Extraction optimization of polyphenols, antioxidant and xanthine oxidase inhibitory activities from Prunus salicina Lindl.

Abstract Optimization of polyphenols extraction from plum (Prunus salicina Lindl.) was evaluated using response surface methodology. The Box-Behnken experimental results showed the optimal conditions involved an extraction temperature of 59 °C, a sonication time of 47 min, and an ethanol concentration of 61% respectively. The maximum extraction yield of total polyphenols was 44.74 mg gallic acid equivalents per gram of dried plum at optimal conditions. Polyphenol extracts exhibited stronger antioxidant activities than Vc by evaluating of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activity. Furthermore, polyphenol extracts (IC50 = 179 g/mL) showed obvious inhibitory effects on xanthine oxidase. These findings suggest that polyphenol extracts from P. salicina can be potentially used as natural antioxidant and xanthine oxidase inhibitory agents.

Distribuição da xantina oxidase no fígado e no sôro de rato

The localization of the xanthine oxidase (X.O.) and xanthine dehydrogenase (X.D.) activities in rat liver have been studied using separation of cytoplasmic particles into fractions by differential centrifugation. The results clearly demonstrate that practically all the enzymic activity is present in the supernatant fluid corresponding to the cell sap containing the soluble proteins of the cell. No activity could be detected for the nuclear, mitocondrial and microsomal fractions. The enzymatic activity of the mixture of the four factions was 102 per cent of that of the original homogenate. The distribution of the xanthine dehydrogenase in the protein fractions of the rat serum was accomplished in preliminary experiments by means of 50% ammonium sulphate precipitation and subsequent dialysis against water. All enzymatic activity was confined to the globulin fractions of the serum. Paper electrophoresis was performed and the protein and lipoprotein fractions determined. A method for the localization of the X.D. activity in the protein fractions separated by paper electrophoresis was developed. The results obtained suggest that xanthine dehydrogenase is localized in the globulin fractions possessing mobilities of [alpha 1], [beta] and [gamma] globulins and are probably bound to the lipoproteins.

Role of nitric oxide and superoxide in Giardia lamblia killing

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (~60% dead trophozoites). This effect was inhibited (>90%) by an NO synthase inhibitor (200 µM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 µM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (~35% dead trophozoites at 1 mM). The mixture of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoite killing and this effect is not mediated by peroxynitrite

Application of various antioxidants in the treatment of influenza

We determined the effect of the antioxidants superoxide dismutase, desferrioxamine and allopurinol on the survival of male CBA mice infected intranasally with 2-5 LD50 lung influenza virus A/Aichi/2/68. Survival for at least 20 days was observed for 45% of the mice that received 1000 U/day superoxide dismutase prepared from red blood cells on days 5, 6, 7 and 8 after infection, and 75% survival was observed for mice that received the same dose on days 4, 5, 6, 7 and 8. Desferrioxamine, 25 mg/kg per day and 100 mg/kg per day injected subcutaneously, resulted in survival rates of 5 and 0%, respectively, compared to 10% survival observed for saline-injected controls. Allopurinol at doses of 5 to 50 mg/kg per day had no effect on mouse survival. These data demonstrate the efficacy of superoxide dismutase for the protection of mice against hemorrhagic lung edema. The ineffectiveness of allopurinol suggests that the xanthine oxidase system does not play a major role in hemorrhage or lung edema and that caution is necessary when desferrioxamine is administered during an acute inflammatory process accompanied by erythrocyte lysis

Arachidonic acid triggers an oxidative burst in leukocytes

The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA) stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells), Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold), whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

Protective effect of N-acetylcysteine against oxygen radical-mediated coronary artery injury

The present study investigated the protective effect of N-acetylcysteine (NAC) against oxygen radical-mediated coronary artery injury. Vascular contraction and relaxation were determined in canine coronary arteries immersed in Kreb's solution (95% O2-5% CO2), incubated or not with NAC (10 mM), and exposed to free radicals (FR) generated by xanthine oxidase (100 mU/ml) plus xanthine (0.1 mM). Rings not exposed to FR or NAC were used as controls. The arteries were contracted with 2.5 µM prostaglandin F2alpha. Subsequently, concentration-response curves for acetylcholine, calcium ionophore and sodium fluoride were obtained in the presence of 20 µM indomethacin. Concentration-response curves for bradykinin, calcium ionophore, sodium nitroprusside, and pinacidil were obtained in the presence of indomethacin plus Nomega-nitro-L-arginine (0.2 mM). The oxidative stress reduced the vascular contraction of arteries not exposed to NAC (3.93 ± 3.42 g), compared to control (8.56 ± 3.16 g) and to NAC group (9.07 ± 4.0 g). Additionally, in arteries not exposed to NAC the endothelium-dependent nitric oxide (NO)-dependent relaxation promoted by acetylcholine (1 nM to 10 µM) was also reduced (maximal relaxation of 52.1 ± 43.2%), compared to control (100%) and NAC group (97.0 ± 4.3%), as well as the NO/cyclooxygenase-independent receptor-dependent relaxation provoked by bradykinin (1 nM to 10 µM; maximal relaxation of 20.0 ± 21.2%), compared to control (100%) and NAC group (70.8 ± 20.0%). The endothelium-independent relaxation elicited by sodium nitroprusside (1 nM to 1 µM) and pinacidil (1 nM to 10 µM) was not affected. In conclusion, the vascular dysfunction caused by the oxidative stress, expressed as reduction of the endothelium-dependent relaxation and of the vascular smooth muscle contraction, was prevented by NAC.

Desbalanço redox: NADPH oxidase como um alvo terapêutico no manejo cardiovascular

Vários estudos destacam as espécies reativas de oxigênio e nitrogênio (ERONs) como importantes contribuintes na patogênese de numerosas doenças cardiovasculares, incluindo hipertensão, aterosclerose e falência cardíaca. Tais espécies são moléculas altamente bioativas e com vida curta derivadas, principalmente, da redução do oxigênio molecular. O complexo enzimático da NADPH oxidase é a maior fonte dessas espécies reativas na vasculatura. Sob condições fisiológicas, a formação e eliminação destas substâncias aparecem balanceadas na parede vascular. Durante o desbalanço redox, entretanto, há um aumento na atividade da NADPH oxidase e predomínio de agentes pró-oxidantes, superando a capacidade de defesa orgânica antioxidante. Além disso, tal hiperatividade enzimática reduz a biodisponibilidade do óxido nítrico, crucial para a vasodilatação e a manutenção da função vascular normal. Apesar de a NADPH oxidase relacionar-se diretamente à disfunção endotelial, foi primeiramente descrita por sua expressão em fagócitos, onde sua atividade determina a eficácia dos mecanismos de defesa orgânica contra patógenos. As sutis diferenças existentes entre as unidades estruturais das NADPH oxidases, a depender do tipo celular que as expressa, podem ter implicações terapêuticas, permitindo a inibição seletiva do desequilíbrio redox induzido pela NADPH oxidase, sem comprometer, entretanto, sua participação nas vias fisiológicas de sinalização celular que garantem a proteção contra microorganismos.

Efeito do calor na atividade da polifenol oxidase e peroxidase em algumas frutas e hortaliças

O objetivo deste trabalho foi estudar o efeito de tratamentos térmicos na atividade da polifenol oxidase e da paroxidade em algumas frutas e hortaliças, bem como estudar a possível regeneração dessas enzimas após apertização. Em termos gerais, nas frutas a polifenol oxidase apresentou maior resistência à inativação pelo calor que a peroxidase e no caso das hortaliças ocorreu o inverso. Quanto à regeneração das enzimas após apertização, o fenômeno foi constatado somente no caso da peroxidase que mostrou assim grande estabilidade às condições adversas durante aquele tratamento térmico. A polifenol oxidase por sua vez, demonstrou ser uma enzima muito sensível, não se regenerando durante o tempo em que os produtos ficaram armazenados.

Efeito do SO2 e do ácido ascórbico na atividade da polifenol oxidase e peroxidase em algumas frutas e hortaliças

No presente trabalho foi estudado o efeito do SO2 e do ácido ascórbico na atividade da polifenol oxidase e peroxidase em algumas frutas e hortaliças, visando determinar as melhores condições para o controle da reação de escurecimento. Para a peroxidase das frutas e hortaliças, o ácido ascórbico foi considerado o melhor método de inativação. Já para a polifenol oxidase das frutas e hortaliças, o SO2 foi mais eficiente, porém mostrou-se inadequado para controlar a atividade da peroxidase em quase todos os produtos estudados. Uma combinação dos compostos utilizados (SQ2 e ácido ascórbico) parece ser o metodo mais indicado para o controle simultâneo da atividade das duas enzimas.

Efeito comparativo do calor, S0(2) e ácido ascórbico na atividade da polifenol oxidase e peroxidase de algumas frutas e hortaliças

O objetivo deste trabalho foi o de comparar o efeito do calor, do SO2 e do ácido ascorbico, visando determinar qual o método mais eficiente para controlar o escurecimento enzímico de cada uma das frutas e hortaliças estudadas. Os resultados mostraram que para a banana, pêssego, maça, cenoura, couve-flor e palmito, o calor foi o melhor agente inativador do sistema enzímico responsável pelo escurecimento. O SO2 foi mais eficiente para a pera, e para o figo e batata, o melhor agente inibidor foi o ácido ascórbico.

Signal transduction and activation of the NADPH oxidase in eosinophils

Activation of the eosinophil NADPH oxidase and the subsequent release of toxic oxygen radicals has been implicated in the mechanism of parasite killing and inflammation. At present, little is known of the signal transduction pathway that govern agonist-induced activation of the respiratory burst and is the subject of this review. In particular, we focus on the ability of leukotrine B4 to activate the NADPH oxidase in guinea-pig peritoneal eosinophils which can be obtained in sufficient number and purity for detailed biochemical experiments to be performed.

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotTManalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.

Mechanisms for suppressing NADPH oxidase in the vascular wall

Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex as expressed in vascular cells differs from that in phagocytic leucocytes both in biochemical structure and functions. The crucial flavin-containing catalytic subunits, Nox1 and Nox4, are not found in leucocytes, but are highly expressed in vascular cells and upregulated with vascular remodeling, such as that found in hypertension and atherosclerosis. The difference in catalytic subunits offers the opportunity to develop "vascular specific" NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic functions carried out by reactive oxygen and nitrogen species. Nitric oxide and targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack, than are conventional antioxidants. The roles of Nox isoforms in other inflammatory conditions are yet to be explored.