Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester

Context and objective: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. Materials and methods: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. Results and discussion: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. Conclusion: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Hydroxyl scavenging activity accounts for differential antioxidant protection of Plantago major against oxidative toxicity in isolated rat liver mitochondria

Objectives The aim of this work was to study the effects of P. major against the oxidative damage of isolated rat liver mitochondria. Methods The extracts were obtained using methanol (MeOH), ethyl acetate (EAc), dichloromethane (DCM), and hexane (Hex) as solvents. Key findings Hex, DCM, and EAc totally, and MeOH partially, inhibited ROS generation and lipid peroxidation of membranes induced by Fe2+ or t-BOOH. However, only MeOH was able to prevent the t-BOOH-induced glutathione and NAD(P)H oxidation. All extracts chelated Fe2+ and reduced DPP Hradicals. EPR analysis revealed that P. major exhibited potent scavenger activity for hydroxyl radicals. Conclusions The potent antioxidant activity exhibited by P. major was able to prevent oxidative mitochondrial damage, contributing to the understanding of its hepatoprotective action against ROS-mediated toxicity.

Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [ reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Analysis of Passion Fruit rinds (Passiflora edulis): Isoorientin quantification by hptlc and evaluation of antioxidant (radical scavenging) capacity

The content of isoorientin in passion fruit rinds (Passiflora Mulls fo. flavicarpa O. Degener) was determined by HPTLC (high performance thin layer chromatography) with densitometric analysis. The results revealed a higher amount of isoorientin in healthy rinds of P edulis (92.275 +/- 0.610 mg L-1) than in rinds with typical symptoms of PWV (Passion fruit Woodiness Virus) infection (28.931 +/- 0.346 mg L-1). The HPTLC data, allied to assays of radical scavenging activity, suggest the potential of P Mulls rinds as a natural source of flavonoids or as a possible functional food.

Antioxidant Activity of Brazilian Vegetables and Its Relation with Phenolic Composition

Vegetables are widely consumed in Brazil and exported to several countries. This study was performed to evaluate the phenolic content and antioxidant activity of vegetables commonly consumed in Brazil using five different methods, namely DPPH and ABTS free radical, beta-carotene bleaching, reduction of Fe3+ (FRAP), oxidative stability in Rancimat, and the chemical composition using gas chromatography-mass spectrometry (GC-MS). The content of phenolic compounds ranged from 1.2 mg GA/g (carrot) to 16.9 mg GA/g (lettuce). Vegetables presenting the highest antioxidant activity were lettuce (77.2 mu mol Trolox/g DPPH center dot; 447.1 mu mol F2+/g FRAP), turmeric (118.6 mu mol Trolox/g ABTS(center dot+); 92.8% beta-carotene), watercress and broccoli (protective factor 1.29-Rancimat method). Artichoke, spinach, broccoli, and asparagus also showed considerable antioxidant activity. The most frequent phenolic compounds identified by GC-MS were ferulic, caffeic, p-coumaric, 2-dihydroxybenzoic, 2,5-dihydroxybenzoic acids, and quercetin. We observed antioxidant activity in several vegetables and our results point out their importance in the diet.

Analysis of passion fruit rinds (Passiflora edulis): isoorientin quantification by HPTLC and evaluation of antioxidant (radical scavenging) capacity

The content of isoorientin in passion fruit rinds (Passiflora edulis fo. flavicarpa O. Degener) was determined by HPTLC (high performance thin layer chromatography) with densitometric analysis. The results revealed a higher amount of isoorientin in healthy rinds of P. edulis (92.275 ± 0.610 mg L-1) than in rinds with typical symptoms of PWV (Passion fruit Woodiness Virus) infection (28.931 ± 0.346 mg L-1). The HPTLC data, allied to assays of radical scavenging activity, suggest the potential of P. edulis rinds as a natural source of flavonoids or as a possible functional food.

Antioxidant, anti-acetylcholinesterase and cytotoxic activities of ethanol extracts of peel, pulp and seeds of exotic Brazilian fruits Antioxidant, anti-acetylcholinesterase and cytotoxic activities in fruits

Ethanol extracts of powdered genipap (Genipa americana L), umbu (Spondia tuberosa A.) and siriguela (Spondia purpurea L) prepared from separate pulp, seeds and peel were investigated for their (i) antioxidant capacity, which was evaluated by various known methods; (ii) acetylcholinesterase (AChE) inhibitory activity; and (iii) cytotoxic effect on corneal epithelial cells of sheep. The highest values of total phenolic content were obtained with peel and seed extracts. Siriguela and umbu (seeds and peel) extracts displayed the highest antioxidant activities. Lipid peroxidation assays using mimetic biomembranes and mouse liver homogenates indicated that genipap pulp is a promising antioxidant. The investigation of phenols and organic acid contents revealed the presence of quercetin, citric and quinic acids, chlorogenic acid derivatives, among others, in several extracts, with the highest amount found in siriguela seeds. Genipap pulp and siriguela seed ethanol extracts presented an AChE inhibition zone similar to that of the positive control, carbachol. AChE inhibition assay with chlorogenic acid, one of the main constituents of siriguela seeds, revealed that this acid showed activity similar to that of the control physostigmine. These data suggest that these extracts are potentially important antioxidant supplements for the everyday human diet, pharmaceutical and cosmetic industries. (C) 2012 Elsevier Ltd. All rights reserved.

Increased Antioxidant Content in Juice Enriched with Dried Extract of Pomegranate (Punica granatum) Peel

Antioxidants are compounds responsible for free radical scavenging in the body. They protect the organism from oxidative modification of cells and tissues. These modifications have been associated with degenerative diseases, atherosclerosis and carcinogenesis. Punica granatum displays high antioxidant potential due to the presence of phenolic compounds, which are capable of disease prevention. The present study showed the highest antioxidant activity in pomegranate peel than in seeds and pulp. Based on these results, pomegranate peel was used to produce dried extract that was added to commercial tomato juice and orange juice with strawberries. Analysis to determine the content of phenolic compounds and antioxidant activity was performed on pomegranate pulp, seeds and peel and in juices enriched with dried extract of pomegranate peel. The dried extract was responsible for a significant increase in antioxidant activity of the juices, proportional to the concentrations added. However, although both flavors of enriched juices displayed high antioxidant levels, the samples with higher dried extract concentrations received the lowest scores from sensory analysis participants due to the characteristic astringent flavor of pomegranate peels. Therefore, to obtain greater acceptance in the consumer market, we concluded that the maximum addition of dried pomegranate peel extract is 0.5% in tomato juice and orange juice with strawberries.

Is cardiac tissue more susceptible than lung to oxidative effects induced by chronic nasotropic instillation of residual oil fly ash (ROFA)?

The current study aimed to determine the role of oxidants in cardiac and pulmonary toxicities induced by chronic exposure to ROFA. Eighty Wistar rats were divided into four groups: G1 (10 mu L Saline), G2 (ROFA 50 mu g/10 mu L), G3 (ROFA 250 mu g/10 mu L) and G4 (ROFA 500 mu g/10 mu L). Rats received ROFA by nasotropic instillation for 90 days. After that, they were euthanized and bronchoalveolar lavage (BAL) was performed for total count of leukocytes, protein and lactate dehydrogenase (LDH) determinations. Lungs and heart were removed to measure lipid peroxidation (MDA), catalase (CAT) and superoxide dismutase (SOD) activity. BAL presented an increase in leukocytes count in G4 in comparison to the Saline group (p = 0.019). In lung, MDA level was not modified by ROFA, while CAT was higher in G4 when compared to all other groups (p = 0.013). In heart, G4 presented an increase in MDA (p = 0.016) and CAT (p = 0.027) levels in comparison to G1. The present study demonstrated cardiopulmonary oxidative changes after a chronic ROFA exposure. More specifically, the heart tissue seems to be more susceptible to oxidative effects of long-term exposure to ROFA than the lung.

Efficacy of Cosmetic Formulations Containing Dispersion of Liposome with Magnesium Ascorbyl Phosphate, Alpha-Lipoic Acid and Kinetin

The present study aimed to evaluate the photoprotective effects of cosmetic formulations containing a dispersion of liposome with magnesium ascorbyl phosphate (MAP), alpha-lipoic acid (ALA) and kinetin, as well as their effects on the hydration and viscoelastic skin properties. The photoprotection was determined in vitro (antioxidant activity) and in vivo on UV-irradiated hairless mouse skin. The hydration effects were performed with the application of the formulations under study on the forearm of human volunteers and skin conditions were analyzed before and after a single application and daily applications during 4 weeks in terms of transepidermal water loss (TEWL), skin moisture and viscoelastic properties. The raw material under study possessed free-radical scavenging activity and the formulation with it protected hairless mouse skin barrier function against UV damage. After 4 weeks of application on human skin, the formulation under study enhanced stratum corneum skin moisture and also showed hydration effects in deeper layers of the skin. Thus, it can be concluded that the cosmetic formulation containing a dispersion of liposome with MAP, ALA and kinetin under study showed photoprotective effects in skin barrier function as well as pronounced hydration effects on human skin, which suggests that this dispersion has potential antiaging effects.

Oral Administration of Oleic or Linoleic Acids Modulates the Production of Inflammatory Mediators by Rat Macrophages

Oleic (OLA) and linoleic (LNA) acids are commonly consumed fatty acids and they can modulate the inflammatory response, in which macrophages play an important role. The aim of this study was to investigate the effects of these two fatty acids on the production of inflammatory mediators by macrophages. Rats received oral administration of water (control), OLA or LNA (0.22 g/kg body weight) daily for 10 days and peritoneal resident macrophages were then isolated. Subsequently, they were seeded in culture plates and the production of various inflammatory mediators was assessed. Oral administration with OLA decreased the production of IL-1 beta, IL-6 and CINC-2 alpha beta by resident macrophages and LNA decreased the production of IL-1 beta, IL-6 and VEGF in the absence of lipopolysaccharide (LPS), although it accelerated IL-1 beta release and decreased IL-10 synthesis when cells were stimulated with LPS. Neither fatty acid affected the production of superoxide anion, hydrogen peroxide, nitrite, TNF-alpha, PGE(2), LTB4 or 15(S)-HETE. Thus, OLA and LNA influence the production of several inflammatory mediators by macrophages.

Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen (1O2). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of 1O2 by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While 1O2 is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of 1O2 we have established a new cytometric method that allows the stage specific quantification of 1O2. Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200600 pixels for rings, 7001,200 pixels for trophozoites and 1,4002,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous 1O2 of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that 1O2 derives predominantly from the digestion of hemoglobin. (c) 2012 International Society for Advancement of Cytometry

The basic antioxidant structure for flavonoid derivatives

An antioxidant structure-activity study is carried out in this work with ten flavonoid compounds using quantum chemistry calculations with the functional of density theory method. According to the geometry obtained by using the B3LYP/6-31G(d) method, the HOMO, ionization potential, stabilization energies, and spin density distribution showed that the flavonol is the more antioxidant nucleus. The spin density contribution is determinant for the stability of the free radical. The number of resonance structures is related to the pi-type electron system. 3-hydroxyflavone is the basic antioxidant structure for the simplified flavonoids studied here. The electron abstraction is more favored in the molecules where ether group and 3-hydroxyl are present, nonetheless 2,3-double bond and carbonyl moiety are facultative.