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In this study, the phytochemical composition, in vitro antioxidant, and anti-inflammatory effects of the aqueous and 60% ethanolic (EtOH) extracts of Santolina rosmarinifolia leaf, flower, and root were examined. The antioxidant activity of S. rosmarinifolia extracts was determined by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. The total phenolic content (TPC) of the extracts was measured by the Folin–Ciocalteu assay. The anti-inflammatory effect of the extracts was monitored by the Griess assay. The chemical composition of S. rosmarinifolia extracts was analysed using the LC-MS technique. According to our findings, 60% EtOH leaf extracts showed the highest Trolox equivalent antioxidant capacity (TEAC) values in both ABTS (8.39 ± 0.43 µM) and DPPH (6.71 ± 0.03 µM) antioxidant activity assays. The TPC values of the samples were in good correspondence with the antioxidant activity measurements and showed the highest gallic acid equivalent value (130.17 ± 0.01 µg/mL) in 60% EtOH leaf extracts. In addition, the 60% EtOH extracts of the leaves were revealed to possess the highest anti-inflammatory effect. The LC-MS analysis of S. rosmarinifolia extracts proved the presence of ascorbic acid, catalpol, chrysin, epigallocatechin, geraniol, isoquercitrin, and theanine, among others, for the first time. However, additional studies are needed to investigate the direct relationship between the chemical composition and physiological effects of the herb. The 60% EtOH extracts of S. rosmarinifolia leaves are potential new sources of natural antioxidants and anti-inflammatory molecules in the production of novel nutraceutical products. [ABSTRACT FROM AUTHOR]

A facile and efficient method for the regioselective [3 + 2] cycloaddition of 4-azidofuroxans to 1-dimethylamino-2-nitroethylene under p-TSA catalysis affording (4-nitro-1,2,3-triazolyl)furoxans was developed. This transformation is believed to proceed via eliminative azide–olefin cycloaddition resulting in its complete regioselectivity. The developed protocol has a broad substrate scope and enables a straightforward assembly of the 4-nitro-1,2,3-triazole motif. Moreover, synthesized (4-nitro-1,2,3-triazolyl)furoxans were found to be capable of NO release in a broad range of concentrations, thus providing a novel platform for future drug design and related biomedical applications of heterocyclic NO donors. [ABSTRACT FROM AUTHOR]

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson's disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Nitrite has been determined using a simple and sensitive spectrophotometric method. The suggested procedure is based on the diazotization reaction of p-bromoaniline in the presence of nitrite ion in a hydrochloric acid media, which produces diazonium salt. This salt is then combined with paracetamol in an ammonium hydroxide medium to produce an orange azo color with a maximum wavelength (λ max.) of 460 nm, molar absorptivity (ε) is 3.8432 x 103 L. mol-l.cm-1, and Beer’s law was established between 0.3-12.0 µg. mL-1. It displays LOD, LOQ, and R2 values of 0.2 µg. mL-1, 0.3 µg. mL-1, and 0.9994 respectively. The dye can be extracted into chloroform. The yellow chloroform extract displays a maximum wavelength (λ max.) of 410 nm, molar absorptivity (ε) is 9.93 x103 L. mol-l.cm-1, and Beer’s law was observed between 0.1-5.0 µg. mL-1. The LOD, LOQ, and R2 values for the suggested extraction procedure were 0.05 µg. mL-1, 0.1 µg. mL-1, and 0.9996, respectively. The suggested method was successfully applied on samples of a human saliva.

A photoactivable NO releasing complex [Ru(L1-2)(PPh 3)(NO)Cl 2 ](PF 6) (1a) have been synthesized by complex [RuL1-2(PPh 3) 2 Cl 2 ](1). Newly designed bidentate ligands, i.e., 4-methoxy- N ′-phenyl- N '-(pyridin-2-ylmethyl)benzohydrazide(L 1 ) and 4-nitro- N ′-phenyl- N '-(pyridin-2-ylmethyl)benzohydrazide (L 2 ) were utilized to synthesize complex (1). Complex (1) was characterized by ESI-MS, and the solid structure of the complex [ 1a ](PF 6) was acquired by X-ray crystallography. Different spectroscopic techniques were employed for the identification of ligands (L 1 and L 2 ) and complexes (1 and [ 1a ](PF 6)). Calculations employing DFT and TD-DFT were made better to understand the electronic properties of the complex [ 1a ](PF 6). The photo liberation experiments were screened in the presence of visible light lamp. Griess assay experiment was used to quantify the photo released amount to NO. The photo liberated NO was successfully transferred to reduced myoglobin (Mb). The complex [ 1a ](PF 6) at 50 μg/mL concentration was used for wound healing and antimicrobial activity on B16F1 mouse skin cells and Escherichia coli bacteria, respectively. In results, we observed a considerable wound healing activity of [ 1a ](PF 6) complex after 36 h of incubation in the light-treated cells compared to the control medium, and also it shows more than 99% inhibition of bacterial cells after 1.5 h of treatment in the presence of light. These study suggested that this complex 1a ](PF 6) could be utilized for topical delivery of NO for combating several dermatological infections. [Display omitted] • Synthesis of visible light activatable RuNO complex, release NO under illumination. • Light-triggered NO has been transported to reduced myoglobin. • The visible-light triggered NO was assessed by the Griess experiment. • Light-triggered NO delivery reveals potential in wound healing and antibacterial treatments. • This type of NO complex can deliver NO to a site specific targets on demand. [ABSTRACT FROM AUTHOR]

• Development and validation of visible light-activatable nitrosyl complexes of ruthenium under the illumination of low intensity light. • Reduced myoglobin has been delivered using light-triggered NO. • The Griess experiment was used to examine the visible-light triggered NO. • Antibacterial activity examined under the exposure of visible light. • This kind of NO complexes may immediately release NO to targets located at a certain spot. In this article, a chloro-bridged dinuclear ruthenium complex Ru-(a) has been synthesized, which was employed for the synthesis of a photolabile NO liberating complex Ru-(b). A newly synthesized tridentate ligand 2-methyl-6-((2-phenyl-2-(pyridin-2-yl)hydrazineylidene)methyl)pyridine with three N-atoms as donating site was used to synthesize the complex Ru-(a). The molecular structure of Ru-(a) was investigated by single-crystal X-ray crystallography and, in solution was characterized by or ESI mass spectrometry. Several spectroscopic techniques were used for the characterization of the ligand as well as complexes. To understand the electronic structure of Ru-(a) and Ru-(b) , DFT computations were utilized. By the utilization of visible light, photo-liberation experiments were examined. The amount of NO released during the photodissociation experiment was measured using the Griess reagent assay technique. The photodissociated NO was fruitfully transported to reduced myoglobin (Mb). The antibacterial activity against Escherichia coli was carried out with 31.2 μg/mL concentration of Ru-(b). In the final results, we detected considerable antibacterial activity against E. coli and showed >99 % inhibition of the bacterial cells after 120 min of the experiment on exposure to visible light. These investigations proposed that Ru-(b) could be employed to treat several topical deliveries of NO for treating various topical and bacterial infections. [Display omitted] [ABSTRACT FROM AUTHOR]

A facile and efficient method for the regioselective [3 + 2] cycloaddition of 4-azidofuroxans to 1-dimethylamino-2-nitroethylene under p-TSA catalysis affording (4-nitro-1,2,3-triazolyl)furoxans was developed. This transformation is believed to proceed via eliminative azide–olefin cycloaddition resulting in its complete regioselectivity. The developed protocol has a broad substrate scope and enables a straightforward assembly of the 4-nitro-1,2,3-triazole motif. Moreover, synthesized (4-nitro-1,2,3-triazolyl)furoxans were found to be capable of NO release in a broad range of concentrations, thus providing a novel platform for future drug design and related biomedical applications of heterocyclic NO donors.

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson’s disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases.

Background: Nitric oxide and reactive nitrogen species play an important role in various pathological conditions like cancer, inflammation and neurodegeneration. As plants and natural compounds have a great potency of discovering lead compounds which might affect NO production during inflammation and various pathologies, we examined the effects of three medicinal plants native to Iran, on NO production during oxidative stress in PC12 cells. Methods: In this study, cell death and NO levels were measured by MTT and by Griess assay, respectively. Oxidative stress was induced by hydrogen peroxide and extracts of Astragalus jolderensis, Convolvulus commutatus and Salvia multicaulis were used as pretreatment in oxidative stressed PC12 cells. Results: A. jolderensis extract significantly suppressed NO production in 150 and 200 μg/ml concentrations and C. commutatus extract in all concentration inhibited NO production in stressed PC12 cells. In addition, the extract of S. multicaulis inhibited NO production during stress at all concentrations above 50 μg/ml. Besides, the extract of S. multicaulis showed protective effect at lower doses in stressed cells. Conclusion: According to the results, S. multicaulis inhibited NO production and protected cells from oxidative stress. Hence, S. multicaulis is a good candidate for further in vitro and in vivo investigations. A. jolderensis and C. commutatus also suppressed NO production during stress. Therefore, they could be noticed in experiments that centralize on the inhibition of NO production and drug discovery studies in the field of neurodegenerative and chronic inflammatory diseases.

The anaerobe Clostridium acetobutylicum belongs to the most important industrially used bacteria. Whereas genome mining points to a high potential for secondary metabolism in C. acetobutylicum, the functions of most biosynthetic gene clusters are cryptic. We report that the addition of supra‐physiological concentrations of cysteine triggered the formation of a novel natural product, clostrisulfone (1). Its structure was fully elucidated by NMR, MS and the chemical synthesis of a reference compound. Clostrisulfone is the first reported natural product with a diphenylsulfone scaffold. A biomimetic synthesis suggests that pentamethylchromanol‐derived radicals capture sulfur dioxide to form 1. In a cell‐based assay using murine macrophages a biphasic and dose‐dependent regulation of the LPS‐induced release of nitric oxide was observed in the presence of 1. [ABSTRACT FROM AUTHOR]

A novel colorimetric sheet based on Griess reagent–doped mesoporous silica nanoparticles was developed for nitrite detection. Griess reagent was adsorbed on long–range ordered hexagonal mesoporous silica nanoparticles and developed ink–bottle pores with some disorder. When the modified nanoparticles were bound using starch to fabricate a thin (~ 313 μm) colorimetric sheet, spherical particles with a rougher surface and some distortion of their mesoporosity were observed. The sheet was used in conjunction with digital image colorimetry (DIC) and provides a wide linear range of 0.05 to 2.50 mg L−1 with a low detection limit (15.0 μg L−1–NO2−, equal to 4.5 μg L−1 NO2−–N), good inter-day precision (1.93%RSD), and excellent precision (2.67% relative error). The colorimetric sensors produced from the sheet costs only 0.04 USD each, while the DIC uses a standard smartphone for photographic detection. The method developed offers an easier and cheaper means of conducting rapid on-site determination of nitrite in water with reliable quantitative results. [ABSTRACT FROM AUTHOR]

Two new benzophenone glycosides, aquilarisides A (1) and B (2), together with six known analogues (3-8) were isolated from the pericarps of Aquilaria yunnanensis S. C. Huang. Their structures were elucidated on the basis of 1D and 2D NMR and mass spectroscopic analyses, and the absolute configuration of compound 1 was determined by experimental and calculated electronic circular dichroism (ECD) spectra. Anti-inflammatory activities of all compounds 1–8 were evaluated for their inhibitory activities against lipopolysaccharide (LPS)-stimulated induced nitric oxide (NO) production in RAW 264.7 cells using the Griess assay. Compound 2 indicated a weak inhibition of NO production. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a key mediator that plays an important role in pathogenesis of various chronic diseases like Alzheimer’s disease and Parkinson’s disease. Additionally, there is a great attitude for finding natural compounds, which could control and inhibit NO production in pathological conditions. Therefore, we were encouraged to investigate the effects of some Lamiaceae species on NO production and cell injury during oxidative stress in PC12 cells. In this study, cell death determined by MTT assay and NO levels were evaluated using Griess assay. PC12 cells were exposed to total metanolic extracts of three Scutellaria and one Nepeta species. The results revealed that Nepeta laxiflora (N. laxiflora) could protect PC12 cells from hydrogen proxide-induced oxidative stress and all of the plants inhibited NO production in that condition except Scutellaria tournefortii (Sc. tournefortii). In addition, Scutellaria multicaulis (Sc. multicaulis) was meanwhile subjected to fractionation using different organic solvents. The dichloromethan and ethyl acetate fractions of Sc. multicaulis could protect PC12 cells from oxidative stress injury. However, NO production was restrained by the hexane and dichloromethane fractions. Considering the results, N. laxiflora, Scutellaria nepetifolia (Sc. nepetifolia), and Sc. multicaulis are good candidates for further investigations in neuroprotection and anti-inflammation studies. [ABSTRACT FROM AUTHOR]

Abstract Mitragyna speciosa Korth., is a controversial plant that possesses various medicinal values and has been used from time immemorial in folk medicine. Recently, it has been widely abused as a narcotic source. The aim of the present study was to characterize the in vitro nitric oxide (NO) inhibition property of the supercritical CO 2 fluid extracts from M. speciosa leaves and to identify the chemical constituents of the extract that exhibited the most inhibition without toxicity effect. Samples were extracted using the green technology, supercritical fluid extraction (SFE) technique via CO 2 as the mobile phase. The NO inhibitory activity was evaluated by Griess assay, which measures the formation of nitrite ion (NO 2 −) in recombinant mouse interferon gamma/lipopolysaccharide (IFN-γ/LPS) stimulated RAW 264.7 cells. Matrix 5 Step-1 (M5S1) that was extracted with pure CO 2 at 3000 psi and 60 °C exhibited the highest NO inhibitory activity (60.08%) without cytotoxicity (cell viability, 91.98%) at a concentration of 100 μg/mL. GC and GC–MS analysis revealed palmitic acid as the major constituent (34.90%) of M5S1. This study provides first evidence that M5S1, the non-alkaloidal extract obtained by supercritical fluid extraction of M. speciosa leaves possesses potential property in preventing inflammatory diseases mediated by excessive production of NO. [ABSTRACT FROM AUTHOR]

The development of solid materials that deliver nitric oxide (NO) are of interest for several therapeutic applications. Nevertheless, due to NO’s reactive nature, rapid diffusion and short half-life, reporting their NO delivery characteristics is rather complex. The full knowledge of this parameter is fundamental to discuss the therapeutic utility of these materials, and thus, the NO quantification strategy must be carefully considered according to the NO-releasing scaffold type, to the expected NO-releasing amounts and to the medium of quantification. In this work, we explore and discuss three different ways of quantifying the release of NO in different biological fluids: haemoglobin assay, Griess assay and NO electrochemical detection. For these measurements, different porous materials, namely zeolites and titanosilicates were used as models for NO-releasing platforms. The oxyhaemoglobin assay offers great sensitivity (nanomolar levels), but it is only possible to monitor the NO release while oxyhaemoglobin is not fully converted. On the other hand, Griess assay has low sensitivity in complex biological media, namely in blood, and interferences with media make NO measurements questionable. Nevertheless, this method can measure micromolar amounts of NO and may be useful for an initial screening for long-term release performance. The electrochemical sensor enabled real-time measurements in a variety of biological settings. However, measured NO is critically low in oxygenated and complex media, giving transient signals, which makes long-term quantification impossible. Despite the disadvantages of each method, the combination of all the results provided a more comprehensive NO release profile for these materials, which will help to determine which formulations are most promising for specific therapeutic applications. This study highlights the importance of using appropriate NO quantification tools to provide accurate reports.

A facile and effective colorimetric-sensing platform based on the diazotization of phenosafranin for the detection of NO 2 − under acidic conditions using the Griess assay is presented. Diazotization of commercial phenosafranin produces a color change from purplish to blue, which enables colorimetric quantitative detection of NO 2 − . Optimal detection conditions were obtained at a phenosafranin concentration of 0.25 mM, HCl concentration of 0.4 M, and reaction time of 20 min. Under the optimized detection conditions, an excellent linearity range from 0 to 20 μM was obtained with a detection limit of 0.22 μM. Favorable reproducibility and selectivity of the colorimetric sensing platform toward NO 2 − were also verified. In addition, testing spiked ham sausage, bacon, and sprouts samples demonstrated its excellent practicability. The presented colorimetric sensing platform is a promising candidate for the detection of NO 2 − in real applications.

Nitrite ions are important markers of inflammation, food safety, and water quality; therefore, various methods are under development to detect nitrite ions. This paper describes a novel method for detecting nitrite ions using polyethylene glycol (PEG) hydrogel superimposed glass fiber membrane strips. A colorimetric Griess assay was performed on the hydrogel modified with N -(1-naphthyl)ethylenediamine by dropping a solution of nitrite and sulfanilamide. This colorimetric hydrogel sensor exhibited a detection limit as low as 10 μM for nitrite ions and the working concentration of nitrite ions was between 10 μM and 5 mM. The colorimetric reaction was completed within a few seconds; the performance test results indicated that the sensor was specific to nitrite ions compared to other anions, and it was suitable to detect nitrite ions in clinical samples. In the future, this sensor platform can be used to detect nitrite ions in various complex solutions including biological and environmental samples. [ABSTRACT FROM AUTHOR]

Three EPR silent dinitrosyl cobalt complexes (DNCCs) containing a {Co(NO) 2 }10 unit were synthesized and characterized by cyclic voltammograms, NMR and X-ray diffraction, etc. The DNCCs have demonstrated significantly enhanced NO release efficacy in DMEM/F-12 medium, enhanced NO cellular uptake by human umbilical vein endothelial cells as well as low toxicity than marketed sodium nitroprusside. [Display omitted] • Dinitrosyl cobalt complexes demonstrate more efficient and sustained NO release than sodium nitroprusside. • Dinitrosyl cobalt complexes exhibit fast and efficient NO cellular uptake by human umbilical vein endothelial cells. • Dinitrosyl cobalt complexes demonstrate low cytotoxicity to human umbilical vein endothelial cells. Three dinitrosyl cobalt complexes (DNCCs) [(Dppe)Co(NO) 2 ]Cl , [(Dppp)Co(NO) 2 ]Cl and [(TMEDA)Co(NO) 2 ]BPh 4 were synthesized using CoCl 2 ·6H 2 O as a starting material and were isolated via fast column chromatography with final yields ranging from 53% to 67%. These EPR silent DNCCs each showed only one pair of redox events ({Co(NO) 2 }10/11) in the cyclic voltammograms. In comparison with sodium nitroprusside (SNP), the DNCCs' NO release amount and duration significantly enhanced, and were related closely to their hydrolytic stability. Additionally, DNCCs have demonstrated significantly higher NO cellular uptake rates in the first 5 h than SNP by human umbilical vein endothelial cells (HUVEC), to which the DNCCs also showed low cytotoxicity. Based on the fact that DNCCs could hydrolyze yielding ligands as the remaining products and the possible formation of [(Dppp)Co(NO) 2 ] and [(Dppp)Co(PPh 3)(NO)] in the reduction of [(Dppp)Co(NO) 2 ]Cl , a plausible NO release mechanism via a one electron reduction process was proposed. [ABSTRACT FROM AUTHOR]

Inflammation is linked to numerous chronic disease states. Phenolic compounds have attracted attention because a number of these compounds possess anti-inflammatory properties. A phenolic crude extract was prepared from pecans and separated by Sephadex LH-20 column chromatography into low- and high-molecular-weight (LMW/HMW) fractions. Anti-inflammatory properties of these fractions were assessed in LPS-stimulated RAW 264.7 murine macrophage cells. NO and reactive oxygen species (ROS) production was monitored after 3 different experimental protocols: (1) pre-treatment with Escherichia coli O111:B4 lipopolysaccharide (LPS); (2) pre-treatment with a pecan crude extract and its fractions; and (3) co-incubation of LPS with a pecan crude extract and its fractions. The LMW fraction displayed a dose-dependent decrease in NO production and a significant decrease from the LPS control in ROS production when cells were either co-incubated with or pre-treated with LPS. The phenolics were characterized by HPLC to help identify those responsible for the observed effect. [ABSTRACT FROM AUTHOR]

Inhibition of cyclooxygenase-2 (COX-2) is a promising anti-inflammatory therapeutic strategy, but long-term medication with COX-2-inhibitors (coxibs) may be associated with adverse cardiovascular effects. Functionalization of existing lead structures with nitric oxide (NO)-releasing moieties is an auspicious approach to minimize these effects. In this regard, an organic nitrate (–O–NO 2 ) substituent was introduced at a (pyrazolyl)benzenesulfonamide lead structure. The novel NO-coxibs selectively inhibited COX-2 in a low micromolar range (IC 50 (COX-2): 0.22–1.27 μM) and are supposed to be promising anti-inflammatory compounds with, in parallel, positive effects on vascular homeostasis. [ABSTRACT FROM AUTHOR]

A procedure for quantitative analysis of nitric oxide production by cultured mammalian cells based on nitrite ion quantitation has been optimized. The Griess assay adapted for microvolumes was found to yield optimal results when the NO ions formed from NO ions in the reaction medium were reduced by nonactivated cadmium prior to the assay to form the analyte NO. The assay was verified for the culture of C6 rat glioma cells. The assay developed is suitable for quantitative determination of nitrogen oxide in 96- and 48-well cell culture plates; the detection limit for NO was 2.1 ± 0.1 μM and that for NO was 2.9 ± 0.1 μM. [ABSTRACT FROM AUTHOR]

The Griess assay is used to measure nitric oxide concentrations in liquid solutions after reaction into nitrite. The assay is challenging when applied to cell culture supernatants. During optimization, we focused on the anti-inflammatory potential of test compounds in murine RAW264.7 macrophages. This led to (i) the required inductivity of cells by lipopolysaccharide (LPS) and allowed (ii) the characterization of putative anti-inflammatory test compounds with high sensitivity. The modifications reported here prominently improved resolution and efficiency of the widely used Griess assay and are of broad interest for studies on the pharmacological modulation of macrophages activation. [ABSTRACT FROM AUTHOR]

The Griess assay has been used to determine the possible changes in the measured NO concentrations induced by TiO nanoparticles in three types of nitrite-containing samples: aqueous NaNO solutions with known concentrations, and two types of cell culture media-Roswell Park Memorial Institute medium (RPMI-1640) and Dulbecco's Modified Eagle Medium (DMEM-F12) used either as delivered or enriched in NO by NaNO addition. We have used three types of titania with average particle sizes between 10 and 30 nm: Degussa P25 and two other samples (undoped and Fe-doped anatase TiO) synthesised by a hydrothermal route in our laboratory. The structural, morphological, optical and physicochemical characteristics of the used materials have been studied by X-ray diffraction, transmission electron microscopy (EDX), Mössbauer spectroscopy, Brunauer-Emmett-Teller nitrogen adsorption, UV-Vis reflectance spectroscopy, dynamic light scattering and diffuse reflectance infrared Fourier transform spectroscopy. The opacity and sedimentation behaviour of the studied TiO suspensions have been investigated by photometric attenuance measurements at 540 nm. To account for the photocatalytic properties of titania in a biologically relevant context, multiple Griess tests have been performed under controlled exposure to laboratory natural daylight illumination. The results show significant variations of light attenuance (associated with NO concentrations in the Griess test) depending on the opacity, sedimentation behaviour, NO adsorption and photocatalytic properties of the tested TiO nanomaterials. These findings identify material characteristics recommended to be considered when analysing the results of Griess tests performed in biological studies involving TiO nanoparticles. [ABSTRACT FROM AUTHOR]

Abstract: A new method for extraction and concentration of organic dyes that uses a reagent composed of a nonionic detergent mixed with an alcohol is described. We have observed that water-soluble organic dyes are also soluble in nonionic detergents and can be extracted by adding salt, which separates the dye–detergent component from the aqueous phase. We have also found that mixing nonionic detergents with alcohols markedly reduces their viscosity and produces stable, free-flowing, and effective reagents for color extraction. On the basis of these observations, we used a mixture of Triton X-100 and 1-butanol and observed that water-soluble natural and synthetic chromophores, as well as dyes generated in biochemical reactions, can be extracted, concentrated, and analyzed spectrophotometrically. Trypan blue and phenol red are used as examples of synthetic dyes, and red wine is used as an example of phenolic plant pigments. Applications for quantification of nitric oxides and sialic acids are described in more detail and show that as little as 0.15nmol of nitric oxide and 0.20nmol of sialic acid can be detected. A major advantage of this method is its ability to concentrate chromophores from dye-containing solutions that otherwise cannot be measured because of their low concentrations. [Copyright &y& Elsevier]

Abstract: Developing an understanding of how chronically elevated levels of nitric oxide at sites of inflammation or infection can lead to cancer and other diseases requires ways to expose cells and biomolecules to controlled concentrations of NO for hours to days. To achieve this, a small (65ml) stirred reactor was fabricated that included a flat, porous poly(tetrafluoroethylene) membrane and a loop of poly(dimethylsiloxane) tubing for NO and O2 delivery, respectively. It was equipped with probes for continuous monitoring of NO and O2 concentrations. Transport through the membrane and tubing was characterized using separate O2 depletion experiments. In experiments using only a 10% NO mixture and a buffer that was initially air-equilibrated, constant rates of accumulation were observed for NO2 − (53±2μM/h; n =8), the end product of NO oxidation, as expected. Simultaneous delivery of NO and O2 yielded steady NO concentrations of 0.7–2.3μM, depending on the tubing length and gas compositions. A model was developed that allows the steady NO and O2 concentrations and the duration of the transients to be predicted to within a few percent. This system should be useful for exposing cells and biomolecules to concentrations of NO that mimic those in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Nitric oxide (NO) plays a role in various physiological and pathophysiological conditions such as immunoregulatory and inflammatory processes. Hence, NO and its generating enzyme, inducible nitric oxide synthase (iNOS) may not only be of diagnostic and prognostic value, but may also serve as targets for novel therapeutic options. In the present investigation, we have screened a phytochemical library by correlating the IC50 values for 531 natural products of 60 cell lines with the microarray-based mRNA expression of 95 genes known to be involved in NO metabolism and signaling with the aim to identify candidate compounds as inhibitors for NO metabolism and signaling. We identified bis(helenalinyl)glutarate (BHG) as putative candidate compound. Indeed, BHG inhibited NO production (IC50 value: 0.90±0.04μM) and down-regulated iNOS protein expression (IC50 value: 1.12±0.16μM) of RAW264.7 mouse macrophages in the presence of lipopolysaccharide and interferon-γ. Performing XTT cytotoxicity assays, we found that BHG inhibited cell growth in a dose-dependent manner with an IC50 value of 5.6μM. To gain insight into molecular pathways involved in NO inhibition and cytotoxicity, we performed microarray experiments which were exemplarily validated by real-time RT-PCR. A total of 227 genes (67 up- and 160 down-regulated) were obtained, which exhibited significant differences in mRNA regulation between BHG-treated and untreated RAW264.7 macrophages. Sixteen of 227 genes are known to be involved in NO-signaling. Pathway analyses revealed that further five and four down-regulated genes belong to the glucocorticoid receptor and interleukin-1 and interleukin-10 pathways, respectively. An interference of these two pathways and NO is known for inflammation and auto-immune diseases. The therapeutic potential of this compound has to be explored in the future. [Copyright &y& Elsevier]

The methanol extracts of 32 plant parts of 19 species of the genus Garcinia (Guttiferae) were collected from rainforests of the Malaysian Peninsula and the island of Sumatra, Indonesia, for evaluation of their in vitro cytotoxic and nitric oxide inhibitory activities. An end-point MTT cell viability assay was used to determine the 50% inhibitory concentration (IC50) of the extracts in three human tumor cell lines representing tumors of the breast (MCF-7), lung (NCI-H460) and prostate (DU-145). Griess assay was performed to assess the nitric oxide (NO) inhibitory activity. Of the 32 extracts, 27 showed cytotoxic activity in at least one of the three tumor cell lines used in this study. Four extracts, Garcinia opaca King (fruit), Garcinia maingayi Hook.f. (stem), Garcinia penangiana Pierre (leaf) and Garcinia urophylla Scortech.ex King (leaf) extracts showed the most potent and selective cytotoxic activity against MCF-7 cells (IC50 3-8 μg/mL). The extracts from Garcinia cowa Roxb. (stem), Garcinia bancana Miq. (stem) and Garcinia malaccensis Hook.f. (leaf) showed moderate activity and selectivity towards non-small lung tumor cells. The extracts from Garcinia bancana (stem), Garcinia malaccensis (stem), Garcinia prainiana King (leaf), Garcinia rostrata Hassk.ex Hook.f. (stem and leaf), Garcinia cowa (stem) and Garcinia nervosa Miq. (leaf) exhibited inhibition against NO production without affecting the viability of LPS and IFN-γ-induced RAW 264.7 macrophage cells. Among these, the most promising extracts were G. bancana (stem) and G. malaccensis (stem), as they showed the highest selectivity indices (> 50) for NO inhibition. In conclusion, these data provide evidence that some of the Garcinia species could potentially contain potent and selective cytotoxic and anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

Abstract: Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure–function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed. [Copyright &y& Elsevier]

Abstract: Mass spectrometry-based approaches are the reference techniques for the determination of nitrite and nitrate in plasma and serum. However, due to their simplicity and rapidity, assays based on the Griess reaction or HPLC are generally used in clinical studies, but they generate diverging values for nitrite/nitrate concentration. In this study, particular attention is paid to the optimization of the deproteinization procedure for plasma and serum samples prior to nitrite/nitrate analysis by an enzymatic batch Griess assay, HPLC and GC–MS. A method is reported to verify completeness of deproteinization and to correct for nonspecific contribution to the absorbance of the diazo dye at 540nm. With the application of such optimized procedures, we were able to significantly improve the correlation between Griess and HPLC method or the GC–MS technique for nitrite+nitrate concentrations in human serum and plasma. Despite remaining potentially interfering pre-analytical and analytical factors, the procedures reported in the present study may be helpful in a critical evaluation of limits and possibilities of the enzymatic batch Griess assay as a large-scale method for nitrite/nitrate determination in human serum in clinical studies. [Copyright &y& Elsevier]

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO˙) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO˙ production by the oxymyoglobin oxidation assay, or electrochemically using an NO˙ electrode. An apparent NO˙ production detected by the Griess assay was identified as an artifact. NO˙ generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO˙. Our data show that NO˙ production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed. [ABSTRACT FROM AUTHOR]

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152 kDa along with a proteolytically cleaved product of 121 kDa. The DNOS heme protein exhibited very low Ca2+/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3–4 M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure–function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant. [Copyright &y& Elsevier]

Recent studies have shown that nitric oxide molecule may have a role in the development of cataract. In this study, we measured the levels of a nitric oxide metabolite (nitrite) in the cataractous and normal human lenses. A modified Griess assay was used to determine the nitrite levels in the lenses as a measure of nitric oxide, based on the spectrophotometric method. Nitrite was detected in 26 (44·1%) cataractous lenses and was found below the detection limit in 33 (55·9%) cataractous lenses. Mean nitrite levels in cataractous lenses (2·77±5·26 nmol/100 mg) was found higher than the normal lenses (0·77±0·79 nmol/100 mg) but this increase was not statistically significant. Comparison of nitrite levels among lenses with various types of cataracts revealed higher levels in lenses with posterior subcapsular cataracts. Hypertensive patients had also significantly higher nitrite levels in their cataractous lenses. The increased levels in the cataractous lenses display a possible role of nitric oxide in the pathogenesis of cataract in human eyes. [Copyright &y& Elsevier]

Introduction: Amperometric sensors that directly measure nitric oxide (NO) are readily employed in pharmacologic research. While several of these sensors have been developed, none has been investigated for use in conscious, freely moving animals. An approach was developed and validated for real-time quantitation of neuronal NO production in rats without restricting locomotor activity or other potentially useful behavioral endpoints. Methods: Male Sprague–Dawley rats were equipped with a femoral vein or intraperitoneal cannula. A guide cannula and an amperometric NO sensor were placed in the left and right hippocampus, respectively. Following recovery, rats received a 6-h intravenous infusion of saline, l-arginine (an NO precursor; 250 or 500 mg/kg/h), or incremental intraperitoneal 7-nitroindazole (an NO synthase inhibitor; 200-mg/kg loading dose and 100 mg/kg every 2 h). The sensor recorded NO production continuously and microdialysis samples were collected incrementally throughout the experiment. Griess assay analysis of microdialysate samples was compared to sensor readings in vivo. In vitro degradation of an NO donor also was used to validate sensor performance. Results: Exogenous administration of l-arginine resulted in incremental increases in the neuronal NO signal. A reduction in NO production was observed during administration of 7-nitroindazole, a selective neuronal NO synthase inhibitor. A significant correlation was observed in vitro between the Griess assay analysis, an indirect analytical approach, and the NO sensor readings. The lack of a strong correlation between these measures in vivo is consistent with the indirect nature of the Griess assay. Discussion: The current approach allows real-time determination of neuronal NO production in unrestrained rats. This model will be invaluable in evaluating pharmacologic issues regarding brain tissue NO synthesis, assessing brain NO synthase as a molecular target, and establishing the effects of pharmacologic agents on neuronal NO production. [Copyright &y& Elsevier]

Background: Danshen (Radix et rhizoma Salviae miltiorrhizae ; Salvia miltiorrhiza Bunge, Lamiaceae) is commonly used in Asia, including China, Japan, and Korea with markets in America and Europe growing substantially. It is included in multiple pharmacopeias and salvianolic acid B and tanshinone IIA are used as quality markers. However, on the markets, substitutes and different processing methods often are a concern. a concern regarding patients' safety and expected outcomes. Aims: This study aims at understanding the quality of Danshen-derived products on the market, and the relationship between the chemistry, biological activity and the processing and storage methods. Methods: For heavy metal analysis, inductively coupled plasma optical emission spectrometry was used. High performance thin-layer chromatography and proton nuclear magnetic resonance coupled with principal component analysis were used to understand the variation of metabolite composition. MTT assay and LPS induced NO production assay were used to evaluate the cytotoxicity effect and anti-inflammatory activity, respectively. Result and Discussion: Six out of sixty samples exceed the limits of cadmium according to the Chinese or United States Pharmacopoeia. Arsenic, lead and copper contents are all below pharmacopoeial thresholds. With more complex processing procedure, the risk of heavy metal contamination increases, especially with arsenic and cadmium. The metabolite compositions show a variability linked to processing and storage methods. Authenticated samples and Vietnamese primary samples contain higher salvianolic acid B, and their chemical compositions are more consistent compared to Chinese online store samples. Overall, a significant chemical variation can be observed in Danshen products directly linked to processing and storage method. In the MTT assay, fourteen samples show cytotoxicity while seven samples increase the proliferation of RAW264.7. In the LPS induced NO production of RAW 264.7, only seven samples show significant inhibitory effects. Conclusion: This is the first interdisciplinary investigation focusing on understanding the current market and the quality of Danshen. The quality of Danshen products on the high street are inferior to the authenticated samples. The results of the bioassays selected is not useful to differentiate the quality and composition according to the current definition in the pharmacopoeias. Overall, this approach highlights the tremendous variability of the products linked to processing and the need for more systematic and stringent quality assurance. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "A simple, fast and inexpensive automated technique for measurement of plasma nitrite," by R. da Silva Pereira in the 2010 issue.

The development of solid materials that deliver nitric oxide (NO) are of interest for several therapeutic applications. Nevertheless, due to NO's reactive nature, rapid diffusion and short half-life, reporting their NO delivery characteristics is rather complex. The full knowledge of this parameter is fundamental to discuss the therapeutic utility of these materials, and thus, the NO quantification strategy must be carefully considered according to the NO-releasing scaffold type, to the expected NO-releasing amounts and to the medium of quantification. In this work, we explore and discuss three different ways of quantifying the release of NO in different biological fluids: haemoglobin assay, Griess assay and NO electrochemical detection. For these measurements, different porous materials, namely zeolites and titanosilicates were used as models for NO-releasing platforms. The oxyhaemoglobin assay offers great sensitivity (nanomolar levels), but it is only possible to monitor the NO release while oxyhaemoglobin is not fully converted. On the other hand, Griess assay has low sensitivity in complex biological media, namely in blood, and interferences with media make NO measurements questionable. Nevertheless, this method can measure micromolar amounts of NO and may be useful for an initial screening for long-term release performance. The electrochemical sensor enabled real-time measurements in a variety of biological settings. However, measured NO is critically low in oxygenated and complex media, giving transient signals, which makes long-term quantification impossible. Despite the disadvantages of each method, the combination of all the results provided a more comprehensive NO release profile for these materials, which will help to determine which formulations are most promising for specific therapeutic applications. This study highlights the importance of using appropriate NO quantification tools to provide accurate reports. [ABSTRACT FROM AUTHOR]

A facile and effective colorimetric-sensing platform based on the diazotization of phenosafranin for the detection of NO 2 − under acidic conditions using the Griess assay is presented. Diazotization of commercial phenosafranin produces a color change from purplish to blue, which enables colorimetric quantitative detection of NO 2 − . Optimal detection conditions were obtained at a phenosafranin concentration of 0.25 mM, HCl concentration of 0.4 M, and reaction time of 20 min. Under the optimized detection conditions, an excellent linearity range from 0 to 20 μM was obtained with a detection limit of 0.22 μM. Favorable reproducibility and selectivity of the colorimetric sensing platform toward NO 2 − were also verified. In addition, testing spiked ham sausage, bacon, and sprouts samples demonstrated its excellent practicability. The presented colorimetric sensing platform is a promising candidate for the detection of NO 2 − in real applications. [ABSTRACT FROM AUTHOR]

In this work, we presented a sensitive and selective colorimetric and fluorescent dual-readout sensor based on Griess assay for nitrite (NO 2 −) detection under acidic condition. The sensor system was constituted of acid-resistant carbon quantum dots (CQDs) and 3-aminophenol (3-Aph) with acidic condition regulated using HCl. During the sensing procedure, reaction of 3-Aph and NO 2 − under acidic condition can yield a yellow-colored azoic compound (AZO), which gives the colorimetric readout; meanwhile, the fluorescence of CQDs (fluorescence reporter) quenched due to the strong absorption of AZO, leading to fluorescent readout. Wherein, CQDs were synthesized via hydrothermal method through using polyacrylamide as precursor and characterized by AFM, XRD and XPS. Under the optimized condition, the sensor exhibit broad linear relationships towards NO 2 − in the range of 10 to 100 nM and 2.5 to 100 μM, with practical detection limits of 10 nM and 2.5 μM for the fluorescent and colorimetric readout, respectively. And the sensor displayed excellent capability of selectivity according to interferences study. Furthermore, testing of sprouts, bacon and ham sausage real samples demonstrated good recoveries and reproducibility of the sensor system. All these results suggested the presented colorimetric and fluorescent dual-readout sensor can be a promising candidate for the NO 2 − detection in real applications. Unlabelled Image • It is the first time to use commercial 3-aminophenol as reaction site to detect nitrite based on Griess assay. • Fluorescent and colorimetric dual-readout sensing platform was simply fabricated. • This work could serve as a good model for the design of new fluorescent and colorimetric dual-readout sensor. • The sensor as-presented display good sensitivity and excellent capability of anti-disturbance, reproducibility and stability. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a key mediator that plays an important role in pathogenesis of various chronic diseases like Alzheimer's disease and Parkinson's disease. Additionally, there is a great attitude for finding natural compounds, which could control and inhibit NO production in pathological conditions. Therefore, we were encouraged to investigate the effects of some Lamiaceae species on NO production and cell injury during oxidative stress in PC12 cells. In this study, cell death determined by MTT assay and NO levels were evaluated using Griess assay. PC12 cells were exposed to total metanolic extracts of three Scutellaria and one Nepeta species. The results revealed that Nepeta laxiflora ( N. laxiflora ) could protect PC12 cells from hydrogen proxide-induced oxidative stress and all of the plants inhibited NO production in that condition except Scutellaria tournefortii ( Sc. tournefortii ). In addition, Scutellaria multicaulis ( Sc. multicaulis ) was meanwhile subjected to fractionation using different organic solvents. The dichloromethan and ethyl acetate fractions of Sc. multicaulis could protect PC12 cells from oxidative stress injury. However, NO production was restrained by the hexane and dichloromethane fractions. Considering the results, N. laxiflora , Scutellaria nepetifolia ( Sc. nepetifolia ) , and Sc. multicaulis are good candidates for further investigations in neuroprotection and anti-inflammation studies.