cyclin-B1 and disruption of the actin cytoskeleton, arresting B16F10 cells in G2/M phase. Taken together, the in vitro and in vivo experiments suggest that DHCB was effective against cancer, however, it remains to be proved if DHCB will be a good candidate for drug development.

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.

Antinociceptive effect of Croton celtidifolius Baill (Euphorbiaceae).

Croton celtidifolius Baill (Euphorbiaceae) is a tree found in the Atlantic forest of southern Brazil. This plant is used in folk medicine for the treatment of several inflammatory diseases, leukaemia, ulcers and other pathologies. Previous studies demonstrated anti-inflammatory and antioxidant activities and the objective of this work was to investigate a possible antinociceptive action of ethanolic extract of Croton celtidifolius bark (EE) and ethyl acetate fraction (EAF), n-butanol fraction (FBuOH), and aqueous fraction (FAq) obtained from EE. Two standard rodent models of pain were employed for this investigation, the writhing test and the formalin test. In the writhing test, the pre-treatment with EE significantly reduced the writhing induced by 0.6% acetic acid injection and its effect persisted for 4 h. In the formalin test, the pre-treatment with EAF caused marked and dose-related inhibition of formalin-induced licking in mice in the first phase, while pre-treatment with EAF, FBuOH and FAq had a similar effect in the second phase, when given by intraperitoneal (i.p.) and orally (p.o.) route. However, given by i.p. route, the effect of fractions was about three to five-fold more potent in inhibiting licking than when administered by p.o. route. EE presented an antinociceptive effect only in the second phase, when given by i.p. or p.o. route. The oedema caused by formalin was significantly reduced in animals treated i.p. with EAF, FBuOH and FAq. Under the same experimental conditions, in animals treated with sub-fractions derived from EAF only the 63 sub-fraction significantly reduced nociception in both phases and oedema caused by formalin. The results obtained suggest that Croton celtidifolius possesses antinociceptive properties since the EE, fractions and a sub-fraction significantly reduced the writhing induced by acetic acid and the nociception in both phases of the formalin test.

An ethyl acetate fraction obtained from a Southern Brazilian red wine relaxes rat mesenteric arterial bed through hyperpolarization and NO-cGMP pathway.

A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease (CAD). In this study, we investigated the effect of a crude extract (CE), as well as an ethyl acetate fraction (EAF) obtained from a Brazilian red wine in the mesenteric arterial bed (MAB) from rats. Our results showed that after the tonus of MAB was increased with phenylephrine (PE), increasing concentrations of CE induced a concentration-dependent relaxation; moreover, EAF was more potent in relaxing the MAB when compared with CE. In vessels depolarized with KCl (80 mM) or treated with the Na(+)/K(+)-ATPase pump inhibitor, ouabain (OUA; 100 microM), or with the K(+) channel blockers: barium (BaCl(2), 100 microM) and tetraethylammonium (TEA; 500 microM), the effect of EAF was significantly reduced. However, this effect was not altered by the ATP-dependent K(+) (K(ATP)) channel blocker, glibenclamide (GLI; 100 microM) as well as Charybdotoxin (ChTx 10 nM), a nonselective inhibitor of K(Ca) channels of large and intermediate conductance plus Apamin (Apamin 100 nM), a specific inhibitor of K(Ca) channels of small conductance. The residual vasodilator effect of EAF observed in vessels pretreated with L-NOARG (100 microM), 1H-[1,2,4,] oxadiazolo[4,3-alfa]quinoxalin, ODQ (10 microM) or KCl (80 mM), given separately, was reduced by the administration of KCl (40 mM) plus L-NOARG (100 microM). The present study demonstrates that the vasodilator effect of EAF is partially dependent upon membrane hyperpolarization in combination with nitric oxide (NO) release.

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.