Superoxide Assays Research Articles

Biochemical evaluation of antioxidant activity and polysaccharides fractions in seaweeds

In the present study ethanol and water extracts of 15 seaweeds, Dictyota dichotoma var. velutricata, Dictyota indica, Iyengaria stellata, Padina pavonia, Sargassum swartzii, Sargassum variegatum, Stoechospermum marginatum, Stokeyia indica, Jolyna laminarioides, Caulerpa taxifolia, Halimeda tuna, Ulva fasciata, Ulva lactuca, Solieria robusta, and Melanothamnus afaqhusainii, were evaluated for their antioxidant potential by ABTS or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), superoxide and total antioxidant capacity (TAC) assays. The activity was concentration dependent and the variation in antioxidant potential was also observed by different assays in both extracts. Ethanol extract of D. dichotoma var. velutricata, D. indica and S. marginatum demonstrated highest activity by TAC assay. The antioxidant potential in organic solvent fractions of seaweeds namely P. pavonia, S. swartzii, S. marginatum and M. afaqhusainii was also determined and chloroform fraction of all the four seaweeds showed highest activity by superoxide assay. Antioxidant activity of extracted fractions of polysaccharides from S. indica, C. taxifolia and D. dichotoma var. velutricata was also evaluated by superoxide method. Polysaccharide fractions of S. indica obtained from HCl (at 700C and room temperature) and water extract demonstrated highest activity respectively. All the polysaccharide fractions of C. taxifolia showed excellent activity except CaClF70°C. Polysaccharide fractions of D. dichotoma var. velutricata also exhibited very good activity.

Antioxidant and proapoptotic activities of Sclerocarya birrea [(A. Rich.) Hochst.] methanolic root extract on the hepatocellular carcinoma cell line HepG2.

The main goal of this study was to characterize the in vitro antioxidant activity and the apoptotic potential of S. birrea methanolic root extract (MRE). Among four tested extracts, obtained with different solvents, MRE showed the highest content of polyphenols, flavonoids, and tannins together with antioxidant activities tested with superoxide, nitric oxide, ABTS, and beta-carotene bleaching assays. Moreover, the cytotoxic effect of MRE was evaluated on the hepatocarcinoma cell line HepG2. In these cells, MRE treatment induced apoptosis and generated reactive oxygen species (ROS) in dose-dependent manner. The cytotoxic effect promoted by MRE was prevented by pretreatment of HepG2 cells with N-acetyl-L-cysteine (NAC), suggesting that oxidative stress was pivotal in MRE-mediated cell death. Moreover, we showed that the MRE treatment induced the mitochondrial membrane depolarization and the cytochrome c release from mitochondria into the cytosol. It suggests that the apoptosis occurred in a mitochondrial-dependent pathway. Interestingly, MRE showed a sensibly lower cytotoxicity, associated with a low increase of ROS, in normal human dermal fibroblasts compared to HepG2 cells. It is suggested that the methanolic root extract of S. Birrea is able to selectively increase intracellular ROS levels in cancer cells, promoting cell death.

Evaluation of Ethanol and Aqueous extracts of Cinnamomum verum Leaf Galls for Potential Antioxidant and Analgesic activity.

In the present study, ethanol and aqueous extracts of leaf galls of Cinnamomum verum were prepared to evaluate the antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay and superoxide radical scavenging assay with ascorbic acid as a standard, and analgesic activity by tail immersion test and acetic acid-induced writhing test methods using diclofenac sodium as the reference drug. Swiss albino mice maintained under standard laboratory conditions were used for analgesic tests. In the 2,2-diphenyl-1-picrylhydrazyl assay it was found that the aqueous and the ethanol extract possessed almost equal capacity to inhibit free radicals (IC50=13.3 and 13.53 µg/ml) but found less than ascorbic acid (IC50=9.96 µg/ml). And in superoxide assay the ethanol extract was found to be more potent in scavenging super oxide radicals when compared to ascorbic acid and the aqueous extract (IC50=237.1 and 197.8 µg/ml) with the IC50=119.7 µg/ml. For analgesic activity, ethanol extract showed the maximum time required for response against thermal stimuli (6.75±0.47 s) and maximum % of writhing inhibition (44.57%) when compared to aqueous extract (5.25±0.48 s and 32.61%), whereas diclofenac showed response in 7.25±0.25 s 67.39% inhibition in tail immersion and writhing tests, respectively. These results demonstrate that the ethanol extracts of leaf galls possessed high antioxidant and analgesic activity.

Glucagon-like peptide-1 protects against cardiac microvascular endothelial cells injured by high glucose.

To investigate the protective effect of glucagon-like peptid-1 (GLP-1) against cardiac microvascular endothelial cell (CMECs) injured by high glucose. CMECs were isolated and cultured. Superoxide assay kit and dihydroethidine (DHE) staining were used to assess oxidative stress. TUNEL staining and caspase 3 expression were used to assess the apoptosis of CMECs. H89 was used to inhibit cAMP/PKA pathway; fasudil was used to inhibit Rho/ROCK pathway. The protein expressions of Rho, ROCK were examined by Western blot analysis. High glucose increased the production of ROS, the activity of NADPH, the apoptosis rate and the expression level of Rho/ROCK in CMECs, while GLP-1 decreased high glucose-induced ROS production, the NADPH activity and the apoptosis rate and the expression level of Rho/ROCK in CMECs, the difference were statistically significant (P<0.05). GLP-1 could protect the cardiac microvessels against oxidative stress and apoptosis. The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-dependent manner, resulting in a subsequent decrease in the expression of NADPH oxidase.

An in vitro study on the effect of vinca alkaloid, vinorelbine, on chromatin histone, HMGB proteins and induction of apoptosis in mice non-adherent bone marrow cells

Context: Vinoreline is a vinca alkaloid anticancer drug widely used in cancer therapy. Drugs are not target specific, therefore might affect normal tissues/cells, in which bone marrow is the important one. Objective: To elucidate the cytotoxic and genotoxic effect of vinca alkaloid anti cancer drug, vinorelbine, on mice non-adherent bone marrow cells in vitro. Materials and methods: Non-adherent bone marrow cells were isolated and exposed to various concentrations (0–160 µg/ml) for 4 h at 23 °C. The chromatin proteins were analyzed by SDS PAGE and western blot. Fluorescent dye staining of the cells, anion superoxide and DNA fragmentations assays were also employed. Result: The results from MTT and trypan blue exclusion assays represented reduction of the cells viability. Extractability of histones and HMG proteins contrasted with difficulty as their content was decreased on SDS-gel upon increasing drug concentration as western blots confirmed it. The amount of degradation form of PARP (89 KD) increased significantly in a dose dependent manner. Increase in anion superoxide production and DNA fragmentation together with cytological detection of chromatin condensation and cellular damage upon exposure of the cells to vinorelbine were indicative of apoptosis induction in these normal cells. Conclusion: Vinorelbine is genotoxic in non-adherent bone marrow cells as affects chromatin components, DNA, histone and HMGB1 proteins and induces apoptosis.

Parmotrema tinctorum exhibits antioxidant, antiglycation and inhibitory activities against aldose reductase and carbohydrate digestive enzymes: an in vitro study

This study evaluated the inhibitory potential of ethyl acetate extract of Parmotrema tinctorum (PTEE), an edible lichen, against aldose reductase (AR) and carbohydrate digestive enzymes such as α-glucosidase and α-amylase. It was also screened for antioxidant activities by using DPPH, ABTS, superoxide and hydroxyl radical-scavenging assays. PTEE exhibited α-glucosidase, α-amylase and AR inhibition along with significant antiglycation potential with an estimated IC50 value of 58.45 ± 1.24, 587.74 ± 3.27, 139.28 ± 2.6 and 285.78 ± 1.287 μg/mL, respectively. Antioxidant activity of PTEE against DPPH (IC50 396.83 ± 2.98 μg/mL), ABTS (151.34 ± 1.79 μg/mL), superoxide (30.29 ± 1.17 μg/mL) and hydroxyl (35.42 ± 1.22 μg/mL) radicals suggests the antioxidant potential of P. tinctorum. Significant antioxidant activity and inhibitory potential against carbohydrate digestive enzymes and AR suggest that P. tinctorum can be developed as functional food/nutraceuticals for diabetes after detailed study.

Comparative analysis of antioxidant and antimelanogenesis properties of three local guava (Psidium guajava L.) varieties of Thailand, via different extraction solvents

The antioxidant and antimelanogenesis potentials of three local varieties of guava (Psidium guajava L.) from Thailand namely: Pansithong, Thai original and Kimju were studied. The extracts were made with water, 40 % ethanol and simulated gastrointestinal digestion (SGD). Antioxidant properties were determined by total polyphenolic compounds, DPPH assay, superoxide anion assay and hydroxyl radical assay. Antimelanogenesis properties were determined by anti-tyrosinase assay, total phenolic acid and total vitamin C contents. The results for antioxidant analysis revealed that Pansithong, when extracted by 40 % ethanol and SGD showed the presence of highest polyphenol compounds, and DPPH antioxidant activity respectively. Kimju exhibited the highest hydroxyl radical antioxidant activity among all extracts and highest superoxide anion antioxidant activity, when extracted by 40 % ethanol. The results for antimelanogenesis determination presented that Kimju showed highest tyrosinase inhibition, total phenolic acid and total vitamin C contents. It was concluded that Pansithong showed highest antioxidant potential while the highest antimelanogenesis potential was found in Kimju. Over all water has been found as the best solvent for antioxidant extraction followed by 40 % ethanol and SGD.

Interleukin-13/Interleukin-4-induced oxidative stress contributes to death of prothrombinkringle-2 (pKr-2)-activated microglia

The present study examined whether Interleukin-13 (IL-13) or IL-4, an anti-inflammatory cytokine, could induce cell death of activated microglia by prothrombin kringle-2 (pKr-2) which is a domain of prothrombin distinct from thrombin. Microglia cell death was detected at eight days after co-treatment of pKr-2 with IL-13/IL-4 in vitro. This cell death was assessed by live assay, dead assay, TUNEL and MTT assay. In parallel, reactive oxygen species (ROS) production was evident as assessed by superoxide assay, WST-1 and analyzing DCF in combination of pKr-2 and IL-13 or IL-4 treated microglia. The IL-13/IL-4-enhanced ROS production and cell death in pKr-2 activated microglia was partially inhibited by an NADPH oxidase inhibitor, apocynin and/or by several antioxidants. Moreover, Western blot analysis showed a significant increase in cyclooxygenase-2 (COX-2) expression in combination of pKr-2 and IL-13 or IL-4 treated microglia, which was partially inhibited by apocynin and an antioxidant, trolox. Additional studies demonstrated that microglia cell death was reversed by treatment with COX-2 inhibitor, NS398. Our data strongly suggest that oxidative stress and COX-2 activation through NADPH oxidase may contribute to IL-13/IL-4 induced cell death of pKr-2 activated microglia.

Development of a Topical Formulation Containing S. Lutea Extract: Stability, In Vitro Studies and Cutaneous Permeation

Flavonoids have been widely incorporated into cosmetic and dermatological formulations, affording benefits such as antioxidant action, improved skin tone and fewer lines and wrinkles. Brazil has a huge biodiversity, with one of the richest flora in the world, and existing studies justify the quest for greater research efforts in this area. The cajazeira (Spondias lutea L.), a plant of the Anacardiaceae family from tropical America, is widely disseminated in Brazil. This plant was chosen because of the presence of flavonoids that exhibit antioxidant activity. The purpose of this research was to develop a stable topical formulation containing Spondias lutea extract with the aim of preventing skin diseases caused by UV radiation. Hydro ethanolic extract of Spondias lutea fruit was prepared and assayed for its the flavonoids content. The antioxidant activity was estimated by DPPH and superoxide assay. The physicochemical stability and skin permeation of the cream containing 8% (w/w) of extract were assessed. The results showed that the S. lutea extract possessed antioxidant activity, and that it is possible to obtain a stable cosmetic containing the extract, which is able to penetrate the skin. Thus, it is possible to use this extract to produce an anti-aging cosmetic.

Phosphorylation of Smooth Muscle 22α Facilitates Angiotensin II–Induced ROS Production Via Activation of the PKCδ-P47 phox Axis Through Release of PKCδ and Actin Dynamics and Is Associated With Hypertrophy and Hyperplasia of Vascular Smooth Muscle Cells In Vitro and In Vivo

We have demonstrated that smooth muscle (SM) 22α inhibits cell proliferation via blocking Ras-ERK1/2 signaling in vascular smooth muscle cells (VSMCs) and in injured arteries. The recent study indicates that SM22α disruption can independently promote arterial inflammation through activation of reactive oxygen species (ROS)-mediated NF-κB pathways. However, the mechanisms by which SM22α controls ROS production have not been characterized. To investigate how SM22α disruption promotes ROS production and to characterize the underlying mechanisms. ROS level was measured by dihydroethidium staining for superoxide and TBA assay for malondialdehyde, respectively. We showed that downregulation and phosphorylation of SM22α were associated with angiotensin (Ang) II-induced increase in ROS production in VSMCs of rats and human. Ang II induced the phosphorylation of SM22α at Serine 181 in an Ang II type 1 receptor-PKCδ pathway-dependent manner. Phosphorylated SM22α activated the protein kinase C (PKC)δ-p47phox axis via 2 distinct pathways: (1) disassociation of PKCδ from SM22α, and in turn binding to p47phox, in the early stage of Ang II stimulation; and (2) acceleration of SM22α degradation through ubiquitin-proteasome, enhancing PKCδ membrane translocation via induction of actin cytoskeletal dynamics in later oxidative stress. Inhibition of SM22α phosphorylation abolished the Ang II-activated PKCδ-p47phox axis and inhibited the hypertrophy and hyperplasia of VSMCs in vitro and in vivo, accompanied with reduction of ROS generation. These findings indicate that the disruption of SM22α plays pivotal roles in vascular oxidative stress. PKCδ-mediated SM22α phosphorylation is a novel link between actin cytoskeletal remodeling and oxidative stress and may be a potential target for the development of new therapeutics for cardiovascular diseases.

Oxidative stress contributes to the cigarette smoke extract induced adverse functional effects of rat cardiac stem cells

Cigarette smoke (CS) is a causative agent in several cardiovascular diseases and premature death. CS is composed of many constituents that can modulate cardiac cell functions. The integrity of cardiac cell functions is crucial in maintaining proper cardiovascular health. The main objective of our study is to investigate whether cigarette smoke extracts (CSE) cause impairment of cardiac stem cell functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of proteins and generation of reactive oxygen species can modulate rat cardiac stem cell (RCSC) functions. This is a novel idea and there are few reports discussing CSE induced impairment of endothelial progenitor cell functions via oxidative stress, but not in the context of cardiac stem cells. In our study c‐kit positive RCSC were treated with cigarette smoke extracts (CSE) for 24 hours, and modulations of RCSC functions were assayed. CSE attenuated RCSC viability, migration, and accelerated apoptosis in a dose dependent manner. Anti‐oxidant vitamin C has prevented CSE induced effects. To find out whether oxidative stress play a role in CSE mediated adverse effects, oxidative modification of proteins due to CSE was detected using Oxiblot ™ assay. Compared to control, in CSE treated cells carbonyl groups of proteins were oxidatively modified. The possibility of vitamin C mediated prevention of CSE induced oxidative modification of proteins is currently under investigation. CSE induced generation of reactive oxygen species will be detected using cytochrome C superoxide assay. Research support was provided by Sullivan University College of Pharmacy faculty development grant awarded to Dr. Wasana Sumanasekera.

Superoxide determination using membrane-engineered cells: An example of a novel concept for the construction of cell sensors with customized target recognition properties

Membrane-engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor molecules on the cell surface. We have previously reported the construction of an ultra-sensitive superoxide anion (O 2 − ) sensor based on immobilized cells, which have been membrane-engineered with superoxide dismutase (SOD). In the present study, we provide evidence that superoxide dismutation triggered changes to the membrane potential of membrane-engineered fibroblast cells, as confirmed by electrophysiological and fluorescence assays. These changes were associated with changes in [Ca 2+ ] cyt , as revealed by the selective inhibition of intracellular calcium ion traffic. In addition, by conducting selective inhibition assays, we show that electroinserted SOD molecules retained their characteristic catalytic properties. We also investigated the effect of the concentration of electroinserted SOD molecules on the performance of the superoxide assay. Finally, we increased the sensitivity of the sensor by hundredfold to a detection limit of 1 pM O 2 − by changing the concentration of immobilized cells on the performance of the biosensor.

Oxygen radical scavenging capacity of phenolic and non-phenolic compounds in red and white wines

Abstract The aim of the present study was the evaluation of the antioxidant content in phenolic and non-phenolic extracts of ten wine samples, trying to elucidate the potential role of unusual antioxidant compounds. Samples of wines processed from red and white grapes (Vitis vinifera L.), deprived of the volatile fraction at low temperature and buffered at physiological pH, were fractionated by C18 into two fractions: FR1 and FR2. Non-phenolics, such as tartaric, malic, lactic, and succinic acids; glucose; fructose; and glycerin were mainly found in FR1, while polyphenols were present exclusively in FR2. Peroxyl radical quenching was assayed by the ORAC method, while superoxide and hydroxyl radical scavenging activity were assayed by electron paramagnetic resonance. In the ORAC and superoxide assays, most of the activity was found in FR2, while in hydroxyl radical assay, the activity was found in FR1. Model solutions were used to attribute a role to the single compounds in the evaluation of wine’s ROS scavenging capacity: the ORAC and superoxide anion scavenging effects were mainly attributed to the polyphenols, averaging 94.8%, with some contribution from glycerin, particularly in white wines. Unexpectedly, the main chemical responsible for hydroxyl radical scavenging activity was glycerin (56.1%), with the polyphenols scavenging at 18.1%.

Antioxidant Activity of Isoflavones and Biflavones Isolated from Godoya antioquiensis

Two biflavones, ochnaflavone (1) and 2",3"-dihydroochnaflavone (2), and two isoflavones, 5,7,4'-trihydroxy-3',5'-dimethoxyisoflavone (piscigenin) (3) and 5,4'-dihydroxy-7,3',5'-trimethoxyisoflavone (4), a new natural compound, were isolated from the leaves of Godoya antioquiensis (Ochnaceae). Their structures were determined on the basis of spectral data and by comparison with data reported in the literature. The isolated compounds were evaluated for their radical scavenging activity using the NBT (nitrobluetetrazolium)/hypoxanthine superoxide and the .OH/luminol chemiluminescence methods. The isolated isoflavones were found to exhibit a strong hydroxyl radical scavenging activity and a moderate inhibition of the superoxide anion, whereas the two biflavones were inactive in the superoxide anion assay and showed a low hydroxyl radical scavenging activity.

Antioxidant activity and free radical-scavenging capacity of a selection of wild-growing Colombian plants

The replacement of some synthetic food antioxidants by safe natural antioxidants has fostered research on the screening of raw materials to find new vegetable sources of antioxidants. In this study the antioxidant activity of eight wild-growing Colombian plants was assessed by four complementary assays. An evaluation of the antioxidant activity of ten ethanolic extracts from Baccharis chilco, Cinnamomum triplinerve, Ilex laurina, Lachemilla orbiculata, Lepechinia conferta, Quercus humboldtii, Rubus urticifolius and Tephrosia cinerea was carried out. Furthermore, the total phenolic content was determined by the Folin-Ciocalteu method, and the relationship between phenolic content and activity was also statistically investigated. Cinnamomum triplinerve, L. conferta and I. laurina were found to have the highest phenolic contents. Baccharis chilco, C. triplinerve, I. laurina, L. conferta, Q. humboldtii and R. urticifolius showed higher radical-scavenging activity (DPPH and superoxide assays) than commercial rosemary oleoresin (reference). Lachemilla orbiculata and R. urticifolius showed higher antioxidant activity (β-carotene-bleaching test) than the reference. The protection factor of all studied plant extracts was below that of the reference according to the Rancimat test. On the basis of the results obtained, C. triplinerve, Q. humboldtii and R. urticifolius seem to be the most promising species for further investigation in order to identify the compounds responsible for their activity.

In Vitro and In Vivo Antioxidant Activity of Bifidobacterium animalis 01 Isolated from Centenarians

Several studies reported the antioxidant activity of bifidobacteria using assays in vitro. In present study, the in vitro and in vivo antioxidant activity of Bifidobacterium animalis 01 was investigated. Culture supernatant, intact cells, and intracellular cell-free extracts of B. animalis 01 were involved in this study. The antioxidant assays in vitro included lipid peroxidation assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, hydroxyl radical ((•)OH) assay and superoxide anion (O₂⁻) assay. The antioxidant assays in vivo were conducted using mice model. Activities of antioxidative enzymes, malondialdehyde (MDA) content in serums and livers of aging mice were evaluated. Monoamine oxidase (MAO) activity and lipofuscin level in brains of aging mice were also characterized. Results showed that culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 could effectively scavenge free radicals, significantly enhance mice's activities of antioxidative enzymes and reduce mice's MDA content, lipofuscin level and MAO activity. Our results indicated that B. animalis 01 has the potential to be developed into a dietary antioxidant supplements.

Evaluation of Antioxidant Properties of Phenolics Extracted from Ananas comosus L.

Phenolics were extracted from the fruit tissues of Ananas comosus L. var. queen, cv. ‘Meitei Keehom’, a variety of pineapple grown in Manipur, India, after skin peeling, purified and their antioxidant properties were analyzed. The antioxidant properties were assessed based on the ability of fruit phenolics in absolute methanol to scavenge DPPH, superoxide anion radicals and hydroxyl radicals and compared to antioxidant compounds like ascorbic acid and pyragallol. Pineapple fruit phenolics scavenged DPPH, superioxide anion radicals and hydroxyl radicals in a dose dependent way. In DPPH assay, the IC50 values of pineapple phenolics, ascorbic acid and pyragallol were 12.2μg/ml, 17.82μg/ml and 15.92μg/ml respectively. In superoxide anion and hydroxyl radical scavenging activities, the IC50 values of pineapple phenolics were 11.42μg/ml and 55.292μg/ml, for ascorbic acid 49.62μg/ml, 48.52μg/ml and that of pyragallol was 15.672μg/ ml and 60.62μg/ml. The IC50 value was lowest in pineapple phenolics than ascorbic acid and pyragallol in DPPH and superoxide anion assays. But it is higher than ascorbic acid and lower than pyragallol in hydroxyl radical assay. The lower the IC50 values, the higher the antioxidant activities. The phenolics extracted from this variety of pineapple exhibit excellent free radical scavenging activity. The result shows that pineapple and its active constituents may be used in further antioxidative therapy.

Free radical scavenging activities and inhibition of inflammatory enzymes of phenolics isolated from Tripodanthus acutifolius

Ethnopharmacological relevance Leaf extracts from Tripodanthus acutifolius (Ruiz and Pavón) Van Tieghem have long been used in argentinean traditional medicine as anti-inflammatory, however, there is no scientific evidence which supports this use in the literature. Aim of the study The present study was conducted to evaluate the ability of five phenolic compounds purified from infusion prepared from Tripodanthus acutifolius leaves to inhibit key enzymes in inflammatory processes. As anti-inflammatory compounds frequently possess free radical scavenging activities, purified substances were comparatively evaluated to asses their free radical scavenging properties. Genotoxic effects were also evaluated. Materials and Methods Compounds were evaluated on their ability to inhibit hyaluronidase and cyclooxygenase-2 (COX-2) activities to assess their anti-inflammatory capacities. Free radical scavenging activity was assessed by: 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH), superoxide anion assay and the inhibition on lipid peroxidation. Genotoxicity was evaluated by Bacillus subtilis rec assay. Results Fractionation of Tripodanthus acutifolius infusion yielded a novel phenylbutanoid derivative (tripodantoside) and four known flavonoid glycosides (rutin, nicotiflorin, hyperoside and isoquercitrin). Flavonoids produced higher inhibition on hyluronidase activity (IC 50 ≈ 1.7 mM) than tripodantoside (IC 50 = 27.90 mM). A similar COX-2 inhibition activity was exerted by tripodantoside and monoglycosilated flavonoids (IC 50 ∼ 50 μM). Compounds were strong radical scavengers, with effective concentration 50 (EC 50) values for DPPH in the range of 2.7–6.3 μg/mL, and for superoxide anion in the range of 3.9–8.7 μg/mL. All compounds scavenged peroxyl radicals in the lipid peroxidation assay. The substances showed no genotoxic effects. Conclusions The anti-inflammatory effects, free radical scavenging activities and lack of genotoxicity of purified compounds may support the folk use of infusion from Tripodanthus acutifolius leaves as anti-inflammatory.

β-Actin Association with Endothelial Nitric-oxide Synthase Modulates Nitric Oxide and Superoxide Generation from the Enzyme

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.