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Combined HPLC-CUPRAC (cupric ion reducing antioxidant capacity) assay of parsley, celery leaves, and nettle

This study aims to identify the essential antioxidant compounds present in parsley (Petroselinum sativum) and celery (Apium graveolens) leaves belonging to the Umbelliferae (Apiaceae) family, and in stinging nettle (Urtica dioica) belonging to Urticaceae family, to measure the total antioxidant capacity (TAC) of these compounds with CUPRAC (cupric ion reducing antioxidant capacity) and ABTS spectrophotometric methods, and to correlate the TAC with high performance liquid chromatography (HPLC) findings. The CUPRAC spectrophotometric method of TAC assay using copper(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline) as the chromogenic oxidant was developed in our laboratories. The individual antioxidant constituents of plant extracts were identified and quantified by HPLC on a C18 column using a modified mobile phase of gradient elution comprised of MeOH-0.2% o-phosphoric acid and UV detection for polyphenols at 280 nm. The TAC values of HPLC-quantified antioxidant constituents were found, and compared for the first time with those found by CUPRAC. The TAC of HPLC-quantified compounds accounted for a relatively high percentage of the observed CUPRAC capacities of plant extracts, namely 81% of nettle, 60-77% of parsley (in different hydrolyzates of extract and solid sample), and 41-57% of celery leaves (in different hydrolyzates). The CUPRAC total capacities of the 70% MeOH extracts of studied plants (in the units of mmol trolox g(-1)plant) were in the order: celery leaves>nettle>parsley. The TAC calculated with the aid of HPLC-spectrophotometry did not compensate for 100% of the CUPRAC total capacities, because all flavonoid glycosides subjected to hydrolysis were either not detectable with HPLC, or not converted to the corresponding aglycons (i.e., easily detectable and quantifiable with HPLC) during the hydrolysis step.

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 Na(2)EDTA 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 1M concentration) with 8M urea buffer adjusted to pH 7 by neutralizing with 6M 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: epsilon(CUPRAC)=7.71x10(3), epsilon(Ellman)=1.37x10(4), epsilon(ABTS)=2.06x10(4), and epsilon(FRAP)=2.98x10(3)L mol(-1)cm(-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.

A comprehensive review of CUPRAC methodology

Measuring the antioxidant activity/capacity levels of food and biological fluids is carried out for the meaningful comparison of the antioxidant content of foodstuffs and for the diagnosis and treatment of oxidative stress-associated diseases in clinical biochemistry. Current literature clearly states that there is no widely adopted/accepted “total antioxidant parameter” as a nutritional index available for the labeling of food and biological fluids due to the lack of standardized quantitation methods. The “parent” CUPRAC (CUPric Reducing Antioxidant Capacity) method of antioxidant measurement, introduced by our research group to world literature, 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; thus this is an electron-transfer (ET)-based method. From the parent CUPRAC method initially applied to food (apricot, herbal teas, wild edible plants, herby cheese etc.) and biological fluids (as hydrophilic and lipophilic antioxidants together or in separate fractions), a number of “daughter” methods have evolved, such as the simultaneous assay of both lipophilic and hydrophilic antioxidants in acetone-water as methyl-β-cyclodextrin inclusion complexes, determination of ascorbic acid alone in the presence of flavonoids (with preliminary extraction of flavonoids as their La(III)-complexes), determination of hydroxyl radical scavenging activity of both water-soluble antioxidants (using benzoate derivatives and salicylate as hydroxylation probes) and of polyphenols using catalase to stop the Fenton reaction so as to prevent redox cycling of antioxidants, measurement of Cu(II)-catalyzed hydrogen peroxide scavenging activity and of xanthine oxidase inhibition activity of polyphenols, TAC measurement of protein thiols in urea buffer, development of a CUPRAC-based antioxidant sensor on a Nafion cation-exchanger membrane, the off-line HPLC-CUPRAC assay and finally the on-line HPLC-CUPRAC assay of antioxidants with post-column detection. The current direction of CUPRAC methodology can be best described as a self-sufficient and integrated train of measurements providing a useful “antioxidant and antiradical assay package”. This review attempts to unify and summarize various methodologies of main and modified CUPRAC procedures that can normally be extracted from quite different literature sources.

Comparison of antioxidant capacity and phenolic composition of peel and flesh of some apple varieties

BACKGROUNDnApples contain a large concentration of phenolic compounds, dependent on factors such as cultivar, harvest, storage conditions, and processing. This study aims to identify the essential phenolic compounds present in various apple varieties, to measure their total antioxidant capacity (TAC) with the CUPRAC (cupric ion reducing antioxidant capacity) and ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) methods, and to correlate their TAC values with HPLC findings.nnnRESULTSnThe order of TAC (mmol Trolox g(-1) fresh weight) of apple peels determined with the CUPRAC method was: Granny Smith > Amasya > Sky Spur > Ervin Spur > King Luscious ≥ Arap Kizi ≥ Lutz Golden. The theoretically calculated TAC values of HPLC-quantified compounds, with the aid of the combined HPLC-CUPRAC method, accounted for 18.4-33.5% of the experimentally observed CUPRAC capacity of peel extracts and 19.5-56.3% of flesh extracts, depending on apple variety.nnnCONCLUSIONnIn synthetic samples of apple antioxidants, the CUPRAC-TAC values of constituents, identified and analyzed by HPLC, proved to be additive, enabling measurement of the cooperative action of antioxidants using the proposed methodology. Apple peel showed higher contents of phenolics and therefore higher TAC than apple flesh, confirming the health benefit of the consumption of apples together with peel.

Protein–Incorporated Serum Total Antioxidant Capacity Measurement by a Modified CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method

Due to inadequacies in analytical methodology of total antioxidant capacity (TAC) measurement, proteins are initially separated from the human serum matrix by precipitation and are left unmeasured, thereby causing an important “antioxidant gap.” The aim of this work is to measure the TAC of serum with the modified CUPric Reducing Antioxidant Capacity (CUPRAC) method, and to identify the contribution of serum proteins, especially thiol-containing proteins, to TAC. CUPRAC results were statistically compared to those found by reference methods, namely ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), and Ellman thiols assay. The curves of absorbance vs thiol concentration, as well as of absorbance vs diluted serum (whole serum, trichloroacetic acid (TCA)−precipitated and redissolved serum protein solution, and TCA supernatant fractions) volume, of three distinct serum samples showed excellent linearity and low intercept values only with the modified CUPRAC method. The proposed method will help characterize the “antioxidant gap” of serum TAC originating from protein components which should not be neglected in future antioxidant measurements.

Spectrophotometric and Chromatographic Assessment of Contributions of Carotenoids and Chlorophylls to the Total Antioxidant Capacities of Plant Foods

Carotenoids and chlorophylls are photosynthetic compounds and also efficient antioxidants. This study aims to identify and quantify carotenoids and chlorophylls in some vegetables (carrot, tomato, spinach), to measure the total antioxidant capacity (TAC) of these samples with two spectrophotometric methods, to correlate TAC data with carotenoid structure, and to compare the TAC results with HPLC findings. Separation of the individual antioxidant pigments was achieved on a C30 column using a developed gradient elution program involving methanol-acetonitrile (50:50, v/v) with 0.1% (v/v) triethylamine (TEA) (A) and acetone (B) mobile phases. Total antioxidant capacities of the acetone extracts of studied samples, in trolox and β-carotene equivalents, were in the order: spinach > tomato > carrot by both CUPRAC and ABTS methods. CUPRAC responded favorably to both chlorophylls a and b. The TAC calculated with aid of combined HPLC-spectrophotometry was very close to the spectrophotometric value (93-108%) for real samples and synthetic mixtures.

Spectrophotometric total reducing sugars assay based on cupric reduction

As the concentration of reducing sugars (RS) is controlled by European legislation for certain specific food and beverages, a simple and sensitive spectrophotometric method for the determination of RS in various food products is proposed. The method is based on the reduction of Cu(II) to Cu(I) with reducing sugars in alkaline medium in the presence of 2,9-dimethyl-1,10-phenanthroline (neocuproine: Nc), followed by the formation of a colored Cu(I)-Nc charge-transfer complex. All simple sugars tested had the linear regression equations with almost equal slope values. The proposed method was successfully applied to fresh apple juice, commercial fruit juices, milk, honey and onion juice. Interference effect of phenolic compounds in plant samples was eliminated by a solid phase extraction (SPE) clean-up process. The method was proven to have higher sensitivity and precision than the widely used dinitrosalicylic acid (DNS) colorimetric method.

Preparation, characterization and usage of molecularly imprinted polymer for the isolation of quercetin from hydrolyzed nettle extract

Quercetin (3,3,4,5,7-pentahydroxyflavone, QC) is a health-beneficial flavonoid, widely occurring in leaves, fruits, and flowers of various plants. In this work aiming isolation, purification and pre-concentration of QC, QC imprinted polymers (QC-MIPs) in different molar ratios {template:monomer:cross-linker (1:4:20, 1:5:30, 1:8:40, 1:10:50)} were prepared thermally through bulk polymerization by using QC as the template molecule, 4-vinylpyridine (4-VP), methacrylic acid (MAA), acrylamide (AA) as the functional monomers, ethylene glycol dimethacrylate (EDMA) as the cross-linker and 2,2-azobisisobutyronitrile (AIBN) as initiator in the porogens of acetone and tetrahydrofuran. Their recognition and selectivity properties were investigated in solutions containing QC and other similar-structure phenolics by equilibrium binding experiments using different proportions of acetonitrile (ACN)-dimethylsulfoxide (DMSO) mixtures and methanol (MeOH) as solvents. The MIP with 1:4:20 molar ratio of QC:4-VP:EDMA was established as the most suitable for recognition of QC. Sorption parameters of the MIP and the NIP (non-imprinted polymer) were calculated by using Freundlich and Langmuir isotherms with QC solutions in ACN:DMSO (98:2, v/v). The mentioned MIP was found to be highly selective for quercetin over other phenolic compounds (rutin, catechin, etc.). Thus, molecularly imprinted solid-phase extraction (MISPE) procedures were applied for selective pre-concentration and purification of QC from synthetic mixtures of phenolic compounds and nettle extract, known as a source of official and folk medicine. The results demonstrated the possibility of direct extraction of certain pharmacophoric constituents such as QC and QC derivatives from nettle by MIP separation.

Assessment of the contributions of anthocyanins to the total antioxidant capacities of plant foods

Anthocyanins widely distributed in plant foods have antioxidant activity and several health-beneficial effects. Anthocyanin contents and their contribution to total antioxidant capacity (TAC) of various plant foods (Angeleno plum, red plum, sour cherry, red grape, beetroot, and red cabbage) were analyzed in this study by using high-performance liquid chromatography (HPLC) and spectrophotometric methods such as pH differential, cupric reducing antioxidant capacity (CUPRAC) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The individual anthocyanin constituents of plant extracts were identified and quantified by HPLC on a C18 column using gradient elution of mobile phase comprising MeOH–0.2xa0% o-phosphoric acid and photodiode array detection. Trolox equivalent antioxidant capacity (TEAC) coefficients of the studied standards by the CUPRAC assay were found in the order: cyanidinxa0>xa0kuromaninxa0>xa0keracyaninxa0>xa0delphinidinxa0>xa0pelargonidinxa0>xa0peonidinxa0>xa0malvidinxa0>xa0peonidin-3-glucoside in accordance with the theoretical expectations of established structure–antioxidant activity relationships. The theoretical TAC values calculated by the HPLC–CUPRAC and HPLC–ABTS methods were very close to the spectrophotometric values (95–103xa0%) for synthetic mixtures. These combined HPLC–spectrophotometric methods using TEAC coefficients of anthocyanins determined by CUPRAC and ABTS methods become possible to evaluate the anthocyanins contribution to TAC values of foods as well as individual anthocyanins in a single chromatographic run.

Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions

Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant negative-biased deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beers law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).