Comparative evaluation of three methods based on high-performance liquid chromatography analysis combined with a 2,2'-diphenyl-1-picrylhydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers

Traditional activity-guided fractionation of natural products is a time-consuming, labor intensive, and expensive strategy, which cannot compete with high-throughput and rapid screening of natural products. Therefore, more efficient approaches are necessary for searching active compounds from natural products. Three main methods based on high-performance liquid chromatography (HPLC) analysis combined with 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, DPPH spiking HPLC analysis, on-line post-column HPLC-DPPH analysis, and HPLC-based DPPH activity profiling, were then developed for the rapid screening of antioxidants from complex mixtures. In the present study, a comparative study of these three methods has been conducted to identify antioxidants from an ethyl acetate fraction of Pueraria lobata flowers. The parameters in HPLC analysis and DPPH assay were optimized. The results indicated that all three methods could achieve similar information with regard to antioxidants, without the need for preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity, and efficiency. DPPH spiking HPLC analysis seemed to be more sensitive and effective with simpler instrumental set-up and easier operation, which could also detect the total antioxidant capacity of color complexes. Eighteen antioxidants were tentatively screened and identified from P. lobata flowers by DPPH spiking HPLC-MS/MS. Among them, ten compounds including one new compound were first isolated from P. lobata flowers, and the DPPH radical scavenging activity of the new compound was reported for the first time.     

The Interaction of Maleic Anhydride,N -Vinylpyrrolidone,and Their Mixture with 2,2-Diphenyl-1-picrylhydrazyl

Spectrophotometry studies have shown that N-vinylpyrrolidone and/or maleic anhydride interact with 2,2-diphenyl-1-picrylhydrazyl radical in a solution. Thermochemical parameters of the most probable reactions involving these compounds have been estimated by means of the PM6 and B3LYP/6-31+G(d) (gas phase) density functional theory methods. It has been suggested that the stable radical is attached to the monomer molecule, the resulting radical being involved in the chain growth reactions. Termination via the oligomeric radicals recombination is the most probable.     

Reactions of unsubstituted hydroxybenzenes with 2,2-diphenyl-1-picrylhydrazyl in the system water-aprotic solvent

The reactivity of natural unsubstituted di- and trihydroxybenzenes in the reaction with a stable free radical 2,2-diphenyl-1-picrylhydrazyl in a mixed solvent water-dimethyl sulfoxide (2:1) was investigated. Kinetic and stoichiometric parameters of the studied reaction were estimated. In the water environment the studied hydroxybenzenes were found to dissociate to form ions, which reacted rapidly with the radical by the mechanism of single electron transfer with subsequent adding a proton. The corresponding rate constants were calculated taking into account the concentration of the ionized form of phenol.     

Potent 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity of novel antioxidants, double-stranded tyrosine residues conjugating pyrocatechol

New potent antioxidants conjugating the catechol (=pyrocatechol; pyrCat) group to two N-termini of modified double-stranded tyrosine residues were synthesized and showed radical scavenging activity with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH˙) as a free radical model, second-order rate constants for the DPPH˙ scavenging reaction, and the results from electron spin resonance (ESR) studies. It was found that the tyrosine (Tyr) residue and pyrCat containing new antioxidants developed in the study have about 3-20 times more potent antioxidative activity than Trolox, pyrCat, and L-ascorbic acid (VC). In order to elucidate the relationship between antioxidant activity and the molecular orbital states, and to design potent antioxidants we present an interesting approach using an absolute hardness (η)-absolute electronegativity (χ) diagram based on chemical hardness. It was shown that quantum chemicals were required to develop potent antioxidants.     

Study of reaction of gossypol and its imino derivatives with 2,2-diphenyl-1-picrylhydrazyl

Structures and state in solutions of natural polyphenol gossypol and four its imino derivatives, three of which were synthesized for the first time, were studied by IR and NMR spectroscopy, and by quantum chemistry. The reaction of these compounds with 2,2-diphenyl-1-picrylhydrazyl (DPPH) in ethanol was examined. The antioxidant activity of the studied compounds in the reaction with DPPH was evaluated using the values of the stoichiometric coefficients of reaction, EC₅₀, T ₁^2/DPPH and AE parameters. Gossypol hydrazones were shown to be 5–10 times more efficient, while Schiff base to be less efficient as antioxidants in comparison with gossypol itself. The influence of metal cations on the antioxidant activity of gossypol derivatives was studied.     

Spin Trapping of the Phosphorus-centered Radicals Generated from Hydrogen Abstraction Reaction by 2,2-Diphenyl-1-picrylhydrazyl

IntroductionSPin trapping technique has been widely used for the detection and identification of unstable radicals. As traps, nitrones and nitroso compounds are most widely usedll--4]. However,the identification of spin adducts by EPR spectroscopy is rather difficult because the variationof the hyperfine coupling constants(hfccs) of spin adducts caused by the structural changes oftrapped radicals is not very large. Recently, the ph osp horns- con t al m ng m t rox id e h a s att ra ctedmuch …     

Synthesis and properties of novel bis(triarylamines) based on a 3,3'-diphenyl-2,2'-bithiophene core

An efficient synthesis of 3,3'-diphenyl-2,2'-bithiophene based bis(triarylamines) and their physical properties are reported.     

Unexpected stereodynamic consequences of the restricted rotations in ortho-acyl- and ortho-vinyl biphenyls

Surprisingly, the aryl-aryl rotation barriers of biphenyl derivatives ortho-substituted by the "small" HC=O and HC=CH2 groups (10.0 and 8.4 kcal mol-1, respectively) were found greater than those observed in biphenyls ortho-substituted by the "large" t-BuC=O and t-BuC=CH2 groups (6.7 and 6.9 kcal mol-1, respectively).     

Selective and low potential electrocatalytic oxidation of NADH using a 2,2-diphenyl-1-picrylhydrazyl immobilized graphene oxide-modified glassy carbon electrode

DPPH (2,2-diphenyl-1-picrylhydrazil), a free radical-containing organic compound, is used widely to evaluate the antioxidant properties of plant constituents. Here, we report an efficient electroactive DPPH molecular system with excellent electrocatalytic sensor properties, which is clearly distinct from the traditional free radical-based quenching mechanism. This unusual molecular status was achieved by the electrochemical immobilization of graphene oxide (GO)-stabilized DPPH on a glassy carbon electrode (GCE). Potential cycling of the DPPH adsorbed-GCE/GO between ??1 and 1 V (Ag/AgCl) in a pH 7 solution revealed a stable and well-defined pair of redox peaks with a standard electrode potential, E⁰′?=?0?±?0.01 V (Ag/AgCl). Several electrochemical characterization studies as well as surface analysis of the GCE/GO@DPPH-modified electrode by transmission electron microscopy, Raman, and infrared spectroscopy collectively identified the imine/amine groups as the redox centers of the electroactive DPPH on GO. The use of different carbon-supports showed that only oxygen-functionalized GO and MWCNTs could provide major electroactivity for DPPH. This highlights the importance of a strong hydrogen-bonded network structure assisted by the concomitant π-π interactions between the organic moiety and oxygen function groups of carbon for the high electroactivity and stability of the GCE/GO@DPPH-NH/NH₂-modified electrode. The developed electrode exhibited remarkable performance towards the electrocatalytic oxidation of NADH at 0 V (Ag/AgCl). The amperometric i-t sensing of NADH showed high sensitivity (488 nA μM^?1 cm^?2) and an extended linear range (50 to 450 μM) with complete freedom from several common biochemical/chemical interferents, such as ascorbic acid, hydrazine, glucose, cysteine, citric acid, nitrate, and uric acid.     

Abnormal solvent effects on hydrogen atom abstractions. 1. The reactions of phenols with 2,2-diphenyl-1-picrylhydrazyl (dpph*) in alcohols

Rate constants, k(ArOH/dpph*)(S), for hydrogen atom abstraction from 13 hindered and nonhindered phenols by the diphenylpicrylhydrazyl radical, dpph*, have been determined in n-heptane and a number of alcoholic and nonalcoholic, hydrogen-bond accepting solvents. Abnormally enhanced k(ArOH/dpph*)(S) values of have been observed in alcohols. It is proposed that this is due to partial ionization of the phenols and a very fast electron transfer from phenoxide anion to dpph*. The popular assessment of the antioxidant activities of phenols with dpph* in alcohol solvents will generally lead to an overestimation of their activities.     

Synthesis,resolution, and applications of 3-amino-2,2-dimethyl-1,3-diphenylpropan-1-ol,a conformationally restricted 1,3-aminoalcohol

Efficient synthetic routes to both syn and anti diastereomers of a conformationally restricted 1,3-aminoalcohol were devised. Resolution of the aminoalcohols was accomplished through diastereomeric salt with R-(?)-O-acetyl mandelic acid. These aminoalcohols were examined as ligands for two standard reactions, namely, enantioselective addition of Et₂Zn to aldehydes and reduction of prochiral ketones with BH₃.     

Key role of chemical hardness to compare 2,2-diphenyl-1-picrylhydrazyl radical scavenging power of flavone and flavonol O-glycoside and C-glycoside derivatives

The antioxidant activities of flavonoids and their glycosides were measured with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH(·)) scavenging method. The results show that free hydroxyl flavonoids are not necessarily more active than O-glycoside. Quercetin and kaempferol showed higher activity than apigenin. The C- and O-glycosides of flavonoids generally showed higher radical scavenging activity than aglycones; however, kaempferol C3-O-glycoside (astragalin) showed higher activity than kaempferol. In the radical scavenging activity of flavonoids, it was expected that OH substitutions at C3 and C5 and catechol substitution at C2 of B ring and intramolecular hydrogen bonding between OH at C5 and ketone at C3 would increase the activity; however, the reasons have yet to be clarified. We here show that the radical scavenging activities of flavonoids are controlled by their absolute hardness (η) and absolute electronegativity (χ) as a electronic state. Kaempferol and quercetin provide high radical scavenging activity since (i) OH substitutions at C3 and C5 strikingly decrease η of flavones, (ii) OH substitutions at C3 and C7 decrease χ and η of flavones, and (iii) phenol or o-catechol substitution at C2 of B ring decrease χ of flavones. The coordinate r(χ,?η) as the electron state must be small to increase the radical scavenging activity of flavonoids. The results show that chemically soft kaempferol and quercetin have higher DPPH radical scavenging activity than chemically hard genistein and daidzein.     

Effects of solvents on the reaction of 2,2-dimethylpropanesulfinyl chloride with activated zinc

Although 2,2-dimethylpropanesulfinyl chloride reacts with zinc in C₆H₆ or CCl₄ to give the corresponding thiosulfonate as major product, the reaction in C₂D₁₀O, C₂H₁₀O, and CD₃CN affords the corresponding thiosulfinate as the major product.     

Synthesis, Crystal Structure and Theoretical Calculation of 1,1',5,5'-Tetramethyl-2,2'-diphenyl-4,4'-[i-phenylene-bis(methylidynenitrilo)] di-1H-pyrazol-3(2H)-one

1,1',5,5'-Tetramethy1-2,2'-diphenyl-4,4'-[i-phenylenebis(methylidynenitrilo)]di1H-pyrazol-3(2H)-one was synthesized and characterized by X-ray single-crystal diffraction analysis. The crystal crystallizes in monoclinic, space group P21/c with a = 6.1375(1), b =24.6571(4), c = 17.7487(3)(A), β = 94.781(1)°, V= 2676.62(8)(A)3, C30H28N6O2, Mr= 504.58, Z = 4,Dc = 1.252 g/cm3, F(000) = 1064, μ = 0.081 mm-1, R = 0.0463 and wR = 0.1153 (I ＞ 2σ(Ⅰ)).Theoretical studies of the title compound were carried out by density functional theory (DFT) BLYP method, using ADF program package. It indicates that N(26) and N(41) are active sites of the title compound.     

Reaction of phenols with the 2,2-diphenyl-1-picrylhydrazyl radical. Kinetics and DFT calculations applied to determine ArO-H bond dissociation enthalpies and reaction mechanism

The formal H-atom abstraction by the 2,2-diphenyl-1-picrylhydrazyl (dpph(*)) radical from 27 phenols and two unsaturated hydrocarbons has been investigated by a combination of kinetic measurements in apolar solvents and density functional theory (DFT). The computed minimum energy structure of dpph(*) shows that the access to its divalent N is strongly hindered by an ortho H atom on each of the phenyl rings and by the o-NO(2) groups of the picryl ring. Remarkably small Arrhenius pre-exponential factors for the phenols [range (1.3-19) x 10(5) M(-1) s(-1)] are attributed to steric effects. Indeed, the entropy barrier accounts for up to ca. 70% of the free-energy barrier to reaction. Nevertheless, rate differences for different phenols are largely due to differences in the activation energy, E(a,1) (range 2 to 10 kcal/mol). In phenols, electronic effects of the substituents and intramolecular H-bonds have a large influence on the activation energies and on the ArO-H BDEs. There is a linear Evans-Polanyi relationship between E(a,1) and the ArO-H BDEs: E(a,1)/kcal x mol(-1) = 0.918 BDE(ArO-H)/kcal x mol(-1) - 70.273. The proportionality constant, 0.918, is large and implies a "late" or "product-like" transition state (TS), a conclusion that is congruent with the small deuterium kinetic isotope effects (range 1.3-3.3). This Evans-Polanyi relationship, though questionable on theoretical grounds, has profitably been used to estimate several ArO-H BDEs. Experimental ArO-H BDEs are generally in good agreement with the DFT calculations. Significant deviations between experimental and DFT calculated ArO-H BDEs were found, however, when an intramolecular H-bond to the O(*) center was present in the phenoxyl radical, e.g., in ortho semiquinone radicals. In these cases, the coupled cluster with single and double excitations correlated wave function technique with complete basis set extrapolation gave excellent results. The TSs for the reactions of dpph(*) with phenol, 3- and 4-methoxyphenol, and 1,4-cyclohexadiene were also computed. Surprisingly, these TS structures for the phenols show that the reactions cannot be described as occurring exclusively by either a HAT or a PCET mechanism, while with 1,4-cyclohexadiene the PCET character in the reaction coordinate is much better defined and shows a strong pi-pi stacking interaction between the incipient cyclohexadienyl radical and a phenyl ring of the dpph(*) radical.     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.