Simultaneous total antioxidant capacity assay of lipophilic and hydrophilic antioxidants in the same acetone-water solution containing 2% methyl-β-cyclodextrin using the cupric reducing antioxidant capacity (CUPRAC) method

Antioxidants are health beneficial compounds that can protect cells from the damage caused by unstable molecules known as reactive oxygen species (ROS). This work reports the capacity assay of both lipophilic and hydrophilic antioxidants simultaneously, by making use of their ‘host-guest’ complexes with methyl-β-cyclodextrin (M-β-CD), a cyclic oligosaccharide, in acetonated aqueous medium using the cupric reducing antioxidant capacity (CUPRAC) method. Thus the order of antioxidant potency of various compounds irrespective of their lipophilicity could be established in the same solvent medium. M-β-CD was introduced as the water solubility enhancer for lipophilic antioxidants. Two percent M-β-CD (w/v) in an acetone-H₂O (9:1, v/v) mixture was found to sufficiently solubilize β-carotene, lycopene, vitamin E, vitamin C, synthetic antioxidants and other phenolic antioxidants. This assay was validated through linearity, additivity, precision, and recovery. The validation results demonstrate that the CUPRAC assay is reliable and robust. In acetonated aqueous solution of M-β-CD, only CUPRAC and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays were capable of measuring carotenoids together with hydrophilic antioxidants. The CUPRAC antioxidant capacities of a wide range of polyphenolics and flavonoids were experimentally reported in this work as trolox equivalent antioxidant capacity (TEAC) in the CUPRAC assay, and compared to those found by reference methods, ABTS/horseradish peroxidase (HRP)-H₂O₂ and ferric reducing antioxidant power (FRAP) assays.     

Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay

We report on the application of a simple and versatile antioxidant capacity assay for dietary polyphenols, vitamin C and vitamin E utilizing the copper(II)-neocuproine (Cu(II)-Nc) reagent as the chromogenic oxidant, which we term the CUPRAC (cupric reducing antioxidant capacity) method. It involves mixing the antioxidant solution (directly or after acid hydrolysis) with solutions of CuCl₂, neocuproine, and ammonium acetate at pH 7, and measuring the absorbance at 450 nm after 30 min. Slowly reacting antioxidants required an incubation at 50 °C for 20 min for color development. The flavonoid glycosides were hydrolyzed to their corresponding aglycones by refluxing in 1.2 M HCl-containing 50% MeOH for fully exhibiting their antioxidant potencies. Certain compounds also needed incubation after acid hydrolysis for color development. The CUPRAC absorbances of mixture constituents were additive, indicating lack of chemical deviations from Beer’s law. The CUPRAC antioxidant capacities of a wide range of polyphenolics are reported in this work and compared to those found by ABTS/persulfate and Folin assays. The trolox-equivalent capacities of the antioxidants were linearly correlated (r = 0.8) to those found by ABTS but not to those of Folin. The highest antioxidant capacities in the CUPRAC method were observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, caffeic acid, epicatechin, gallic acid, rutin, and chlorogenic acid in this order, in accordance with theoretical expectations. The experiences of other CUPRAC users also are summarized. Correspondence: Reşat Apak, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, TR-34320 Istanbul, Turkey     

Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols

Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na₂EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1 M concentration) with 8 M urea buffer adjusted to pH 7 by neutralizing with 6 M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: ?_CUPRAC = 7.71 × 10³, ?_Ellman = 1.37 × 10⁴, ?_ABTS = 2.06 × 10⁴, and ?_FRAP = 2.98 × 10³ L mol⁻¹cm⁻¹. Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses.     

Classification of Wines Based on Different Antioxidant Responses to Spectrophotometric Analytical Methods

Six well established spectrophotometric assays (Folin-Ciocalteu, DPPH, ABTS^?+, FRAP, CUPRAC, and the o-diphenols method) have been complementary implemented in order to estimate the total phenolic and o-diphenolic content as well as the free radical scavenging and reducing antioxidant capacities of 40 white and 10 red wines of different varieties and geographic origin. In white wines, the results were weakly correlated; whereas, the results in red wines were strongly correlated, therefore, postulating their separate use in order to estimate efficiently the correlation between results of these methods. The results were subjected to the unsupervised PCA pattern recognition method to investigate the possible classification of white wines. PC analysis framed the wine samples in clear clusters, when the extracted PCA model was based on the results of all six spectrophotometric assays. The usefulness of implementing assays based on different antioxidant mechanisms is discussed. To our knowledge, this is the first time different responses of antioxidants to these spectrophotometric assays have been used to classify white wines according to their variety.     

Concerted Determination of the Hydrogen Atom and Electron Transfer Capacity of Lipid Soluble Reducing Agents

This paper describes a method based on square wave voltammetry to evaluate either the electron transfer or the hydrogen atom transfer of lipid soluble antioxidants such as dl ‐mix‐tocopherol, BHT, ethoxyquin and retynil acetate. The electron transfer (ET) capacity was evaluated by the peak current, peak potential and the area under the anodic wave, whereas the hydrogen atom transfer (HAT) capacity by the kinetic rate of the reaction between antioxidants and 2,2‐Azobis(2‐methylpropionamidine) dihydrochloride (AAPH). The results indicate that ethoxyquin and tocopherol have the highest ET and HAT capacity. However, HAT capacity of tocopherol, BHT and retinyl acetate depend on the concentration. The approach has the advantage to assess HAT and ET capacity of lipid soluble antioxidant in a single concerted protocol.     

Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods

Thiol-type compounds are an important class of strong antioxidants and main determinants of total antioxidant capacity (TAC) of cellular homogenates. The TAC of thiol mixtures and the corresponding TEAC (trolox equivalent antioxidant capacity) values of individual thiols were determined by the CUPRAC (CUPric Reducing Antioxidant Capacity) method, and the results were compared with those found by reference assays for method validation. Synthetic mixtures of thiols were prepared, and the expected and found TAC values (in mM trolox (TR) equivalents) of these mixtures showed a good agreement. The technique of standard additions was performed for thiol mixtures and human serum, and the absorbance results confirmed that apparent chemical deviations from Beer's law were absent in the system. The CUPRAC results were compared with those of reference methods, namely 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/persulphate and Ferric Reducing Antioxidant Power (FRAP). As being a most important thiol (-SH) peptide at in vivo conditions, glutathione (GSH) showed a TEAC value of 0.57 in the CUPRAC method, as opposed to the corresponding value (1.51) in the ABTS/persulphate method. The ABTS/persulphate result was not in accordance with the reversible 1-e oxidation of GSH to the corresponding disulfide that is expected to occur under physiological conditions. FRAP did not give consistent results, and even at relatively high concentrations of GSH, the TEAC_FRAP value was only 0.07. The thiol-type antioxidant-bearing pharmaceuticals of Brunac eye drop, Trom and Mentopin effervescent tablets containing N-acetyl-l-cysteine (NAC) were assayed with HPLC for comparison, and the obtained results for NAC were in accordance with those found with CUPRAC.     

Phytochemical and antioxidant properties of some Cassia species

In this study, the antioxidant activity of aqueous and ethanol extracts of four plants from the genus Cassia were evaluated by various antioxidant assays, including ferric reducing antioxidant power (FRAP), DPPH free radical scavenging, metal chelating activity, phosphomolybdenum reducing power, hydrogen peroxide radical scavenging, hydroxyl radical scavenging, deoxyribose degradation and β-carotene bleaching assay. The various antioxidant activities were compared to standard antioxidant such as ascorbic acid. All the extracts showed antioxidant activity in the tested methods. Among the four species, Cassia auriculata has been found to possess highest activity in most of the tested models. In addition to the antioxidant activity, the total phenolics and flavonoids were measured in the extracts. The ethanolic extract exhibited highest phenolics and flavonoid contents and had also shown potent antioxidant activity in comparison to the aqueous extracts. The possible antioxidant mechanism of the ethanol extract can be due to its hydrogen or electron donating and direct free radical scavenging properties. Hence, the ethanol extract represents a source of potential antioxidants that could be used in pharmaceutical industries.     

The main and modified CUPRAC methods of antioxidant measurement

The antioxidant activity/capacity levels of biological fluids and foods are measured for the diagnosis and the treatment of oxidative stress-associated diseases in clinical biochemistry, and for meaningful comparison of the antioxidant content of foods. Currently, there is no “total antioxidant” as a nutritional index available for food labeling and biological fluids due to the lack of standardized quantitative methods.The CUPRAC (CUPric Reducing Antioxidant Capacity) method of antioxidant measurement, introduced by our research group, is based on the absorbance measurement of Cu(I)-neocuproine (Nc) chelate formed as a result of the redox reaction of chain-breaking antioxidants with the CUPRAC reagent, Cu(II)-Nc, where absorbance is recorded at the maximal light-absorption wavelength of 450 nm.We introduce the main CUPRAC method and describe modifications to it in the past six years.     

Evaluation of antioxidant capacity of endemic plant Marrubium astracanicum subsp. macrodon: Identification of its phenolic contents by using HPLC-MS/MS

Antioxidant properties of Marrubium astracanicum subsp. macrodon solvent extracts were measured by both cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) methods. According to the results, ethanol extract of the plant has high potential of reducing antioxidant activity on CUPRAC method. However, water extract of the plant has lower antioxidant potential. Furthermore, both water and ethanol extracts showed lower reducing antioxidant activity compare to standards on FRAP method. Moreover, the composition and content of plant leaves were detected by UHPLC-ESI-MS/MS. High concentrations of quinic acid, p-coumaric acid and malic acid were determined.     

Comparative evaluation of Fe(III) reducing power-based antioxidant capacity assays in the presence of phenanthroline, batho-phenanthroline, tripyridyltriazine (FRAP), and ferricyanide reagents

The chemical diversity of antioxidants in complex matrices such as plant extracts makes it difficult to separate and quantify antioxidants from these solutions. Therefore it is desirable to establish methods that can measure the total antioxidant capacity (TAC) levels directly from plant extracts. Iron(III)-based TAC assays, especially the most widely used FRAP (ferric-reducing antioxidant power), play an important role in this regard. However, many problems have been reported in the application of the FRAP assay, the most serious one being the incomplete oxidation of a number of antioxidants during the time protocol of the assay. Thus, six different ferric ion-based total antioxidant capacity (TAC) assays have been comparatively tested, modified, and improved so as to obtain more sensitive and precise results for complex mixtures, namely: 1,10-phenanthroline (o-phen) method (with incubation), batho-phenanthroline method (with incubation), original FRAP method, modified FRAP method (with incubation), original ferricyanide method, and modified ferricyanide method (with incubation). Two new assays in this regard (i.e., o-phen and batho-phen) have been established, and the existing assays (FRAP and ferricyanide) have been modified so as to let the oxidation reactions of antioxidants reach completion. The molar absorptivity for a variety of antioxidants was highest for modified FRAP, batho-phen, and original FRAP methods. The absorption maximum wavelength shifted batochromically to a higher extent for modified ferricyanide, FRAP, and batho-phen procedures, decreasing the possibility of interferences due to organics absorbing in the near-UV range of the visible spectrum where most antioxidant assays are performed. The linear concentration ranges were shown to be further extended and linear correlation coefficients improved with respect to the most widely used ferric-based assay, FRAP. Of the six assays tested and developed, only the modified ferricyanide procedure gave high intercept values and low addivitity of TAC values of constituents in complex mixtures, requiring further attention of method optimization. Thus, it was shown that the most widely used FRAP could be effectively modified, and o-phen, batho-phen, and ferricyanide methods constitute cheaper alternatives to FRAP under certain conditions, with partly improved molar absorptivity (and thus sensitivity) for antioxidants, lower intercept values (and higher precision), broader linear range (and higher flexibility), and better additivity of TAC values of antioxidant constituents in mixtures.     

Total Phenolics and Anthocyanins Contents and Antioxidant Activity in Four Different Aerial Parts of Leafy Sweet Potato (Ipomoea batatas L.)

Leafy sweet potato (Ipomoea batatas L.) is an excellent source of nutritious greens and natural antioxidants, but reports on antioxidants content and activity at buds, leaves, petioles, and stems are scarce. Therefore, the total phenolics content (TPC), total anthocyanins content (TAC), and antioxidant activity (assessed by DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power (FRAP)) were investigated in four aerial parts of 11 leafy sweet potato varieties. The results showed that varieties with pure green aerial parts, independently of the part analyzed, had higher TPC, FRAP, and ABTS radical scavenging activities. The green-purple varieties had a significantly higher TAC, while variety GS-17-22 had the highest TAC in apical buds and leaves, and variety Ziyang in petioles and stems. Among all parts, apical buds presented the highest TPC and antioxidant capacity, followed by leaves, petioles, and stems, while the highest TAC level was detected in leaves. The TPC was positively correlated with ABTS radical scavenging activity and FRAP in all parts studied, whereas the TAC was negatively correlated with DPPH radical scavenging activity. Collectively, the apical buds and leaves of sweet potato had the higher levels of nutritional values. These results would provide reference values for further breeding of leafy sweet potatoes.     

Physicochemical,Antioxidant, Microstructural Properties and Bioaccessibility of Dark Chocolate with Plant Extracts

In this study, dark chocolates (DCh) containing zinc lactate (ZnL) were enriched with extracts from elderberries (EFrE), elderflowers (EFlE), and chokeberries (ChFrE) to improve their functional properties. Both dried plant extracts and chocolates were analyzed for antioxidant capacity (AC) using four different analytical methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), cupric ion-reducing antioxidant capacity (CUPRAC), and ferric-reducing antioxidant power (FRAP), while total phenolic content (TPC) was determined by Folin–Ciocalteu (F–C) assay. An increase in antioxidant properties of fortified chocolates was found, and the bioaccessibility of their antioxidants was evaluated. The highest AC and TPC were found in ChFrE and chocolate with chokeberries (DCh + ChFrE) before and after simulated in vitro digestion. Bioaccessibility studies indicated that during the simulated digestion the AC of all chocolates reduced significantly, whereas insignificant differences in TPC results were observed between chemical and physiological extracts. Moreover, the influence of plant extracts on physicochemical parameters such as moisture content (MC), fat content (FC), and viscosity of chocolates was estimated. Furthermore, scanning electron microscopy with dispersive energy spectroscopy (SEM-EDS) was used to analyze surface properties and differences in the chemical composition of chocolates without and with additives.     

Antioxidant compounds from propolis collected in Anhui, China

The antioxidant activities of the chloroform, ethyl acetate and n-butanol extract fractions from propolis collected in Anhui, China were evaluated in this study. The ethyl acetate fraction contained the highest amount of total phenolics and total flavonoids, and showed the greatest 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging capacities and Ferric Reducing/Antioxidant Power (FRAP). The antioxidant activity of twenty-two compounds isolated from the ethyl acetate fraction was also evaluated using the above-mentioned three assays. The results indicated that phenolics contributed to the antioxidant activity of propolis collected in Anhui, China. Therefore, propolis collected in Anhui, China and its phenolics might be used as a natural antioxidant.     

Identifying Major Drivers of Antioxidant Activities in Complex Polyphenol Mixtures from Grape Canes

Grape canes represent a valuable source of numerous polyphenols with antioxidant properties, whose compositions vary depending on the genotype and environmental factors. Antioxidant activities of pure molecules are often reported without considering possible interactions that may occur in complex polyphenol mixture. Using UPLC-MS-based metabolomics and unsupervised classification, we explored the polyphenol variations in grape cane extracts from a collection of European varieties. Antioxidant activities were assessed using ORAC, ABTS, DPPH, FRAP, CUPRAC and chelation assays. Pairwise correlations between polyphenols and antioxidant capacities were performed to identify molecules that contributed more to the antioxidant capacities within a complex mixture of polyphenols.     

Antioxidant capacity and sensory quality of soy-based powder drink mix enriched with functional hydrolysates of swiftlet (Aerodramus fuciphagus)

The enrichment with low amount of bioactive protein of spray-dried edible bird’s nest hydrolysates (EBNH) (3.0 %) in view of its cost and high solubility provided significant value added to the overall in vitro antioxidant capacity of soy-based powder drink mix (PDM). Its beverage (12.5 % concentration, consistency index 0.39 Pa.sⁿ) antioxidant capacity as measured by ABTS and FRAP was comparable (p > 0.05) but significantly higher than antioxidant assays of FCR and DPPH. The respective antioxidant capacity of the PDM beverage in terms of trolox equivalent (TE) and gallic acid equivalent (GAE) were 21.95 TE mg/g, 20.75 TE mg/g, 2.93 TE mg/g and 14.72 GAE mg/g for FRAP, ABTS, DPPH and FCR. Depending on antioxidant assay, EBNH in beverage of PDM contributed an increase in the range of 3.7–9.3 % (which was significant (p < 0.05) according to ABTS and FCR assays) or about 6.0 % to its overall antioxidant capacity. The interaction among the antioxidant activity of all the food product’s ingredients is antagonistic since the difference between the expected and observed total antioxidant potential is significantly higher (p < 0.05) for all antioxidants assays, except FCR. The beverage of PDM has excellent sensory quality. It is sugar free and high protein PDM that has excellent cocoa flavour and possesses sufficient sweetness with acceptable beany aroma and taste when served as hot beverage.     

Assessment of antioxidant activities of three wild medicinal plants from Bahrain

This study reports the antioxidant properties of three wild medicinal plants from Bahrain, namely Aizoon canariense L., Asphodelus tenuifolius Cav., and Emex spinosus (L.) Campdera. Antioxidant and antiradical activities of dried materials of these plants were investigated using FRAP, DPPH and ABTS assays. Total phenolics, free phenolics and total flavonoids were also determined. E. spinosus was ranked by the assays as the plant possessing the highest antioxidant and antiradical activities with an average FRAP value of 1.84 mmol/g and IC₅₀ of 10.7 and 7.75 mg/ml for DPPH and ABTS assays, respectively. A. tenuifolius ranked second with a mean FRAP value of 0.69, IC₅₀ DPPH of 1.72 and ABTS of 0.36. A. canariense possessed the lowest activities with a mean FRAP value of 0.6, and averaged IC₅₀ of 103.8 and ABTS of 14.6. E. spinosus possessed the highest content of free phenolics (mg/100 g) (64.64) followed by A. tenuifolius (45.21) and A. canariense (32.23). E. spinosus also exhibited the highest total flavonoids with an average 82.71 mg/100 g followed by A. canariense (55.92) and A. tenuifolius (49.10). The studied medicinal plants possess considerable antioxidant activities and may contribute to the well-being of individuals who consume them.     

Antioxidant capacity and antibacterial activity from Annona cherimola phytochemicals by ultrasound-assisted extraction and its comparison to conventional methods

In recent years, the food, pharmacy, and cosmetic industries have focused on the search of natural compounds with antimicrobial and antioxidant properties; commonly, these compounds are obtained from Kingdom plantae. The aim of the present work is comparing antibacterial and antioxidant capacity of Annona cherimola Mill leaves, using different extraction methods. The ultrasound assisted extraction technique (UAE) was compared with conventional techniques: Soxhlet and maceration. Water and ethanol were used as solvents for leaves extractions performed with these three methods. The main acetogenins reported in Annona cherimola Mill and Annona muricata L. species were simulated using the functional hybrid B3LYP and to confirm its presence, analysis of the compound composition was performed using FT-IR, UV–Vis and HPLC. Total phenolics (TP) and flavonoids (TF) were determined by spectroscopy techniques and novel Differential Pulse Voltammetry (DPV) electrochemical technique. Total Antioxidant Capacity (TAC) of the extracts was measured, using the DPPH, FRAP and CUPRAC techniques. The highest antioxidant content was found in the Soxhlet water extracts; even so, the UAE technique presented an attractive alternative due to considerable reduction in extraction time, which was greater than 99%, and possible selectivity in compounds extraction. Finally, antibacterial activity of the extracts was evaluated, obtaining the best results against gram-positive bacteria using UAE water extract. In this way, the UAE technique presents an excellent extraction option due to the considerable reduction in time and energy, as well as the increase in antibacterial activity.     

Effects of Orange Leaves Extraction Conditions on Antioxidant and Phenolic Content: Optimization Using Response Surface Methodology

In the last few years, bioactive components or their extraction techniques are gaining special interest in scientific areas. In this framework, orange leaves were used for preparation of extracts with high content of biologically active compounds. To optimize the extraction process, three levels and three variables of Box–Behnken design with response surface methodology were applied. Investigated responses were the total phenolic content (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, cupric ion reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP). Independent variables were methanol concentration (10–90%), temperature (20–60°C), and extraction time (60–180?min). Experimentally obtained results were fit into a second-order polynomial model with multiple regression. Analysis of variance was used to estimate model fitness and determine optimal conditions for processing. Estimated optimal conditions were 90% methanolic solution, 60°C and 180?min using these parameters; the predicted values of investigated responses were 43.19?mg GAE/g (GAE: gallic acid equivalents), 43.04?mg TE/g (TE: trolox equivalents), 139.34 and 93.76?mg TE/g for TPC, DPPH, CUPRAC, and FRAP, respectively. The obtained optimal conditions could be considered as an alternative strategy for developing novel functional products.     

Synthesis, characterization, acetylcholinesterase inhibition, molecular modeling and antioxidant activities of some novel Schiff bases derived from 1-(2-ketoiminoethyl)piperazines

Some novel Schiff bases derived from 1-(2-ketoiminoethyl)piperazines were synthesized and characterized by mass spectroscopy, FTIR, UV-Visible, 1H and 13C-NMR. The compounds were tested for inhibitory activities on human acetylcholinesterase (hAChE), antioxidant activities, acute oral toxicity and further studied by molecular modeling techniques. The study identified the compound (DHP) to have the highest activity among the series in hAChE inhibition and DPPH assay while the compound LP revealed the highest activity in the FRAP assay. The hAChE inhibitory activity of DHP is comparable with that of propidium, a known AChE inhibitor. This high activity of DHP was checked by molecular modeling which showed that DHP could not be considered as a bivalent ligand due to its incapability to occupy the esteratic site (ES) region of the 3D crystal structure of hAChE. The antioxidant study unveiled varying results in 1,1-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. This indicates mechanistic variations of the compounds in the two assays. The potential therapeutic applications and safety of these compounds were suggested for use as human acetylcholinesterase inhibitors and antioxidants.     

A silver nanoparticle-based method for determination of antioxidant capacity of rapeseed and its products

A novel silver nanoparticle-based (AgNP) method and two modified procedures, ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH), were used for determination of antioxidant capacities of the ethanolic, methanolic, methanolic-aqueous (1?:?1 v/v) and aqueous extracts of rapeseed and its products. The AgNP method based on the electron-transfer reaction between silver ions and antioxidants in an optimized ammonium buffer medium (pH = 8.4) and determination of silver nanoparticle formation has been elaborated. The novel AgNP method was validated using sinapic acid, gallic acid, caffeic acid, ascorbic acid and quercetin as standard antioxidant solutions in concentration ranges of 0.03-0.21 μmol mL(-1), 0.02-0.20 μmol mL(-1), 0.01-0.18 μmol mL(-1), 0.03-0.30 μmol mL(-1) and 0.001-0.009 μmol mL(-1). The calculated detection (DL = 0.01, 0.02, 0.009, 0.02 and 0.0004 μmol mL(-1) for sinapic, gallic, caffeic, ascorbic acids and quercetin, respectively) and quantification limits (QL = 0.04, 0.06, 0.03, 0.08 and 0.001 μmol mL(-1) for sinapic, gallic, caffeic, ascorbic acids and quercetin, respectively) confirm linearity concentration ranges for determination of antioxidant capacity by AgNP assay. The average antioxidant capacities of the studied rapeseed samples ranged between 14.7 and 126.2 μmol sinapic acid per gram for the proposed AgNP method, 7.4-112.7 μmol sinapic acid per gram for the FRAP method and 39.1-339.8 μmol sinapic acid per gram for DPPH assay. The methanol-water mixture (1:1 v/v) was the most efficient solvent for extraction of antioxidants from the studied rapeseed samples. There are significant, positive correlations between the novel AgNP and the modified FRAP, DPPH and FC methods for all extracts of the studied rapeseed samples (r = 0.7564-0.8516, p < 0.001). Satisfactory values of precision (RSD = 1.2-4.4%) and accuracy (recovery = 95.6-104.6%, except methanolic extracts) demonstrate the benefit of the proposed AgNP method for analysis of the antioxidant capacity of rapeseed samples. Results of the principal component analysis (PCA) indicate that there are differences between the total amounts of antioxidants in rapeseed samples extracted by different solvents.