Peroxynitrite-Scavenging Glycosides from the Stem Bark of Catalpa ovata.

Ten new glycosides, 6,10-O-di-trans-feruloyl catalpol (1), 6,6'-O-di-trans-feruloyl catalpol (2), 3,4-dihydro-6-O-di-trans-feruloyl catalpol (10), (8R,7'S,8'R)-lariciresinol 9'-O-ß-d-(6-O-trans-feruloyl)glucopyranoside (17), and ovatosides A-F (18-22, 24), were isolated from the stem bark of Catalpa ovata along with 19 known compounds. All isolates, except 6 (catalposide) and 9 (6-O-veratroyl catalpol), were found to scavenge peroxynitrite (ONOO-) formed by 3-morpholinosydnonimine. In particular, 12 compounds showed potent activity, with IC50 values in the range 0.14-2.2 µM.

Molecular visualizing and quantifying immune-associated peroxynitrite fluxes in phagocytes and mouse inflammation model.

Reactions of peroxynitrite (ONOO-) with biomolecules can lead to cytotoxic and cytoprotective events. Due to the difficulty of directly and unambiguously measuring its levels, most of the beneficial effects associated with ONOO- in vivo remain controversial or poorly characterized. Recently, optical imaging has served as a powerful noninvasive approach to studying ONOO- in living systems. However, ratiometric probes for ONOO- are currently lacking. Herein, we report the design, synthesis, and biological evaluation of F482, a novel fluorescence indicator that relies on ONOO--induced diene oxidation. The remarkable sensitivity, selectivity, and photostability of F482 enabled us to visualize basal ONOO- in immune-stimulated phagocyte cells and quantify its generation in phagosomes by high-throughput flow cytometry analysis. With the aid of in vivo ONOO- imaging in a mouse inflammation model assisted by F482, we envision that F482 will find widespread applications in the study of the ONOO- biology associated with physiological and pathological processes in vitro and in vivo.

A fluorescent carbon-dots-based mitochondria-targetable nanoprobe for peroxynitrite sensing in living cells.

Mitochondria, the power generators in cell, are a primary organelle of oxygen consumption and a main source of reactive oxygen/nitrogen species (ROS/RNS). Peroxynitrite (ONOO-), known as a kind of RNS, has been considered to be a significant factor in many cell-related biological processes, and there is great desire to develop fluorescent probes that can sensitively and selectively detect peroxynitrite in living cells. Herein, we developed a fluorescent carbon-dots (C-dots) based mitochondria-targetable nanoprobe with high sensitivity and selectivity for peroxynitrite sensing in living cells. The C-dots with its surface rich in amino groups was synthesized using o-phenylenediamine as carbon precursor, and it could be covalently conjugated with a mitochondria-targeting moiety, i.e. triphenylphosphonium (TPP). In the presence of peroxynitrite, the fluorescence of the constructed nanoprobe (C-dots-TPP) was efficiently quenched via a mechanism of photoinduced electron transfer (PET). The nanoprobe exhibited relatively high sensitivity (limit of detection: 13.5nM) and selectivity towards peroxynitrite in aqueous buffer. The performance of the nanoprobe for fluorescence imaging of peroxynitrite in mitochondria was investigated. The results demonstrated that the nanoprobe showed fine mitochondria-targeting ability and imaging contrast towards peroxynitrite in living cells. We anticipate that the proposed nanoprobe will provide a facile tool to explore the role played by peroxynitrite in cytobiology.

Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies.

Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.

A ratiometric fluorescent probe based on a coumarin-hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite.

In the study described herein, the red emitting probe CHCN, which possesses a linked coumarin-hemicyanine scaffold, was developed for detection of peroxynitrite (ONOO(-)) under physiological conditions. The studies show that CHCN displays a dual ratiometric and colorimetric response to ONOO(-) that is caused by an oxidation process. A possible mechanism of this oxidation process was proposed and confirmed by ESI-MS spectra for the first time. CHCN shown highly selective and sensitive towards ONOO(-) with a low limit of detection LOD (49.7 nM). Moreover, CHCN has appreciable cell permeability and, as a result, it is applicable to ratiometric detection of exogenous and endogenous ONOO(-) in living cells during phagocytic immune response. We anticipate that, owing to their ideal properties, probes of this type will find great use in explorations of the role played by ONOO(-) in biology.

Simultaneous quantification and validation of new peroxynitrite scavengers from Artemisia iwayomogi.

CONTEXT: Artemisia iwayomogi Kitamura (Compositae) has been very widely used for the treatment of acute or chronic hepatitis, jaundice, and gastritis. In the course of our continuing efforts to identify and quantify peroxynitrite scavengers from Compositae plants, A. iwayomogi was used in this study. OBJECTIVE: The present study was aimed to identify and quantify the peroxynitrite scavengers of A. iwayomogi. MATERIALS AND METHODS: Silica gel and ODS were used for column chromatography. The isolated compounds were quantified using an HPLC equipped with a Capcell Pak C18 column (5 µm, 250 mm × 4.6 mm i.d.), and the method was validated for the quality control. Peroxynitrite (ONOO(-))-scavenging activities of the compounds and extracts were evaluated on the measurement of highly fluorescent rhodamine 123 converted from non-fluorescent dihydrorhodamine (DHR)-123 under the presence of peroxynitrite. RESULTS: Based on the spectroscopic evidences, a new compound, 2"-O-caffeoylrutin (2"-O-trans-caffeic acid ester of quercetin 3-O-α-L-rhamnopyranosyl(1 → 6)-ß-D-glucopyranoside) was isolated and determined together with patuletin 3-O-glucoside, scopolin, scopoletin, rutin, 3,4-dicaffeoylquinic acid, and chlorogenic acid. All of them were potent peroxynitrite scavengers (IC50 ≤ 1.88 µg/mL). DISCUSSION AND CONCLUSION: The peroxynitrite scavengers were mainly distributed in the EtOAc fraction rather than the ether and BuOH fractions. The 70% MeOH extract exhibited a high peroxynitrite-scavenging activity. Through the validation, the present HPLC method was verified to be sufficiently sensitive, accurate, precise, and stable. Therefore, this method can be used for the quality control of A. iwayomogi.

Tryparedoxin peroxidases from Trypanosoma cruzi: high efficiency in the catalytic elimination of hydrogen peroxide and peroxynitrite.

During host cell infection, Trypanosoma cruzi parasites are exposed to reactive oxygen and nitrogen species. As part of their antioxidant defense systems, they express two tryparedoxin peroxidases (TXNPx), thiol-dependent peroxidases members of the peroxiredoxin family. In this work, we report a kinetic characterization of cytosolic (c-TXNPx) and mitochondrial (m-TXNPx) tryparedoxin peroxidases from T. cruzi. Both c-TXNPx and m-TXNPx rapidly reduced hydrogen peroxide (k=3.0 x 107 and 6 x 106 M⁻¹ s⁻¹ at pH 7.4 and 25 °C, respectively) and peroxynitrite (k=1.0 x 106 and k=1.8 x 107 M⁻¹ s⁻¹ at pH 7.4 and 25 °C, respectively). The reductive part of the catalytic cycle was also studied, and the rate constant for the reduction of c-TXNPx by tryparedoxin I was 1.3 x 106 M⁻¹ s⁻¹. The catalytic role of two conserved cysteine residues in both TXNPxs was confirmed with the identification of Cys52 and Cys173 (in c-TXNPX) and Cys81 and Cys204 (in m-TXNPx) as the peroxidatic and resolving cysteines, respectively. Our results indicate that mitochondrial and cytosolic TXNPxs from T. cruzi are highly efficient peroxidases that reduce hydrogen peroxide and peroxynitrite, and contribute to the understanding of their role as virulence factors reported in vivo.

Separation and detection of peroxynitrite using microchip electrophoresis with amperometric detection.

Peroxynitrite (ONOO(-)) is a highly reactive species implicated in the pathology of several cardiovascular and neurodegenerative diseases. It is generated in vivo by the diffusion-limited reaction of nitric oxide (NO(*)) and superoxide anion ((*)O(2)(-)) and is known to be produced during periods of inflammation. Detection of ONOO(-) is made difficult by its short half-life under physiological conditions (approximately 1 s). Here we report a method for the separation and detection of ONOO(-) from other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (approximately 25 s for ONOO(-)) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOO(-) to be detected before substantial degradation occurs, and the increased temporal resolution permits more accurate tracking of dynamic changes in chemical systems.

Separation and detection of peroxynitrite and its metabolites by capillary electrophoresis with UV detection.

A method for the separation and direct detection of peroxynitrite (ONOO(-)) and two of its degradation products, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), using capillary electrophoresis with ultraviolet detection is described. The separation parameters were optimized and included electrokinetic injection, a run buffer consisting of 25 mM K(2)HPO(4) 7.5 mM DTAB, pH 12, and a field strength of -323 V/cm. A diode array UV detector was employed in these studies as it allowed the determination of all three species simultaneously. Nitrate and nitrite provided the maximum response at 214 nm while peroxynitrite generated the best response at 302 nm. All three species could be detected at 214 nm, while simultaneous detection at 214 and 302 nm positively identified each peak.

Optimization of peroxynitrite-luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide.

We established a peroxynitrite-luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide (CS(2)). Three factors, including exposure time to ozone (Factor A), volume of peroxynitrite (ONOO(-)) solution (Factor B) and luminol concentrations (Factor C) at three levels were selected and the combinations were in accordance with orthogonal design L(9) (3(4)). Peroxynitrite was generated from the reaction of ozone and 0.01 mol/L sodium azide (NaN(3)) dissolved in carbonic acid buffer solution (pH 11), and it was reacted with luminol to yield chemiluminescence. The peak value, peak time and kinetic curve of the light emission were observed. The selected combination conditions were 50 s ozone, 800 micro L peroxynitrite and 0.001 mol/L luminol solution. Cell culture solution with CS(2) enhanced the emission intensity of chemiluminescence (F = 8.38, p = 0.018) and shortened the peak time to chemiluminescence (F = 139.00, p = 0.0001). The data demonstrated that this luminol chemiluminescence system is suitable for detecting peroxynitrite in cell culture solutions for evaluating the effect of CS(2) on endothelial cells.

Further isolation of peroxynitrite and 1,1-diphenyl-2-picrylhydrazyl radical scavenging isorhamnetin 7-O-glucoside from the leaves of Brassica juncea L.

From the leaves of Brassica juncea, an radical scavenging isorhamnetin 7-O-glucoside on peroxynitrite and 1,1-diphenyl-2-picrylhydrazyl (DPPH) was isolated and characterized based on the spectroscopic evidence. The compound showed the peroxynitrite and DPPH scavenging activities with IC50 values of 2.07 +/- 0.17 and 13.3 microM, respectively. Penicillamine and L-ascorbic acid as positive control exhibited radical scavenging activities with IC50 values of 3.17 +/- 0.39 and 12.78 microM, respectively.