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Authors: J. Pokorná, P. R. Venskutonis, V. Kraujalyte, P. Kraujalis, P. Dvořák, B. Tremlová, V. Kopřiva and M. Ošťádalová

. , B RAND -W ILLIAMS , W. & B ERSET , C. ( 1997 ): Kinetics and mechanisms of antioxidant activity using the DPPH˙ free radical method . Lebensm. Wiss. Technol ., 30 , 609 – 615

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formic acid (>98%, MS-grade) for liquid chromatography (LC)–MS were purchased from J.T. Baker (Deventher, Netherlands). Ultra-pure water was prepared in-house with a Milli-Q water purification system (Millipore Co.). DPPH · was obtained from Sigma

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compounds with a wide range of polarities from the column [ 24 , 25 ]. The present paper describes an efficient method by coupling of DPPH radical scavenging activity and dual-mode HSCCC experiments to screen and purify antioxidant from E. densa

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DPPH scavenging assay is the widely used method for evaluation of in vitro anti-oxidant activity. Anovel, rapid, and precise online highperformance thin-layer chromatographic (HPTLC) method has been successfully developed for evaluation of DPPH reduction. In this paper, chemistry-based assays, like reduction or scavenging of DPPH with ascorbic acid, are performed and discussed. In its radical form, DPPH has strong absorption in the regions around 330 nm and 520 nm, but if it is reduced, there is no change in the first peak, but the intensity of the second peak (around 520 nm) decreases steadily with the increase in ascorbic acid concentration. The method has been combined with online planar chromatography (HPTLC) technique for rapid observation and quantitative estimation of reduction of DPPH by ascorbic acid. For scavenging of 660 ng to 1320 ng of DPPH, around 120 ng to 160 ng of ascorbic acid or its equivalent is the suitable concentration. The limit of detection (LOD) and limit of quantification (LOQ) of DPPH are 3.9 ng and 13 ng, respectively, while that of ascorbic acid are 4.5 ng and 15 ng, respectively. The focus is on the mechanisms involved rather than on the matrix. It can be used for rapid screening and evaluation of potential natural products for their anti-oxidant activity and will have wider applications.

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Authors: J. Piljac-žegarac, S. Martinez, L. Valek, T. Stipčević and K. Kovačević-Ganić

The Folin-Ciocalteu (FC) test and cyclic voltammetry (CV) at a glassy carbon electrode were used to quantify phenolic antioxidants in a set of 17 Croatian wines and express them in gallic acid (GAE) and catechin equivalents (CE). The total phenolic index (TPI) values for red wines expressed in GAE ranged from 18.851 to 26.905 mM, while TPI for white wines ranged from 1.722 to 2.869 mM. The levels of phenolics derived from CV measurements were markedly lower than those of TPI, since these values include only those phenolic compounds that get oxidised up to 500 mV and contain ortho -diphenol and triphenol groups.The free radical scavenging ability of the same set of wines was evaluated according to the Brand-Williams assay and expressed in equivalents of catechin, gallic acid, vitamin C and Trolox. Ivan Dolac barrique 2002 exhibited the highest antioxidant activity. The DPPH radical scavenging ability of the wines was also evaluated and correlated to the TPI values. Better correlation was observed between the TPI and the antioxidant activity for red wines (r 2 =0.826) as opposed to white wines (r 2 =0.686). The highest correlation (r 2 =0.970) was found between the TPI and the antioxidant activity measured when the whole set of samples was considered.

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Many phytochemical investigations have been focused recently on the antioxidant activity of herbal extracts which can be used in phytotherapy. The previous study revealed antioxidative properties of Mutellina purpurea extract, but the constituents responsible for this action were not described yet. The aim of this study was activityguided separation and identification of antioxidant compounds from M. purpurea herb. Thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl (TLC-DPPH) assay was used to detect compounds of interest; liquid chromatography-diode array detection-mass spectrometry (LC-DAD-MS) analysis allowed to identify antioxidants. The active fractions analyzed with LC-DAD-MS contained as a main compound chlorogenic acid accompanied with p-coumaric acid, ferulic acid, three dicaffeoylquinic acids, and caffeoylferuloylquinic acid. The fast TLC-DPPH assay with LC-DAD-MS identification enabled the accurate identification of antioxidants in M. purpurea herb, which was done for the first time.

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Abstract  

The present paper reports the regioselective [15NO2]-labeling of N-methoxy-2,4,6-trinitroaniline and 2,2-diphenyl-1-picrylhydrazine (reduced DPPH). Starting from N-methoxy-2,6-dinitroaniline, or N-methoxy-2,4-dinitroaniline, nitration in methylene chloride with solid sodium [15N]nitrite and 15-crown-5-ether afforded N-methoxy-2,6-dinitro-4-[15N]nitroaniline and N-methoxy-2,4-dinitro-6[15N]nitroaniline, respectively. The same compounds could be prepared in higher purity by nitrodecarboxylation (ipso-substitution) under the same conditions starting from N-methoxy-4-carboxy-2,6-dinitroaniline (4-methoxyamino-3,5-dinitrobenzoic acid) and N-methoxy-2-carboxy-4,6-dinitroaniline (2-methoxyamino-3,5-dinitrobenzoic acid). Similarly,ipso-substitution of 2,2-diphenyl-1-(4-carboxy-2,6-dinitrophenyl)-hydrazine afforded, under the same reaction conditions, 2,2-diphenyl-1-(2,6-dinitro-4-[15N]nitrophenyl)-hydrazine. By1H-NMR and13C-NMR it was also observed that under these reaction conditions a14NO2 group can be replaced by a15NO2 group.

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Natural compounds possess multiple biological properties, among others antioxidant activity. Phenolic compounds which exhibit excellent antiradical activity may act as scavengers of free radicals or prevent their formation. The main objective of the present research was the examination of the free radical scavenging activity of instant corn gruels with 0%, 1%, 3%, 5%, and 10% addition of dried cranberry fruits. The extrusion of gruels was carried out at varying rotational speeds of the plasticising system (80, 100, and 120 rpm). The study was developed using two methods: thin-layer chromatography–2,2-diphenyl-1-picrylhydrazyl (DPPH) test and spectrophotometric analysis. The findings confirm that the use of higher screw rotations during the extrusion entailed a higher antioxidant activity of the obtained extrudates. Also, a high activity was reported for the extruded corn gruels without additives. This, in turn, demonstrates an insignificant influence of the extrusion process on the biological value of the obtained products.

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The aim of this study was to identify and quantitatively determine kaempferol, quercetin, and gallic acid in the methanol–water extract of the herb Euphorbia humifusa Willd. using high-performance thin-layer chromatography (HPTLC). The antioxidant compounds in E. humifusa Willd. were screened by thin-layer chromatography—1,1-diphenyl-2-trinitrobenzene hydrazine (TLC—DPPH). Kaempferol, quercetin, and gallic acid were used as the reference substances to optimize a highly suitable deployment system. Analysis was performed on silica gel G F254 plates with chloroform—ethyl acetate—formic acid with 5.2:4.4:1 proportion as the mobile phase. Antioxidant activity was screened preliminarily by spraying 10% DPPH ethanol solution on the developed silica gel plate. The quantification of E. humifusa Willd. was done at 300 nm for gallic acid and at 400 nm for quercetin and kaempferol. The linearity ranges of gallic acid, quercetin, and kaempferol were 0.168–0.448, 0.179–0.47, and 0.183–0.488 μg spot−1, respectively, with correlation coefficients of 0.9997, 0.9995, and 0.9995, respectively. Intra-day and inter-day precision studies showed a relative standard deviation (RSD) of <3.5%. Stability studies showed that gallic acid, quercetin, and kaempferol can be stable for at least 24 h at room temperature. The limit of detection (LOD) and limit of quantification (LOQ) were within the nanogram range for all three compounds. Average recovery was >96%. The developed method is simple and robust, with good stability and reproducibility. This method satisfies the requirements for quantitative determination and is effective in the quality control and evaluation of E. humifusa Willd.

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Bandonienė, D., Murkovic, M., Pfannhauser, W., Venskutonis, P.R. & Gruzdienė, D. (2002): Detection and activity evaluation of radical scavenging compounds by using DPPH free radical and on-line HPLC-DPPH methods. Eur. Fd Res. Technol. , 214, 143

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