Recovery of Biologically Active Compounds from Stinging Nettle Leaves Part II: Processing of Exhausted Plant Material after Supercritical Fluid Extraction.

Stinging nettle (Urtica dioica L.) is one fantastic plant widely used in folk medicine, pharmacy, cosmetics, and food. This plant's popularity may be explained by its chemical composition, containing a wide range of compounds significant for human health and diet. This study aimed to investigate extracts of exhausted stinging nettle leaves after supercritical fluid extraction obtained using ultrasound and microwave techniques. Extracts were analyzed to obtain insight into the chemical composition and biological activity. These extracts were shown to be more potent than those of previously untreated leaves. The principal component analysis was applied as a pattern recognition tool to visualize the antioxidant capacity and cytotoxic activity of extract obtained from exhausted stinging nettle leaves. An artificial neural network model is presented for the prediction of the antioxidant activity of samples according to polyphenolic profile data, showing a suitable anticipation property (the r2 value during the training cycle for output variables was 0.999).

Electrochemical Determination of Morphine in Urine Samples by Tailoring FeWO4/CPE Sensor.

Morphine (MORPH) is natural alkaloid and mainly used as a pain reliever. Its monitoring in human body fluids is crucial for modern medicine. In this paper, we have developed an electrochemical sensor for submicromolar detection of MORPH. The sensor is based on modified carbon paste electrode (CPE) by investigating the FexW1-xO4 ratio in iron tungstate (FeWO4), as well as the ratio of this material in CPE. For the first time, the effect of the iron-tungsten ratio in terms of achieving the best possible electrochemical characteristics for the detection of an important molecule for humans was examined. Morphological and electrochemical characteristics of materials were studied. The best results were obtained using Fe1W3 and 7.5% of modifier in CPE. For MORPH detection, square wave voltammetry (SWV) was optimized. Under the optimized conditions, Fe1W3@CPE resulted in limit of detection (LOD) of the method of 0.58 µM and limit of quantification (LOQ) of 1.94 µM. The linear operating range between 5 and 85 µM of MORPH in the Britton-Robinson buffer solution (BRBS) at pH 8 as supporting electrolyte was obtained. The Fe1W3@CPE sensor resulted in good selectivity and excellent repeatability with relative standard deviation (RSD) and was applied in real-world samples of human urine. Application for direct MORPH detection, without tedious sample pretreatment procedures, suggests that developed electrochemical sensor has appeared to be a suitable competitor for efficient, precise, and accurate monitoring of the MORPH in biological fluids.

Evaluation of novel green walnut liqueur as a source of antioxidants: Multi-method approach.

In this study, a novel green walnut liqueur (GWL) based on green walnuts, as the main ingredient, with the addition of fruits, aromatic plants, chocolate and honey, was produced at the pilot-scale. Antioxidant activity (AO) of the obtained GWL was determined using multiple AO assays in parallel, standard spectrophotometric (FC, DPPH and FRAP) and recently developed electrochemical ones, HydroxoPerhydroxoMercury(II) Complex and Mercury Reduction Antioxidant Power, and compared to similar commercial alcoholic beverages. Characterization of the GWL in terms of volatile and polyphenolic components was performed using SPE-GC-MS and HPLC-DAD-MS/MS, respectively. Sensory quality assessment was performed by experts in the field of sensory analysis of alcoholic beverages, using a scoring method. According to all AO assays applied, AO activity and total phenolics content of GWL were superior in comparison to commercial spirits considered. The volatile fraction of GWL was mainly composed of eugenol, α-terpineol and vanillin, while the most prevalent phenolic compounds were gallic (5.054 mg/L) and chlorogenic acid (1.307 mg/L) and flavonoids such as catechine (0.882 mg/L), quercetin (0.499 mg/L) and its sugar-conjugated derivatives, quercetin 3-O-glucoside (0.774 mg/L) and quercetin-3-O-rhamnoside (0.614 mg/L). Gallic acid is the major contributor to total AO activity, especially determined by DPPH and FRAP, followed by catechine, quercetin and chlorogenic acid. Among the terpenoids, contribution of eugenol to total AO activity is estimated as the highest. Excellent sensory quality (18.52 of maximum 20 scores) was ascribed to GWL. Consequently, the presence of bioactive compounds and high AO activity of GWL, in addition to high sensory quality score, indicates a high market potential for this high-value product.

Polarography as a technique of choice for the evaluation of total antioxidant activity: The case study of selected Coprinus Comatus extracts and quinic acid, their antidiabetic ingredient.

This study was focused on in vitro screening of the total antioxidant activity of the selected extracts of the mushroom Coprinus comatus and quinic acid, one of their antidiabetic ingredients, by an uncommon electrochemical assay. Indeed, direct current (DC) polarographic HydroxoPerhydroxo Mercury(II) Complex (HPMC) assay based on decrease of anodic limiting current originating from HPMC formation in alkaline solutions of hydrogen peroxide at potential of mercury dissolution, observed upon gradual addition of antioxidants, was applied herein for the estimation of the natural products' antioxidativity. Quinic acid was found to exhibit most promising antioxidant potential (4.0 ± 0.2%µL-1) being ≈ 2-fold more active than the screened C. comatus extract samples. Actually, such a finding puts some light on the antioxidativity of cyclic polyols, well understimated class of organic compounds, compared to aromatic (poly)phenolics. As a low cost, easy-to handle and accurate this polarographic assay may be thoroughly recommended for much broader use. [Formula: see text].

Evaluation of Apple Pomace Flour Obtained Industrially by Dehydration as a Source of Biomolecules with Antioxidant, Antidiabetic and Antiobesity Effects.

Apple pomace flour (APF) obtained at industrial scale level by the application of innovative technological process (dehydration (5 h, T ≤ 55 °C), grinding (300 µm)) was evaluated as a source of bioactive compounds with antioxidative, antiobesity and antidiabetic effects. Proximate composition, individual (HPLC-DAD-MS/MS) and total phenols (TPC) as well as flavonoids content (TFC), antioxidant (AO) activity (DPPH, ABTS, HPMC), water and oil holding capacity (WHC and OHC) of APFs obtained from apple pomace from mixed and individual apple cultivars grown conventionally and organically were compared. The effect of APF supplementation on the glycaemic status and glucose tolerance (oral glucose tolerance test (OGTT)) of C57BL/6J mice exposed to high-fat and sucrose diet was examined. High K content (4.2-6.4 g/kg), dietary fibres (35-45 g/100 g), TPC (4.6-8.1 mg GAE/g), TFC (18.6-34.6 mg QE/g), high water and oil holding capacity (4.7-6.4 and 1.3-1.6 g/g) were observed in the APFs. Content of major phenols (phlorizin, chlorogenic acid, quercetin), TPC and TFC correlated highly with prominent AO activity. APF supplementation lowered the increase of body weight gain and blood glucose, and improved glucose tolerance significantly. Health-promoting biomolecules, AO activity, functional properties and prevention of diet-driven glucose metabolism disorders pave the way to APF exploitation in human nutrition.

Trametes versicolor ethanol extract, a promising candidate for health-promoting food supplement.

This study aimed to estimate antiradical, antioxidant (AO) and cytotoxic activities of the fungus Trametes versicolor ethanol fruiting body extract. The extract was found to effectively scavenge both O2•- and NO• (29.62 and 52.48 µg/mL, respectively). It also showed a good AO activity in the polarographic HPMC assay (950%/mL). p-Hydroxybenzoic acid may be one of the responsible compounds for the afore-mentioned activities. The same extract also exhibited a concentration-dependent cytotoxicity against MCF-7 and HepG2 tumour cell lines reaching IC50 values of 123.51 and 134.29 µg/mL, respectively with no cytotoxic activity against normal MRC-5 cells. Gentisic, syringic and protocatechuic acids may be among the bioactive principles for the observed cytotoxicity. Taken all together, T. versicolor ethanol extract can be considered as a promising candidate for development of health promoting food supplement.

Antioxidant efficiency of polyphenols from coffee and coffee substitutes-electrochemical versus spectrophotometric approach.

Antioxidant (AO) capacity of instant, espresso, filter and Turkish/Greek coffee brews, coffee substitutes (roasted chicory root, barley, pea, chickpea, carob and dried fig) and individual compounds (phenolic acids, flavonoids, methylxanthines, N-methyl pyridinium and HMW melanoidins) was assessed using DC polarographic assay based on decrease of anodic current originating from hydroxo-perhydroxo mercury complex formed in alkaline solutions of H2O2 at potential of mercury dissolution, as well as three spectrophotometric assays (DPPH, ABTS and FRAP). A large difference between applied assays ability to recognize various types of individual AOs was noticed. Only according to DC polarographic assay significant AO activity was ascribed to methylxanthines and N-methyl pyridinum. The total content of phenolics (TPC) present in complex samples was determined by FC assay. The highest TPC was ascribed to instant coffees and coffee substitutes while the lowest to decaffeinated filter coffee. Complex samples were grouped based on principal components analysis, phenolics AO coefficient, calculated as the ratio between AO capacity and TPC, and relative AO capacity index (RACI), calculated by assigning equal weight to all applied assays including FC. The highest values of RACI were ascribed to instant coffee brews, followed by substitutes while the lowest to the decaffeinated espresso coffee.

The interactions of vanadate monomer with the mycelium of fungus Phycomyces blakesleeanus: reduction or uptake?

The possibility of reduction of vanadate monomer in the mycelium of fungus Phycomyces blakesleeanus was investigated in this study by means of polarography. Control experiments were performed with vanadyl [V(IV)] and vanadate [V(V)] in 10 mM Hepes, pH 7.2. Addition of P. blakesleeanus mycelium resulted in disappearance of all V(IV) polarographic waves recorded in the control. This points to the uptake of all available V(IV) by the mycelium, up to 185 µmol/gFW, and suggests P. blakesleeanus as a potential agent in V(IV) bioremediation. Polarographic measurements of mycelium with low concentrations (0.1-1 mM) of V(V), that only allows the presence of monomer, showed that fungal mycelia removes around 27% of V(V) from the extracellular solution. Uptake was saturated at 104 ± 2 µmol/gFW which indicates excellent bioaccumulation capability of P. blakesleeanus. EPR, 51V NMR and polarographic experiments showed no indications of any measurable extracellular complexation of V(V) monomer with fungal exudates, reduction by the mycelium or adsorption to the cell wall. Therefore, in contrast to vanadium oligomers, vanadate monomer interactions with the mycelium are restricted to its transport into the fungal cell, probably by a phosphate transporter.

Content of Total Phenolics, Flavan-3-Ols and Proanthocyanidins, Oxidative Stability and Antioxidant Capacity of Chocolate During Storage.

Antioxidant (AO) capacity of chocolates with 27, 44 and 75% cocoa was assessed after production and during twelve months of storage by direct current (DC) polarographic assay, based on the decrease of anodic current caused by the formation of hydroxo-perhydroxyl mercury(II) complex (HPMC) in alkaline solutions of hydrogen peroxide at potentials of mercury oxidation, and two spectrophotometric assays. Relative antioxidant capacity index (RACI) was calculated by taking the average value of the AO assay (the sample mass in all assays was identical). Oxidative stability of chocolate fat was determined by differential scanning calorimetry. Measured parameters and RACI were correlated mutually and with the content of total phenols (Folin-Ciocalteu assay), flavan-3-ols (vanillin and p-dimethylaminocinnamaldehyde assay) and proanthocyanidins (modified Bate-Smith assay). During storage, the studied functional and health-related characteristics remained unchanged. Amongst applied AO assays, the DC polarographic one, whose validity was confirmed by two-way ANOVA and F-test, correlated most significantly with oxidative stability (oxidation onset temperature and induction time). In addition, principal component analysis was applied to characterise chocolate types.

Antioxidant Capacity Determination of Complex Samples and Individual Phenolics - Multilateral Approach.

Antioxidant (AO) capacity of various medicinal plants extracts and phenolic compounds was assessed by the most widely used spectrophotometric assays such as ferric reducing antioxidant power (FRAP) and scavenging of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). In addition, two direct current (DC) polarographic assays, one based on a decrease of anodic current of [Hg(O2H)(OH)] - HydroxoPerhydroxoMercury(II) Complex (HPMC) formation in alkaline solution of H2O2, at the potential of mercury dissolution and another recently developed Mercury Reduction Antioxidant Power (MRAP), based on a a decrease of cathodic current of Hg(II) reduction were employed. Percentage of both currents decrease was plotted versus the volume of gradually added complex samples or the amount of individual ones and the slopes of these plots were used to express AO capacity. Total phenolic content (TPC) of extracts was determined by Folin- Ciocalteu (FC) assay. Correlations between applied assays were calculated by regression analysis. Relative Antioxidant Capacity Index (RACI), calculated by assigning equal weight to all applied assays and Phenolic Antioxidant Coefficients (PAC), calculated as a ratio between particular AO capacity and TPC, were used to achieve more comprehensive comparison between analyzed samples, as well as applied assays.

Electrochemical versus spectrophotometric assessment of antioxidant activity of hop (Humulus lupulus L.) products and individual compounds.

Antioxidant (AO) activity of extracts of hop cones (Serbian domestic varieties) and commercial hop products (Saaz, Spalter, Spalter select, and Magnum pellets) was determined by parallel application of recently developed direct current (DC) polarographic and widely used DPPH assay. Correlations between 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging and total phenolics (TPC) determined by the Folin-Ciocalteu assay (FC) (0.99), and between H2O2 scavenging (HPS) and humulone content (H) determined by conductometric method (0.94), total resins (TR) (0.85), and hop storage index (HIS) (-0.90), were found statistically significant at p < 0.05 level while complete lack of HPS correlation with TPC and DPPH was observed. To obtain an insight into differences between results of AO assays applied, activity of individual compounds, prevalent hop phenolics, and bitter acids was determined. By far superior HPS activity of humulone was followed by catechin, quercetin, xanthohumol, lupulone, and rutin. In contrast, DPPH scavenging activity of phenolics (quercetin > catechin > rutin > xantohumol) was found substantially higher than activity of bitter acids. According to ferric reducing antioxidant power (FRAP) and scavenging of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), higher AO activity was ascribed to phenolics, while almost neglecting humulone. Besides reliability, low cost, and an easy-to-handle procedure, an ability to recognize humulone as the major contributor of hop AO activity could allow DC polarographic assay to be applied in analysis of various hop-derived products.

Antioxidant capacity of teas and herbal infusions: polarographic assessment.

Hydrogen peroxide scavenging (HPS) activity of unfermented (green, yellow, and white), partially fermented (oolong), and completely fermented (black) tea ( Camellia sinensis ), maté ( Ilex paraguariensis ), and various herbal infusions, as well as individual compounds (flavan-3-ols, flavonols, cinnamic and benzoic acids, and methylxanthines), was assessed by recently developed direct current (DC) polarographic assay. Correlations of tea and herbal infusion HPS activity with total phenolic content determined using the Folin-Ciocalteu assay (FC-GAE) (0.81 and 0.93), ferric reducing/antioxidant power (FRAP) (0.97 and 0.92), 1,1-diphenyl-2-picrylhydrazyl (DPPH) (0.77 and 0.80), and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging (0.86 and 0.86) were statistically significant. Correlations between relative antioxidant capacity index (RACI), calculated by assigning all applied assays equal weight, and HPS (0.98), FRAP (0.97), ABTS (0.89), and DPPH (0.89) confirmed DC polarographic assay reliability when applied individually. Correlation analysis, ANOVA, and Levene and Tukey's HSD tests unequivocally confirmed this reliable, rapid, and low-cost assay validity, clearly demonstrating its advantages over spectrophotometric assays applied.

Polarographic study of hydrogen peroxide anodic current and its application to antioxidant activity determination.

Behavior of hydrogen peroxide in alkaline medium has been studied by direct current (DC) polarography with dropping mercury electrode (DME) aiming to apply it in antioxidant (AO) activity determination. Development of a peroxide anodic current having form of a peak, instead of common polarographic wave, has been investigated. As a base for this investigation the interaction of H(2)O(2) with anodically dissolved mercury was followed. Formation of mercury complex [Hg(O(2)H)(OH)] has been confirmed. The relevant experimental conditions, such as temperature, concentration and pH dependence, as well as time stability of hydrogen peroxide anodic current, have been assessed. Development of an AO assay based on decrease of anodic current of hydrogen peroxide in the presence of antioxidants (AOs) has been described. Under optimized working conditions, a series of benzoic acids along with corresponding cinnamate analogues have been tested for hydrogen peroxide scavenging activity. In addition, the assay versatility has been confirmed on various complex samples.

Polarographic assay based on hydrogen peroxide scavenging in determination of antioxidant activity of strong alcohol beverages.

Total antioxidant (AO) activity of strong alcohol beverages such as wine and plum brandies, whiskeys, herbal and sweet fruit liqueurs have been assessed using a polarographic assay based on hydrogen peroxide scavenging (HPS). Rank of order of total AO activity, expressed as percentage of decrease of anodic oxidation current of hydrogen peroxide, was found analogous with total phenolic content estimated by Folin-Ciocalteau (FC) assay and radical scavenging capacity against the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). Application of the assay for surveying of a quarter century long maturation of plum brandy in oak barrel was demonstrated. In addition, influence of different storage conditions on preservation of AO activity of some herbal liqueurs was surveyed. Wide area of application of this simple, fast, low cost and reliable assay in analysis and quality monitoring of various strong alcohol beverages was confirmed.

Kinetic method for the determination of traces of thyroxine by its catalytic effect on the Mn(III) metaphosphate-As(III) reaction.

A new, highly sensitive and simple kinetic method for the determination of thyroxine was proposed. The method was based on the catalytic effect of thyroxine on the oxidation of As(III) by Mn(III) metaphosphate. The kinetics of the reaction was studied in the presence of orthophosphoric acid. The reaction rate was followed spectrophotometrically at 516 nm. It was established that orthophosphoric acid increased the reaction rate and that the extent of the non-catalytic reaction was extremely small. A kinetic equation was postulated and the apparent rate constant was calculated. The dependence of the reaction rate on temperature was investigated and the energy of activation and other kinetic parameters were determined. Thyroxine was determined under the optimal experimental conditions in the range 7.0 x 10(-9) to 3.0 x 10(-8) mol L(-1) with a relative standard deviation up to 6.7% and a detection limit of 2.7 x 10(-9) molL(-1). In the presence of 0.08 mol L(-1) chloride, the detection limit decreased to 6.6 x 10(-10) mol L(-1). The proposed method was applied for the determination of thyroxine in tablets. The accuracy of the method was evaluated by comparison with the HPLC method.