Free radical scavenging profile and myeloperoxidase inhibition of extracts from antidiabetic plants: Bauhinia forficata and Cissus sicyoides

There is abundant evidence that reactive oxygen species are implicated in several physiological and pathological processes. To protect biological targets from oxidative damage. antioxidants must react with radicals and other reactive species faster than biological substrates do. The aim of the present study was to determine the in vitro antioxidant activity of aqueous extracts from leaves of Bauhinia forficata Link (Fabaceae - Caesalpinioideae) and Cissus sicyoides L. (Vitaceae) (two medicinal plants used popularly in the control of diabetes mellitus), using several different assay systems, namely, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decolorization. superoxide anion radical (O-2 center dot-) scavenging and myeloperoxidase (MPO) activity. In the ABTS assay for total antioxidant activity, B. forficata showed IC50 8.00 +/- 0.07 mu g/mL, while C. sicyoides showed IC50 13.0 +/- 0.2 mu g/mL. However, the extract of C. sicyoides had a stronger effect on O-2 center dot- (IC50 60.0 +/- 2.3 mu p/mL) than the extract of B. forficata (IC50 90.0 +/- 4.4 mu g/mL). B. forficata also had a stronger inhibitory effect on MPO activity, as measured by guaiacol oxidation, than C. sicyoides. These results indicate that aqueous extracts of leaves of B. forficata and C. sicyoides are a potential source of natural antioxidants and may be helpful in the prevention of diabetic complications associated with oxidative stress.

Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 muM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.

Antioxidant and free radical scavenging activity of isoflavone metabolites

1. Soy isoflavones have been extensively studied because of their possible health-promoting effects. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which might affect their biological properties. 2. The antioxidant activity, free radical-scavenging properties and selected cellular effects of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene were investigated in comparison with their parent aglycones, genistein and daidzein. 3. Electron spin resonance spectroscopy indicated that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher antioxidant activity than parent compounds in standard antioxidant (FRAP and TEAC) assays. However, for the suppression of nitric oxide production by activated macrophages, genistein showed the highest potency, followed by equol and daidzein. 4. The metabolism of isoflavones affects their free radical scavenging and antioxidant properties, and their cellular activity, but the effects are complex.

Antioxidant and free radical scavenging activity of isoflavone metabolites

1. Soy isoflavones have been extensively studied because of their possible health-promoting effects. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which might affect their biological properties. 2. The antioxidant activity, free radical-scavenging properties and selected cellular effects of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene were investigated in comparison with their parent aglycones, genistein and daidzein. 3. Electron spin resonance spectroscopy indicated that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher antioxidant activity than parent compounds in standard antioxidant (FRAP and TEAC) assays. However, for the suppression of nitric oxide production by activated macrophages, genistein showed the highest potency, followed by equol and daidzein. 4. The metabolism of isoflavones affects their free radical scavenging and antioxidant properties, and their cellular activity, but the effects are complex.

The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2•−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

Chemical composition and radical scavenging activity of melanin from Auricularia auricula fruiting bodies

Melanin extracted from Auricularia auricula fruiting bodies (AAFB) was examined by element analyzer, amino acid analyzer, inductively coupled plasma-optical emission spectrometry. Elemental composition analysis revealed that main component of AAFB melanin was pheomelanin. Amino acid analysis showed that 16 amino acids were found in AAFB melanin and total amino acid content was 321. 63 mg/g. There were 13 detectable metal elements in AAFB melanin, which was rich in Ca, Fe, Cu and Zn. In addition, AAFB melanin exhibited stronger scavenging activities on 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical, superoxide radical and hydroxyl radical with IC50 values of 0.18, 0.59 and 0.34 mg/mL, respectively. These results indicated that AAFB melanin might be potentially used as a natural antioxidant.

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.

Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.

HPLC microfractionation of flavones and antioxidant (radical scavenging) activity of Saccharum officinarum L.

The antioxidant activity of sugarcane (Saccharum officinarum L.) juice towards DPPH reagent was determined (EC50) and the main compounds with radical scavenging activity in juice and leaves extracts were identified by HPLC-UV/PAD analysis combined with HPLC microfractionation monitored by TLC using β-carotene and DPPH as the detection reagents. In sugarcane leaves, luteolin-8-C-(rhamnosylglucoside) (1) was the most important compound with radical scavenging activity; in sugarcane juice, the flavones diosmetin-8-C-glucoside (2), vitexin (3) schaftoside (9), isoschaftoside (10) and 4',5'-dimethyl-luteolin-8-C-glucoside (11) were the most relevant compounds. The content of juice flavonoids (0.241 ± 0.001 mg total flavonoids/mL juice), comparable to other food sources of flavonoids, suggest the potential of sugarcane as a dietary source of natural antioxidants. However, the low antioxidant ability of sugarcane juice (EC50 = 100.2 ± 2.6 g L-1) also points to the need for further studies about the dietary intake of sugarcane flavonoids and its effects on human health.

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus ( SLE), the superoxide anion (O(2)(-)) production by neutrophils, mediated by Fc gamma R and Fc gamma R/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: ( 1) manifestations associated with the occurrence of cytotoxic antibodies ( SLE-I group); ( 2) manifestations associated with circulating immune complexes ( IC; SLE-II group), and ( 3) manifestations associated with IC and cytotoxic antibodies ( SLE-III group). O(2)(-) production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with ICIgG opsonized or not with complement. No difference in O(2)(-) production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: ( 1) SLE-I neutrophils showed lower O(2)(-) production mediated by the IgG receptor ( Fc gamma R) with the cooperation of complement receptors ( Fc gamma R/ CR) than observed in the SLE-II, SLE-III, and healthy groups; ( 2) neutrophils from the SLE-II group showed a decreased O(2)(-) production mediated by Fc gamma R/ CR compared to the SLE-III group, ( 3) SLE-III neutrophils produced more O(2)(-) than neutrophils from the SLE-II and control groups, and ( 4) CR showed inefficiency in mediating the O(2)(-) production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence ( CL; T(max) and CL(max)) disclosed differences only for the SLE- I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by Fc gamma R/ CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.

The antioxidant effect of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. β-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 μm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

In vitro radical-scavenging activity, toxicity against A. salina, and nmr profiles of extracts of lichens collected from Brazil and Antarctica

Extracts of six lichen species collected from Brazil and Antarctica were investigated for their potential toxicity and radical-scavenging properties. The composition of the extracts was investigated using TLC and NMR, leading to identification of atranorin (1), along with salazinic (2), barbatic (3), α-alectoronic (4), α-collatolic (5), cryptochlorophaeic (6), caperatic (7), lobaric (8), and protolichesterinic (9) acids. All acetone extracts were evaluated for their 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and subjected to Artemia salina bioassay. The free-radical-scavenging activities of each extract (100 µg) ranged from 8.9 ± 0.1% to 38.7 ± 2.5% and the EC50 values ranged from 0.24 ± 2.10 to 3.54 ± 0.28 mg mL-1, while the toxicity of the extracts against A. salina were low (151.0 to >600 µg mL-1).

HPLC-DAD based method for the quantification of flavonoids in the hydroethanolic extract of Tonina fluviatilis Aubl. (Eriocaulaceae) and their radical scavenging activity

This article describes the isolation and identification of flavonoids in the hydroethanolic extract of the aerial parts from Tonina fluviatilis and evaluation of their antiradical activity. A method based on HPLC-DAD was developed and validated for detecting and quantifying flavonoids in hydroethanolic extracts. The flavonoids identified and quantified in the extract were 6,7-dimethoxyquercetin-3-O-β-D-glucopyranoside (1), 6-hydroxy-7-methoxyquercetin-3-O-β-D-glucopyranoside (2), and 6-methoxyquercetin-3-O-β-D-glucopyranoside (3). The developed method presented good validation parameters, showing that the results obtained are consistent and can be used in ensuring the quantification of these constituents in the extracts. Compounds 2 and 3 showed strong antiradical activity when compared with the positive controls (quercetin and gallic acid).

Peroxyl radical-scavenging activity of coffee brews

The ORAC(FL) assay was used in non-automated mode to evaluate the specific peroxyl radical scavenging properties of the aqueous soluble components of green and roasted Arabica and Robusta coffee samples. A relationship between ORAC(FL) and the concentration of CQAs (caffeoyl quinic acids) was found for the extracts from green coffee beans. Aqueous extracts from roasted coffee beans possessed equal or stronger scavenging power than that obtained for the green coffee beans extracts and the scavenging activity depended on the variety of coffee and the roasting conditions. Brews from Robusta coffee beans showed the highest ORAC(FL). The best scavenging properties for the brews from Arabica coffee beans were detected in samples prepared from coffee beans roasted under light conditions. The data indicate that, during roasting, a complex network of reactions takes place leading to the formation of a wide number of compounds possessing specific scavenging properties. Under mild roasting conditions, caffeoyl quinic acids appear to be the main components responsible for the free radical scavenging power of coffee brews. In contrast, Maillard reaction products may be the principal components with free radical scavenging activity in more severely (medium and dark) roasted coffees.