ABSTRACT
Three berberine-containing plant extracts were investigated for their pharmacological properties. The stems and leaves of Berberis vulgaris, Mahonia aquifolium, and Phellodendron amurense were characterized through scanning electron microscopy. The plant extracts obtained from fresh stem barks were further analyzed through high-performance liquid chromatography, revealing berberine concentrations, among berbamine and palmatine. The plant extracts were further tested for their anticancer potential against 2D and 3D human skin melanoma (A375) and lung adenocarcinoma (A549) cell lines. The concentrations at which 50% of the cells are affected was determined by the viability assay and it was shown that B. vulgaris, the plant extract with the highest berberine concentration, is the most efficient inhibitor (0.4% extract concentration for the 2D model and 3.8% for the 3D model). The membrane integrity and nitrate/nitrite concentration assays were consistent with the viability results and showed effective anticancer potential. For further investigations, the B. vulgaris extract was used to obtain silver nanoparticles, which were characterized through transmission electron microscopy, energy dispersive spectroscopy, and X-ray diffraction. The formed nanoparticles have a uniform size distribution and are suited for future investigations in the field of biomedical applications, together with the B. vulgaris plant extract.
ABSTRACT
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.
Subject(s)
Arabidopsis , Nitrites , Animals , Nitrites/chemistry , Nitric Oxide/metabolism , Hemoglobins/chemistry , Nitrite Reductases/chemistry , Hypoxia , Arabidopsis/metabolism , Oxidation-Reduction , Mammals/metabolismABSTRACT
Allicin is a major thiosulfinate found in garlic and other Allium sp. and it is responsible for their pungent aroma. It is formed during the tissue lysis of garlic, by the initial action of alliinase upon alliin, the major cysteine sulfoxide. Simultaneous detection of these two analytes is usually performed using HPLC. Contrary to the most important phytoconstituents in other samples, allicin is scarcely detected using the simple HPTLC technique, due to challenges caused by its unique structure, despite its simplicity and high needs in the analytical monitoring of the Allium sp. In this work, a cost-effective, simple, sensitive and accurate method was developed for the determination of allicin together with alliin, using HPTLC. Allicin is quickly pre-derivatised with cysteine in excess to the stable S-allylmercaptocysteine that is then simultaneously detected with alliin, using ninhydrin reagent. The method was validated in terms of accuracy (recoveries of 90-120 %), precision (RSD% of 4-12 %), selectivity, robustness, peak purity and limit of detection (LOD = 0.05 µg/band for allicin and LOD = 0.10 µg/band for alliin). The method was successfully applied using real Allium sp. samples and the results were in good agreement with HPLC data.
Subject(s)
Cysteine , Garlic , Garlic/chemistry , Sulfinic Acids , Disulfides , AntioxidantsABSTRACT
Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.
Subject(s)
Blood Substitutes , Blood Substitutes/chemistry , Blood Substitutes/metabolism , Hemerythrin/chemistry , Hemerythrin/metabolism , Polyethylene Glycols/chemistry , Cysteine/chemistry , Hemoglobins/genetics , Hemoglobins/chemistry , Hemoglobins/metabolism , Oxygen/metabolismABSTRACT
Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities.
Subject(s)
Liposomes/administration & dosage , Melanoma, Experimental/drug therapy , Melanoma/drug therapy , Simvastatin/pharmacology , Skin Neoplasms/drug therapy , Tumor Microenvironment/drug effects , Xanthones/pharmacology , Angiogenesis Inhibitors/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Progression , Macrophages/drug effects , Macrophages/metabolism , Male , Melanoma/metabolism , Melanoma, Experimental/metabolism , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Oxidative Stress/drug effects , Skin Neoplasms/metabolism , Melanoma, Cutaneous MalignantABSTRACT
Herein, a method based on selective piazselenol formation is applied for total selenium determination in biofortified Allium species. Piazselenol is formed by reacting Se(IV) with an aromatic diamine, namely 4-nitro-1,2-phenylenediamine, in acidic medium. Samples were digested in a nitric acid/hydrogen peroxide open system, followed by selenate reduction in hydrochloric acid. Reaction conditions were optimized in terms of pH, temperature, reaction time, and other auxiliary reagents for interference removal, namely, EDTA and hydroxylamine. For the extraction of the selectively formed 4-nitro-piazselenol, micro-solid-phase extraction (µSPE) was applied, and the analysis and detection of the corresponding complex was performed by HPLC coupled with DAD. An external standard calibration curve was developed (R2 = 0.9994) with good sensitivity, and was used to calculate the total selenium content from several Allium plants material, with good intermediate precision (RSD% < 16%). The accuracy of the method was evaluated using both, a comparison with an accepted reference method from our previously published data, as well as three certified reference material with recoveries between 84-126%. The limit of detection was determined to be 0.35 µg/g (in solids) and 1.1 µg/L (in solution), while the limit of quantification was 1.07 µg/g and 3.4 µg/L (in solution). Using the proposed method, selenium content can be quickly and accurately determined in several types of samples. In addition, this study present experimental conditions for overcoming the interferences that might be encountered in selenium determination using piazselenol.
Subject(s)
Allium/chemistry , Azoles/chemistry , Organoselenium Compounds/chemistry , Selenium/analysis , Solid Phase Microextraction , Drug Compounding , Molecular StructureABSTRACT
Advanced chemometric methods, such as fuzzy c-means (FCM), a fuzzy divisive hierarchical clustering algorithm (FDHC), and fuzzy divisive hierarchical associative-clustering (FDHAC), which offer the excellent possibility to associate each fuzzy partition of samples with a fuzzy set of characteristics (features), have been successfully applied in this study. FDHAC, a method that utilizes specific regions of chromatographic fingerprints or specific peaks as a fuzzy set of characteristics, was effectively applied to the characterization and classification of medicinal plant extracts according to their antioxidant capacities, using their chromatographic profiles monitored at 242, 260, 280, 320, 340, and 380 nm via HPLC with a multistep isocratic and gradient elution system and diode array detection (HPLC-DAD). What is quite new is the partitioning of the chromatographic retention time ranges and peaks (markers) and their association with different plant extract samples with high, moderate or low antioxidant capacity. Furthermore, the degrees of membership of fingerprints (fuzzy markers) are highly relevant with respect to the (dis)similarity of samples because they indicate both the positions and degrees of association of chromatographic peaks from different classes or individual samples. The obtained results clearly demonstrate the efficiency and information power of these advanced fuzzy methods for medicinal plant characterization and authentication, and this study generates the premise for a new chemometrics approach with high-impact for use in analytical chemistry and other fields.
Subject(s)
Plant Extracts , Plants, Medicinal , Algorithms , Chromatography, High Pressure Liquid , Cluster AnalysisABSTRACT
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.
Subject(s)
Glycosides/chemistry , Quercetin/analogs & derivatives , Rutin/chemistry , Antioxidants/chemistry , Electron Spin Resonance Spectroscopy , Electrons , Free Radical Scavengers/chemistry , Hydrogen/chemistry , Lipids/chemistry , Oxidants/chemistry , Oxidation-Reduction , Quercetin/chemistry , Rutin/pharmacologyABSTRACT
Excess ascorbate (as expected in intravenous treatment proposed for COVID-19 management, for example) oxidizes and/or degrades hemoglobin and albumin, as evidenced by UV-vis spectroscopy, gel electrophoresis, and mass spectrometry. It also degrades hemoglobin in intact blood or in isolated erythrocytes. The survival rates and metabolic activities of several leukocyte subsets implicated in the antiviral cellular immune response are also affected. Excess ascorbate is thus an unselective biological stress agent.
ABSTRACT
The morphophysiological response of Phaseolus vulgaris L. to low-power electromagnetic radiation was investigated in order to assess the potential harmful effects of long-term continuous exposure. The plants were grown in two separate electromagnetic field (EMF) shielded rooms, in a controlled, greenhouse-like environment. One batch was continuously irradiated during the growth period (from sowing to maturity) and the other one was used as a reference. An unmodulated signal at 915 MHz (the central frequency between the uplink and downlink of the GSM900 mobile communications band) was used, with a maximum power density of 10 mW/m2 measured near the plants. The plants were analyzed using ultraviolet-visible, statistical, morphometric, and electron microscopy methods. Significant differences were observed regarding the height of the plants, number of inflorescences, and chlorophyll and carotenoid content, all closely connected with the ultrastructural changes observed in the leaves. The irradiated batch grew higher (19% increase in plant height, 20% increase in stem and leaves' dry mass), with 18% fewer inflorescences, and extremely long roots (34% increase in dry mass). The ultrastructure of the irradiated leaves showed irregular cells and a higher content of plastoglobules in the chloroplasts. All results indicate that the irradiated plants suffered significant morphological modifications during their long-term exposure to the specific EM radiation. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.
Subject(s)
Chlorophyll , Electromagnetic Fields , Phaseolus/physiology , Plant Leaves , Carotenoids/analysis , Carotenoids/metabolism , Chlorophyll/analysis , Chlorophyll/metabolism , Inflorescence , Phaseolus/chemistry , Plant Leaves/chemistry , Plant Leaves/growth & development , Plant Leaves/ultrastructure , Ultrasonic WavesABSTRACT
BACKGROUND: Pruritus and insomnia are common disorders in hemodialysis (HD) patients, with a major clinical impact as they are associated with poor quality of life and increased mortality. Their coexistence and impact on survival in HD patients have rarely been investigated. Our aim is to investigate the survival of HD patients presenting either none, one, or both disorders and to compare certain features between these groups. METHODS: After the inclusion/exclusion criteria, 170 patients treated by HD or online hemodiafiltration were assigned in 4 study groups depending on the presence of either, neither, or both pruritus and insomnia. We analyzed the survival difference between groups after 20 months, and we searched if there were significant differences in terms of clinical and laboratory features. RESULTS: Survival at 20 months was lower in patients with both pruritus and insomnia. Patients with pruritus alone had a lower Kt/V than those with no complaints or insomnia alone. Those with no complaints had lower C-reactive protein and higher albumin levels than patients with insomnia alone or both conditions. CONCLUSION: Pruritus and insomnia should be actively investigated and correlated with some clinical and laboratory features as they have a significant impact on survival in HD patients.
Subject(s)
Kidney Failure, Chronic/therapy , Pruritus/complications , Renal Dialysis , Sleep Initiation and Maintenance Disorders/complications , Adolescent , Adult , C-Reactive Protein/analysis , Child , Humans , Kaplan-Meier Estimate , Kidney Failure, Chronic/blood , Kidney Failure, Chronic/complications , Longitudinal Studies , Middle Aged , Prospective Studies , Pruritus/blood , Renal Dialysis/adverse effects , Risk Factors , Sleep Initiation and Maintenance Disorders/blood , Young AdultABSTRACT
Yellow laccases lack the typical blue type 1 Cu absorption band around 600 nm; however, multi-copper oxidases with laccase properties have been reported. We provide the first evidence that the yellow laccase isolated from Sclerotinia sclerotiorum is obtained from a blue form by covalent, but nevertheless reversible modification with a phenolic product. After separating the phenolics from the extracellular medium, a typical blue laccase is obtained. With ABTS as model substrate for this blue enzyme, a non-natural purple adduct is formed with a spectrum nearly identical to that of the 1:1 adduct of an ABTS radical and Tyr. This modification significantly increases the stability and substrate affinity of the enzyme, not by acting primarily as bound mediator, but by structural changes that also alters the type 1 Cu site. The HPLC-MS analyses of the ABTS adduct trypsin digests revealed a distinct tyrosine within a unique loop as site involved in the modification of the blue laccase form. Thus, S. sclerotiorum yellow laccase seems to be an intrinsically blue multi-copper oxidase that boosts its activity and stability with a radical-forming aromatic substrate. This particular case could, at least in part, explain the enigma of the yellow laccases.
Subject(s)
Ascomycota/enzymology , Laccase/metabolism , Tyrosine/metabolism , Biocatalysis , Color , Hydrogen-Ion Concentration , Phenols/metabolism , Protein BindingABSTRACT
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.
Subject(s)
Polyphenols , Superoxides , Free Radical Scavengers , Hydroxyl Radical , Phenols , Reactive Oxygen SpeciesABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Hypericum/chemistry , Inflammation/drug therapy , Plant Extracts/administration & dosage , Rutin/administration & dosage , Animals , Anti-Inflammatory Agents/chemistry , Catalase/metabolism , Female , Humans , Inflammation/chemically induced , Inflammation/metabolism , Male , Malondialdehyde/metabolism , Oxidative Stress/drug effects , Plant Extracts/chemistry , Rats , Rats, Wistar , Rutin/analysis , Superoxide Dismutase/metabolism , Turpentine/adverse effectsABSTRACT
Hemoglobin in its ferryl form oxidizes hydrogen sulfide and is transformed to sulfhemoglobin, where the sulfur is inserted covalently at the heme edge. Shown here is evidence that-as previously proposed by others-this process involves oxidation of hydrogen sulfide to a sulfanyl radical detectable by spin-trapping in electron paramagnetic resonance (EPR) spectroscopy. The yields and rates of formation of sulfhemoglobin as well as of the sulfanyl radical are affected by the same factors that affect the reactivity of hemoglobin ferryl, in bovine hemoglobin and in phytoglobins as well. A freely-diffusing sulfanyl radical is thus proposed to be involved in sulfhemoglobin formation. Catalase is shown to accelerate this process due to a previously described hydrogen sulfide oxidase activity, within which EPR evidence for sulfanyl generation is shown here for the first time. The reaction of preformed ferryl with hydrogen sulfide-in absence of hydrogen peroxide-is studied by stopped-flow at several pH values and explained in light of reactivity and redox potential control.
Subject(s)
Heme/metabolism , Hemoglobins/metabolism , Sulfhemoglobin/metabolism , Animals , Catalase/metabolism , Cattle , Electron Spin Resonance Spectroscopy , Free Radicals , Hemoglobins/chemistry , Hydrogen Peroxide/chemistry , Hydrogen Sulfide/chemistry , Hydrogen-Ion Concentration , Oxidation-Reduction , Sulfhemoglobin/chemistry , Sulfhydryl CompoundsABSTRACT
Despite a recent increase in interest towards phytoglobins and their importance in plants, much is still unknown regarding their biochemical/biophysical properties and physiological roles. The present study presents data on three recombinant Arabidopsis phytoglobins in terms of their UV-vis and Raman spectroscopic characteristics, redox state control, redox potentials and autoxidation rates. The latter are strongly influenced by pH for all three hemoglobins - (with a fundamental involvement of the distal histidine), as well as by added anion concentrations - suggesting either a process dominated by nucleophilic displacement of superoxide for AtHb2 or an inhibitory effect for AtHb1 and AtHb3. Reducing agents, such as ascorbate and glutathione, are found to either enhance- (presumably via direct electron transfer or via allosteric regulation) or prevent autoxidation. HbFe3+ reduction was possible in the presence of high (presumably not physiologically relevant) concentrations of NADH, glutathione and ascorbate, with differing behaviors for the three globins. The iron coordination sphere is found to affect the autoxidation, redox state interconversion and redox potentials in these three phytoglobins.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis/physiology , Hemoglobins/metabolism , Ascorbic Acid/metabolism , Glutathione/metabolism , Hydrogen-Ion Concentration , NAD/metabolism , Oxidation-Reduction , Superoxides/metabolismABSTRACT
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.
Subject(s)
Antioxidants/pharmacology , Darkness/adverse effects , Galium/chemistry , Plant Extracts/pharmacology , Reactive Oxygen Species/pharmacology , Restraint, Physical/psychology , Stress, Psychological/metabolism , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Cholesterol/metabolism , Corticosterone/blood , Dose-Response Relationship, Drug , Epinephrine/blood , Female , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Polyphenols/pharmacology , Rats , Rats, Wistar , Stress, Psychological/blood , Stress, Psychological/enzymology , Stress, Psychological/etiologyABSTRACT
Oxidative stress and inflammation are interlinked processes. The aim of the study was to perform a phytochemical analysis and to evaluate the antioxidant and anti-inflammatory activities of ethanolic Mahonia aquifolium flower (MF), green fruit (MGF), and ripe fruit (MRF) extracts. Plant extract chemical composition was evaluated by HLPC. A DPPH test was used for the in vitro antioxidant activity. The in vivo antioxidant effects and the anti-inflammatory potential were tested on a rat turpentine oil-induced inflammation, by measuring serum nitric oxide (NOx) and TNF-alpha, total oxidative status (TOS), total antioxidant reactivity (TAR), oxidative stress index (OSI), 3-nitrothyrosine (3NT), malondialdehyde (MDA), and total thiols (SH). Extracts were administrated orally in three dilutions (100%, 50%, and 25%) for seven days prior to inflammation. The effects were compared to diclofenac. The HPLC polyphenol and alkaloid analysis revealed chlorogenic acid as the most abundant compound. All extracts had a good in vitro antioxidant activity, decreased NOx, TOS, and 3NT, and increased SH. TNF-alpha was reduced, and TAR increased only by MF and MGF. MDA was not influenced. Our findings suggest that M. aquifolium has anti-inflammatory and antioxidant effects that support the use in primary prevention of the inflammatory processes.
Subject(s)
Anti-Inflammatory Agents/chemistry , Antioxidants/chemistry , Flowers/chemistry , Fruit/chemistry , Mahonia/chemistry , Plant Extracts/chemistry , Animals , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Biphenyl Compounds/chemistry , Chromatography, High Pressure Liquid , Male , Malondialdehyde/metabolism , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Picrates/chemistry , Plant Extracts/pharmacology , Rats , Rats, Wistar , Sulfhydryl Compounds/metabolism , Toluene/analogs & derivatives , Toluene/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Hemoglobin has previously been shown to display ascorbate peroxidase and urate peroxidase activity, with measurable Michaelis-Menten parameters that reveal a particularly low Km for ascorbate as well as for urate - lower than the respective in vivo concentrations of these antioxidants in blood. Also, direct detection of a hemoglobin-ascorbate interaction was possible by monitoring the 1H-NMR spectrum of ascorbate in the presence of hemoglobin. The relative difference in structures between ascorbate and urate may raise the question as to exactly what the defining structural features would be, for a substrate that binds to hemoglobin with high affinity. Reported here are Michaelis-Menten parameters for hemoglobin acting as peroxidase against a number of other substrates of varying structures - gallate, caffeate, rutin, 3-hydroxyflavone, 3,6-dihydroxyflavone, quercetin, epicatechin, luteolin - all with high affinities (some higher than those of physiologically-relevant redox partners of Hb - ascorbate and urate). Moreover, this high affinity appears general to animal hemoglobins. 1H-NMR and 13C-NMR spectra reveal a general pattern wherein small hydrophilic antioxidants appear to all have their signals affected, presumably due to binding to hemoglobin. Fluorescence and calorimetry measurements confirm these conclusions. Docking calculations confirm the existence of binding sites on hemoglobin and on myoglobin for ascorbate as well as for other antioxidants. Support is found for involvement of Tyr42 in binding of three out of the four substrates investigated in the case of hemoglobin (including ascorbate and urate, as blood-contained relevant substrates), but also for Tyr145 (with urate and caffeate) and Tyr35 (with gallate).
Subject(s)
Antioxidants/chemistry , Antioxidants/metabolism , Hemoglobins/chemistry , Hemoglobins/metabolism , Animals , Cattle , Molecular Docking Simulation , Oxidation-ReductionABSTRACT
The bioactive compounds and "in vitro" antioxidant activity measured by three antioxidant assays of some traditional and non-traditional cold-pressed edible oils from Macedonia were object of this study. The fatty acid composition showed dominance of monounsaturated oleic acid in "sweet" and "bitter" apricot kernel oils with percentages of 66.7 ± 0.5 and 57.8 ± 0.3%, respectively. The most dominant fatty acid in paprika seed oil was polyunsaturated linoleic acid with abundance of 69.6 ± 2.3%. The most abundant tocopherol was γ-tocopherol with the highest quantity in sesame seed oil (57.6 ± 0.1 mg/100 g oil). Paprika seed oil, sesame seed oil and sweet apricot oil were the richest source of phytosterols. DPPH assay was the most appropriate for the determination of the antioxidant activity of cold-pressed sunflower oil due to high abundance of α-tocopherol with a level of 22.8 ± 1.1 mg/100 g of oil. TEAC assay is the best for the determination of the antioxidant activity of sesame seed oil and paprika seed oils as the richest sources of phenolic compounds. ß-carotene assay was the most suitable assay for oils obtained from high pigmented plant material. Triacylglycerols and phytosterol profiles can be used as useful markers for the origin, variety and purity of the oils.