Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function

Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological proper-ties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4'-sulfate and genistein-4'-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4'-sulfate was less potent than genistem, and genistein-4'-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis. (C) 2004 Elsevier B.V All rights reserved.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function

Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological proper-ties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4'-sulfate and genistein-4'-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4'-sulfate was less potent than genistem, and genistein-4'-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis. (C) 2004 Elsevier B.V All rights reserved.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.

Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation

BACKGROUND: Peroxisome proliferator-activated receptor-(gamma) (PPAR(gamma)) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. OBJECTIVE: In the present study, we aimed to demonstrate the ability of PPAR(gamma) ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. METHODS: Washed platelets were stimulated with PPAR(gamma) ligands in the presence and absence of PPAR(gamma) antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR(gamma) agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. RESULTS: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPAR(gamma) ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed. Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR(gamma) was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR(gamma) agonists, indicating a potential mechanism for PPAR(gamma) function in collagen-stimulated platelet activation. CONCLUSIONS: PPAR(gamma) agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5

Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.

Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function

BACKGROUND: Flavonoids may be partly responsible for some health benefits, including antiinflammatory action and a decreased tendency for the blood to clot. An acute dose of flavanols and oligomeric procyanidins from cocoa powder inhibits platelet activation and function over 6 h in humans. OBJECTIVE: This study sought to evaluate whether 28 d of supplementation with cocoa flavanols and related procyanidin oligomers would modulate human platelet reactivity and primary hemostasis and reduce oxidative markers in vivo. DESIGN: Thirty-two healthy subjects were assigned to consume active (234 mg cocoa flavanols and procyanidins/d) or placebo (< or = 6 mg cocoa flavanols and procyanidins/d) tablets in a blinded parallel-designed study. Platelet function was determined by measuring platelet aggregation, ATP release, and expression of activation-dependent platelet antigens by using flow cytometry. Plasma was analyzed for oxidation markers and antioxidant status. RESULTS: Plasma concentrations of epicatechin and catechin in the active group increased by 81% and 28%, respectively, during the intervention period. The active group had significantly lower P selectin expression and significantly lower ADP-induced aggregation and collagen-induced aggregation than did the placebo group. Plasma ascorbic acid concentrations were significantly higher in the active than in the placebo group (P < 0.05), whereas plasma oxidation markers and antioxidant status did not change in either group. CONCLUSIONS: Cocoa flavanol and procyanidin supplementation for 28 d significantly increased plasma epicatechin and catechin concentrations and significantly decreased platelet function. These data support the results of acute studies that used higher doses of cocoa flavanols and procyanidins.

Inhibition of platelet aggregation by omega-3 polyunsaturated fatty acids is gender specific — redefining platelet response to fish oils

Existence of gender differences in cardiovascular disease (CVD) following long-chain omega-3 polyunsaturated fatty acid (LCn-3 PUFA) supplementation have suggested that sex hormones play a role in cardio-protection. The objective of this study was to determine gender specific responses in the efficacy of LCn-3 PUFA to inhibit platelet aggregation in vitro. Blood was analyzed for collagen-induced platelet aggregation following pre-incubation with LCn-3 PUFA in healthy adults (n=42). Eicosapentaenoic acid (EPA) was significantly more effective in reducing platelet aggregation compared with docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). When grouped by gender, this differential pattern was followed in males only. In females, DHA, DPA and EPA were all equally effective. Between group analyses (LCn-3 PUFA vs. gender) showed that both DHA and DPA were significantly less effective in males compared with females. EPA was equally effective in reducing platelet aggregation in both groups. These findings show that significant gender differences exist in platelet aggregation in response to various LCn-3 PUFA treatments.

Efeito do Fucoidam de Fucus vesiculosus em um modelo experimental de artrite reumatóide

Rheumatoid arthritis (RA) is systemic auto imune disorder. It is caracterized by chronic inflammation of joints leading to progressive erosion of cartilage and bone. We investigated the effect of the administration of fucoidan, sulfated polysaccharides, from algae Fucus vesiculosus in the acute (6h) in zymosan-induced arthritis (AZy). Wistar rats (180-230 g) were used for all groups experimental. Non-treated animals received just intraarticular injection of 1 mg the zymosan, control group received intraarticular injection of 50 µL the saline, groups received either fucoidan of Fucus vesiculosus (15, 30, 50 or 70 mg/Kg) or parecoxib (1 mg/Kg) 1 hour after injection of zymosan. After 6 h, the articular exudates were collected for evaluation of the cell influx and nitrite (Griess reaction) release. The sinovial membranes and articular cartilages were excised for histopathological analysis and by determination of the glycosaminoglycan (GAG), respectively. ZyA led to increased NO and cell influx into the joints. Therapeutic administration of the fucoidan or parecoxib did significantly inhibited the cell influx and the synovitis, as compared to non-treated rats (p<0,05), though being able to reduced NO release. Representative agarose gel electrophoresis of the GAGs, the content of condroitin-sulphate was observed during the process. These findings suggest that the fucoidan from Fucus vesiculosus has potential anti-inflammatory activity

Efeito de frações obtidas da fucoidana de Fucus vesiculosus em modelo experimental de artrite induzida por Zymosan

The fucoidan from Fucus vesiculosus is known for having diverse biological properties. This study analyzed the therapeutic action of populations of commercial fucoidan (F. vesiculosus) on zymosan-induced arthritis. Three populations of fucoidan were obtained after acetone fractionation; these were denominated F1 (52.3%), F2 (36.7%) and F3 (10.7%). Chemical analyses showed that F1 contained the largest amount of sulfate ion. The electrophoretic profile shows that the commercial or total fucoidan (TF), different from the other fucoidans and from glycosaminoglycan patterns, is quite polydisperse, which indicates that it is composed of a mixture of sulfate polysaccharides. On the other hand, the fucoidans obtained from TF showed only an electrophoretic band with much lower polydispersion than that observed for TF. Fucoidan F2 showed a migration between fucoidans F1 and F3. Owing to the small amount of mass obtained from F3, we used only fucoidans F1 and F2 in the induced arthritis tests. After 1 hour of induction, we administered F1 or F2 (10, 25 and 50 mg/kg i.p.) or diclofenac sodium (10 mg/kg i.p.) or lumiracoxib (5 mg/kg o.a.) or L-NAME (30 mg/kg i.p.). After 6 hours, we performed analyses of cell influx and nitrite levels in addition to histopathological analysis. Fucoidans F1 and F2 were more potent both in decreasing the number of leukocytes and the amount of nitric oxide found in the synovial fluid. This indicates that the anti-inflammatory mechanism of these fucoidans is not only related to selectin block, but also to nitric oxide synthesis inhibition

Avaliação de uma fração polissacaridica da alga Lobophora variegata (Lamouroux) em modelo de artrite induzida em ratos por Zymosan

Fucans seaweed Lobophora variegata estructures are known for their chemical and biological properties. In this study, we analyzed, the action of fucans L. variegata and the fractions purified with acetone in Zymosan-induced arthritis. After differential fractionation with acetone, six fractions were obtained and named F0.3, F0.5, F0.8, F1, F1.5 and F2. The results showed that the fraction F1 showed high yield (51.9%) and was chosen for studies of antioxidant activity and induced arthritis. Nuclear magnetic resonance (NMR) of 13C showed signals at 103.3 and 15.78 ppm that are assigned to links β13 galactose and of the C6 methyl fucose, respectively. The infrared (IR) showed absorbance at 1238 and 850 cm-1 which are attributed to sulfate. The fraction F1 showed antioxidant activities in vitro. For analysis of inflammatory parameters chosen the polysaccharide was administered in different doses (25, 50 and 75 mg / kg ip, per body weight) and diclofenac sodium (5 mg / kg ip) and L-NAME (25 mg / kg ip) in groups of animals (n = 6). After 6 h, were analyzed for cellular influx and levels of nitrite. In experiment five days, were made analysis of swelling and serum TNF-α. Histopathological analysis were performed for confirmation of results. The fraction F1 (25, 50 and 75 mg / kg ip) reduced the cellular influx (52.1 to 96.7%) and nitric oxide levels (27.2 - 39%) compared to control group. The reduction of edema (63.4 - 100%) and serum TNF-α (p <0.001) were observed when the polysaccharide F1 administered at a dose (50 mg / kg) These results suggest that these heterofucanas of Lobophora variegata have besides the activity antioxidant and potential anti-inflammatory activity in arthritis induced by zymosan

Effect of bovine bone morphogenetic proteins on radius fracture healing in rabbits

OBJETIVO: Investigar a influência de Proteínas Morfogenéticas Ósseas de origem bovina (bBMPs) ligadas a hidroxiapatita mais colágeno na consolidação de fraturas instáveis do rádio. MÉTODOS: em 15 coelhos com aproximadamente 5,5 meses de idade e peso médio de 3,5kg foi realizada uma fratura transversa na porção média da diáfise do rádio de ambos os membros. Na fratura do rádio direito foi aplicada mistura de bBMPs ligadas à hidroxiapatita (bBMP-HA) e colágeno bovino como aglutinante e na do rádio esquerdo, considerada controle, nenhum tratamento foi usado. Os coelhos (cinco por período) foram submetidos à eutanásia aos 30, 60 e 90 dias após a cirurgia para realização do processamento histológico e análise microscópica. RESULTADOS: A análise histológica descritiva revelou que a consolidação foi similar para os membros tratado e controle. Pela análise histomorfométrica, a área de novo osso foi em média 867442,16 mm², 938743.00 mm² e 779621,06 mm² para os membros controles e 841118,47 mm², 788038,76mm² e 618587,24 mm² para os membros tratados, aos 30, 60 e 90 dias, respectivamente. Desta forma, aos 60 dias de pós-operatório a área de novo osso foi 12.17% maior no membro tratado com bBMP-HA/colágeno em relação ao membro controle (p<0.05, teste de Tukey). em ambos os membros a área de novo osso aumentou durante o período experimental até a total consolidação da fratura. CONCLUSÃO: Baseado nos resultados obtidos foi possível concluir que a mistura de bBMP-HA/colágeno induziu pequena, porém significante melhora na consolidação da fratura.

Commercial Bovine Proteoglycan Is Highly Arthritogenic and Can Be Used as an Alternative Antigen Source for PGIA Model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of dipyrone, meloxicam, or the combination on hemostasis in conscious dogs

To compare the effects of dipyrone, meloxicam, and of the combination of these drugs on hemostasis in dogs. Prospective, blinded, randomized crossover study. Research laboratory at a veterinary teaching hospital. Six adult dogs. Animals received 4 intravenous treatments with 15-day washout intervals: control (physiological saline, 0.1 mL/kg), meloxicam (0.2 mg/kg), dipyrone (25 mg/kg), and dipyrone-meloxicam (25 and 0.2 mg/kg, respectively). A jugular catheter was placed for drug injection and for collecting samples for whole blood platelet aggregation (WBPA) and thromboelastometry assays at baseline, 1, 2, 3, 5, and 8 hours after treatment administration. The percent change from baseline of lag time and of the area under the curve (AUC) of impedance changes in response to collagen-induced platelet activation were recorded during WBPA. Thromboelastometry-derived parameters included clotting time, clot formation time, alpha-angle, and maximum clot firmness. The buccal mucosal bleeding time was evaluated by a blinded observer at baseline, 1, 3, and 5 hours after treatment injection. No significant changes in WBPA and thromboelastometry were recorded in the control treatment. Dipyrone significantly (P < 0.05) increased the lag time for 2 hours and decreased the AUC for 3 hours after injection. Meloxicam did not alter WBPA. Dipyrone-meloxicam significantly increased lag time for 2 hours and decreased the AUC for 5 hours after treatment injection. Experimental treatments did not differ from the control treatment for thromboelastometry and buccal mucosal bleeding time. While meloxicam does not alter hemostasis by the methods evaluated, dipyrone inhibits platelet aggregation for up to 3 hours. Meloxicam-dipyrone combination causes more prolonged inhibition of platelet function than dipyrone alone. Decreased platelet aggregation induced by dipyrone and dipyrone-meloxicam does not appear to impact the viscoelastic properties of the blood clot nor increase the risk of bleeding in dogs without preexisting hemostatic disorders.