Redox Reactivity of Nonsymbiotic Phytoglobins towards Nitrite.

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins.

The Phytochemical Analysis of Vinca L. Species Leaf Extracts Is Correlated with the Antioxidant, Antibacterial, and Antitumor Effects.

The phytochemical analysis of Vinca minor, V. herbacea, V. major, and V. major var. variegata leaf extracts showed species-dependent antioxidant, antibacterial, and cytotoxic effects correlated with the identified phytoconstituents. Vincamine was present in V. minor, V. major, and V. major var. variegata, while V. minor had the richest alkaloid content, followed by V. herbacea. V. major var. variegata was richest in flavonoids and the highest total phenolic content was found in V. herbacea which also had elevated levels of rutin. Consequently, V. herbacea had the highest antioxidant activity followed by V. major var. variegata. Whereas, the lowest one was of V. major. The V. minor extract showed the most efficient inhibitory effect against both Staphylococcusaureus and E. coli. On the other hand, V. herbacea had a good anti-bacterial potential only against S. aureus, which was most affected at morphological levels, as indicated by scanning electron microscopy. The Vinca extracts acted in a dose-depended manner against HaCaT keratinocytes and A375 melanoma cells and moreover, with effects on the ultrastructure, nitric oxide concentration, and lactate dehydrogenase release. Therefore, the Vinca species could be exploited further for the development of alternative treatments in bacterial infections or as anticancer adjuvants.

Remarkable rutin-rich Hypericum capitatum extract exhibits anti-inflammatory effects on turpentine oil-induced inflammation in rats.

BACKGROUND: Natural extracts with beneficial biological activities are nowadays of high interest, in various treatment or prophylaxis. Hypericum capitatum has been known for its curative effects for centuries and its extracts have become of interest due to their distinct activity among other Hypericaceae members. In this study, further light is aimed to be shed on the secondary-metabolites composition of H. capitatum extracts, using chromatographic techniques and Electron paramagnetic resonance profiles in alkaline medium. Considering that no previous works explored the anti-inflammatory activity of H. capitatum, here, an in vivo study is also designed in order to evaluate this property by assessing the impact of one of H. capitatum extracts in ameliorating turpentine oil-induced inflammation on rats and to quantify their blood antioxidants level. METHODS: Chromatographic techniques and Electron paramagnetic resonance spectroscopy were used in order to describe the chemical profile in different parts of the plant. The in vivo study on turpentine-oil induced inflammation in rats included three doses of H. capitatum extract expressed in rutin concentration. Oxidative stress was measured using total oxidative status, total antioxidant capacity, oxidative stress index, 3-nitrotyrosine, nitric oxide, malondialdehyde, superoxide dismutase, catalase and the inflammatory response was evaluated by performing a complete blood cells count and C reactive protein. RESULTS: The extract was remarkably rich in rutin; however, other polyphenolic-like minor components appeared important in explaining the observed biological properties. The tested extract prevents the increase of inflammation-induced white blood cell count, number of neutrophils, and serum nitric oxide, and did so in a dose-dependent manner, similarly to the positive control-diclofenac. In addition, the same extract appeared to be a good alternative to diclofenac to restore total oxidative status, thiobarbituric active reactive species, total proteins and C reactive proteins. Moreover, antioxidant enzymes such as catalase, superoxide dismutase and total serum thiol concentration were significantly increased by the tested extract. CONCLUSIONS: Due to its powerful reservoir rich in rutin, H. capitatum extract depicted its in vivo antioxidant and anti-inflammatory effects indicating it to be a good alternative to conventional drugs for oxidative stress protection.

A biomimetic assay for antioxidant reactivity, based on liposomes and myoglobin.

Antioxidant assays are typically based on non-physiologically relevant reagents. We describe here a quantitative assay based on the inhibition of the liposome autooxidation in the presence of myoglobin (ILA-Mb), an oxidative process with direct biomedical relevance. Additional advantages of the assay include the use of standard and readily available reagents (lecithin and myoglobin) and the applicability to lipophilic antioxidants. The ILA-Mb assay is based on previously reported qualitative or semi-quantitative ones that employed cytochrome c instead of myoglobin. A number of antioxidants are tested, and their IC50 parameters are discussed and interpreted to involve direct interaction with both myoglobin and the liposomes.

Transient radical species and oxygen colorimetric indicators grounded on phenothiazinium dyes.

This study investigates four phenothiazinium dyes including methylene blue (MB) and three analogues containing auxochrome variation 2-4 as oxygen colorimetric indicators prepared by their incorporation into two types of oxygen permeable materials containing: a) polyvinyl alcohol substrate with additional TiO2 photocatalysts (PhOxIn), and b) carboxymethylcellulose polymer matrix containing glucose and KOH (ChOxIn). In vacuum packages where volumetric concentrations of oxygen were below 0.01 %, the leuco forms of the tested phenothiazinum dyes were readily oxidised, initiating the colour turn from white to blue. The redox processes involved were explored by experimental electron paramagnetic resonance EPR and UV-vis spectroscopic methods and further supported by theoretical quantum chemistry. The EPR experiments showed that the chemical oxidation of the leuco-form of the phenothiazinium dyes 1-4 produced transient aminyl radical species with the single electron mainly located at the nitrogen atom of the heterocyclic core. The formation of these transient aminyl species was also perceived by UV-vis spectroscopy, their absorption maxima situated in the 552-592 nm range being also supported by TD-DFT theoretical calculations.

The strange case of polyphenols inhibiting the Briggs-Rauscher reaction: pH-modulated reactivity of the superoxide radical.

Superoxide radical is one of the main players when it comes to oxidative stress. Even if in itself is moderately reactive and can cause the degradation of very few biologically relevant macromolecules, it can dismutate to hydrogen peroxide followed by a possible conversion to hydroxyl radical. In order to protect the internal environment against reactive oxygen species, plants have evolved a line of defence made from secondary metabolites with versatile redox properties, such as flavonoids and phenolic acids. Their characteristics are highly modulated by pH, as they turn into prooxidant compounds as it increases. Reported here are the behaviour and clear patterns in reactivity towards superoxide anion radical of four classes of plant phenolics as a pH function. The reactivity towards superoxide radical in acidic conditions has been studied by use of oscillating Briggs-Rauscher reaction with a new spectroelectrochemical experimental setup, by recording the absorbance in high quality for the first time. Some mechanistic intricacies have also been explored with regard to this method. Reactivity modulation at neutral and slightly basic pH has been assayed by superoxide radical scavenging ability using nitroblue tetrazolium as a substrate. For stronger alkaline pHs studies, Electron Paramagnetic Resonance was exploited. Hydroxybenzoic acids tend to be the least reactive species at all tested pH values. Hydroxycinnamic acids have their activity towards superoxide radical decreased as the pH increases, whereas flavonoids act vice versa.

Comprehensive evaluation of radical scavenging, reducing power and chelating capacity of free proteinogenic amino acids using spectroscopic assays and multivariate exploratory techniques.

Among others, amino acids can inhibit oxidative processes through multiple pathways including inactivation of reactive oxygen species, scavenging free radicals, chelation of pro-oxidative transition metals, as well as reduction of hydroperoxides. Moreover, they can be a major target for oxidants as a result of their abundance in biological systems. The purpose of the present study is to examine the radical scavenging, reducing power and chelating capacity of the free proteinogenic amino acids, using several analytical assays based on different mechanisms and monitored via molecular UV-vis absorption spectrophotometry. In most of the assays, the highest antioxidant activity is rendered by Cys and Trp, known for their involvement in physiological radical reactions, followed by Tyr, Asn and Arg. The least reactive amino acids with respect to both radical scavenging and reducing power principle were Ala, Met, Thr and Val. The hierarchy of the amino acids based on their antioxidant activity is shifting, depending on the analytical assay employed. Results from the multivariate exploratory techniques-hierarchical cluster analysis and principal component analysis-could point out the methods that are most similar and best connected to each other, thus classifying the investigated amino acids according to their activity. Considering the results of chemometric techniques, the antioxidant methods that best discriminate between amino acids are DPPH bleaching and SORS assays. Not only the comprehensive study of antioxidant activity of amino acids is novel in this study but also the application of the sum of ranking differences to compare and rank the various antioxidant capacity assays. The obtained results will support the development of amino acids based multifunctional formulations with important physiologically relevant implications for both humans and animals, with a possible future use in the food industry.

Sugar matters: sugar moieties as reactivity-tuning factors in quercetin O-glycosides.

Quercetin, one of the most abundant flavonoids in plant-based foods, commonly occurs in nature in various glycosylated forms. There is still a less explored aspect regarding the cause of diversity of its glycosides, depending on the sugar moiety attached. This work focuses on four wide-spread quercetin glycosides-hyperoside, isoquercitrin, quercitrin and rutin-by testing the property-tuning capacity of different sugar moieties and thus explains and predicts some of their functions in plant-based foods. The electron paramagnetic spectra of the semiquinone anion radicals of these glycosides were interpreted in terms of hyperfine coupling constants and linewidths, highlighting a clear link between spin density trends, the identity of the bound sugar, and their reactivity corroborated with their modelled structures. Redox potential and lipophilicity were connected to a specific flavonoid-enzyme interaction and correlated with their prooxidant reactivity assessed by oxidation of ferrous hemoglobin. Hyperoside and isoquercitin-galactose and glucose glycosides-exhibit the highest prooxidant reactivity owing to their lowest redox potential and lipophilicity whereas rutin and quercitrin-rutinose and rhamnose glycosides-behave vice versa. The ability of the tested glycosides to undergo HAT or SET-type reactions has also been tested using five different analytical assays, including inhibition of cytochrome c-triggered liposome peroxidation. In most cases, rutin proved to be the most unreactive of the four tested glycosides considering either steric or redox reasons whereas the reactivity hierarchy of the other three glycosides were rather assay dependent.

Chemo-mapping and biochemical-modulatory and antioxidant/prooxidant effect of Galium verum extract during acute restraint and dark stress in female rats.

Galium verum is a well-known medicinal plant which is used in various pathologies. G. verum extracts are characterized here using chromatography, where among the rich pool of phenolic acids of flavonoids two known anti-stress modulators, chlorogenic acid and rutin are identified in high quantities. Additionally, the extracts are characterized using a series of in vitro assays (EPR, DPPH, TPC and TEAC). Considering the chemical findings, the potential beneficial effects of the G. verum extract are explored here in a living organism exposed to stress induced oxidative damages. Thus, the biochemical-modulatory and antioxidant roles of two doses of G. verum extract are examined in animals exposed to acute restraint and dark stress (S). The animals were divided in groups [control, S, SG1 (exposed to 25 mg G. verum extract), SG2 (50 mg extract)]. Increased levels of lipid peroxidation (TBARS from 4.43 to 8.06 nmol/mL), corticosterone from 0.43 to 1.96 µg/dL and epinephrine from 44.43 to 126.7 µg/mL, as well as decreased antioxidant enzymes activities (SOD/CAT) were observed in the S group. The G. verum extract afforded a near-normal equilibrium within the biochemical parameters of animals exposed to RS, by reducing oxidative damage (TBARS at a 3.73 nmol/mL; CS at 0.90 µg/dL; EP at 63.72 µg/mL) and by restoring the antioxidant balance.