As a result, these methods have the disadvantage that they do not reflect the situation in an oxidant food or an in vivo case. The results obtained were compared to the antioxidant activity of the same antioxidants, measured by means of the FRAP, ABTS and DPPH tests [53]. Schlesier K., Harwat M., Bhm V., Bitsch R. Assessment of Antioxidant Activity by Using Different In Vitro Methods. (2020) examined the applicability of the reduction reaction of Fe(III) to Fe(II) by the antioxidants in order to develop the electrochemical method of determining antioxidant activity, applying direct current polarography and cyclic voltammetry. The aim of the present research was to obtain a supramolecular complex between a strong antioxidant compound previously reported by our group, in order to extend its antioxidant activity. Puangbanlang et al. Physiol. The end result is the same, regardless of the reaction mechanism involved, although the kinetics and reaction stages are different [8]. Polyphenol-rich foods exhibit DNA antioxidative properties and protect the glutathione system in healthy subjects. (2019). In the biomonitoring and supplementary studies, the FRAP test was used in human investigations, as well as on samples from various animals, insects, and marine organisms. Chung W.Y., Chung J.K.O., Szeto Y.T., Tomlinson B., Benzie I.F. The authors questioned the application of the DPPH test to classify antioxidants, and suggested it was possible to use it to distinguish the reaction mechanism of an antioxidant, i.e., SET and HAT, which display different reaction rates and models in various solvents [109]. ) radical scavenging activity is generally quantified in terms of inhibition percentage of the pre-formed free radical by antioxidants, and the EC(50) (concentration required to obtain a 50% antioxidant effect) is a typically employed parameter to express the antioxidant capacity and . The antioxidant activity was ascribed to either active principles like methylsalicylate or vehicles like ethanol. Concentration of the phytochemicals studied varied greatly between the apple peel and the cortex region. (1) Dihydrofluorescein diacetate; (2) Luminol. Structure of a heteropoly blue. To test the possible borate interference on the DPPH test, X. Chen et al. Peels showed ~ 2.8 . The paper-based tests were successfully applied to detect the antioxidant activity and total phenolic content in 10 beverages with Gallic acid equivalent values (GAE) similar to those obtained in traditional tests, with a confidence interval of 95%. These assays were successfully applied in antioxidant analysis or the determination of the antioxidant capacity of complex samples. If modifications are operated at the level of the reaction conditions or duration, standardisation or the manner of expressing the results, then it is important that they should be justified and clearly described, and the modified method should be validated in comparison to the standard procedure. For example, Milardovic et al. ; WritingReview and editing, C.A. Plants have a large number of bioactive compounds with high antioxidant activity. This can negatively influence selectivity for genuine antioxidant substances. Favourable redox potential: the CUPRAC reagent is selective, because it has a lower redox potential than that of the ferricferrous couple in the presence of phenanthrolineor similar ligands. The ORAC test describes the antioxidants ability to yield the hydrogen atom and, consequently, it is a HAT-based assay. Quantification of the Antioxidant Activity of Plant Extracts: Analysis The present paper is a critical presentation of the most important tests used to determine the antioxidant activity, detection mechanism, applicability, advantages and disadvantages of these methods. eki S.D., etinkaya A., Avan A.N., Apak R. Correlation of Total Antioxidant Capacity with Reactive Oxygen Species (ROS) Consumption Measured by Oxidative Conversion. Screening for Antioxidant Activity: Diphenylpicrylhydrazine (DPPH The site is secure. Re-evaluation of the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay for antioxidant activity. Overall, the proposed kinetic-based DPPH method is simple, rapid, and suitable for studying the activity and capacity of different molecules, and food samples rich in fast antioxidants, like fruit . Arnao M.B., Casas J.L., Ro J.A., Acosta M., Garca-Cnovas F. An enzymatic colorimetric method for measuring naringin using 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of peroxidase. Zheng L., Zhao M., Xiao C., Zhao Q., Su G. Practical problems when using ABTS assay to assess the radical-scavenging activity of peptides: Importance of controlling reaction pH and time. Milardovi S., Ivekovic D., Grabari B.S. Various solvents were used, like methanol, ethanol, acetone and dimethylsulfoxide, subsequently establishing the kinetics of the reaction and also the stability of the radical. The .gov means its official. The first structural data showed that its unique low reactivity was mainly influenced by the efficient screening of the hydrazyl structure by the molecules surrounding parts and less by extended conjugation [90]. They react with ROS to eliminate or to inhibit them. [108]. High-performance thin-layer chromatography method - Springer Nevertheless, the data regarding the hydrogen atom transfer or the data regarding the donating capacity of the electrons obtained by these methods provide important information on their intrinsic antioxidant potential with minimal environment interference. Determination of Antioxidant Activity Using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Method. In order to improve the method, a high-throughput assay has been developed using a multichannel liquid handling system coupled with a microplate fluorescence reader [12]. Abuin E., Lissi E., Ortiz P., Henrquez C. Uric Acid Reaction with Dpph Radicals at the Micellar Interface. A colorimetric method for the determination of total antioxidant activity in a variety of foods and beverages was validated in both a single-laboratory validation and a collaborative laboratory validation study. Studies for the determination of the antioxidant activity of different plant species could contribute to revealing the value of these species as a source of new antioxidant compounds. (A) The -Keggin structure of the anionic derivative [PM12O40]3, where M stands for molybdenum (Mo) or tungsten (W); (B) the big wheel structure of the blue complex [Mo1266+Mo285+O462H14(H2O)70]14. While the discolouration test may solve this problem, other disadvantages occur because of the problems of procedure and mechanism [87]. The pharmacopoeia includes the FolinCiocalteu test [9], and Europe adopted it as an official procedure of measuring the total phenol contents in wines (European Community 1990). In another modified CUPRAC test, an optical sensor was modified to contain immobilised chromogenic redox reagent to measure the reduction power of liquid samples without any pretreatment [25]. Regoli F., Winston G.W. Tufan A.N., elik S.E., zyrek M., Gl K., Apak R. Direct measurement of total antioxidant capacity of cereals: QUENCHER-CUPRAC method. High molecular weight plant polyphenolics (tannins) as biological antioxidants. Benzie I.F.F. The antioxidant action in these tests is often simulated with a suitable fluorescent or coloured sample instead of peroxyl radicals. There is a large variety of in vitro methods to quantify antioxidant activity, and it is important to select the proper method to . The FRAP test may be also used to detect water contamination, and was used to study the effect of radiations, pollution, climate change and space travel on living organisms. Several antioxidant procedures should be performed in vitro to determine antioxidant activities for the sample of interest. the contents by NLM or the National Institutes of Health. HA represents an antioxidant molecule and A+ an oxidised antioxidant molecule (a); Colour change in the assay (b). The TRAP test is based on the antioxidants capacity to inhibit the reaction between peroxyl radicals and a target molecule, which initially represented the O2 consumption (as a sample) in the peroxidation process triggered by the thermal decomposition of 2,2 azobis(2-amidinopropane)dihydrochloride (ABAP). Hegerman A.E., Riedl K.M., Jones G., Sovik K.N., Rechard N.T., Hartzfeld P.W., Reichel T.L. Numerous reviews and research articles mention analytical details, protocol variations and innovations like changing the reaction monitoring from 515 to 580 nm in order to minimise interferences due to carotenoids [. Abstract. Furthermore, the ABTS radical is soluble in water and organic solvents, enabling the determination of antioxidant capacity of both lipophilic and hydrophilic compounds. Figure 1 illustrates the classification of antioxidants whereas Figure 2 indicates the application scope of antioxidants. Gl K., Kbrslolu G., zyrek M., Apak R. Development of a Fluorescent Probe for Measurement of Peroxyl Radical Scavenging Activity in Biological Samples. Sensitivity and linear response: the absorbance vs. concentration curves are linear in the CUPRAC method over a wide range, unlike those of other methods yielding polynomial curves. According to the chemical reactions that may be involved, these tests are divided into two categories: hydrogen atom transfer (HAT) and single electron transfer (SET) reaction-based methods. Bener M., zyrek M., Gl K., Apak R.A. Development of a Low-Cost Optical Sensor for Cupric Reducing Antioxidant Capacity Measurement of Food Extracts. Another advantage was improved cognitive performance, which was associated to an increased supply of polyphenol-rich foods [64]. One of the important selection parameters of the antioxidant test is the working pH. Azo-initiators produce ROO by heating, which harms the fluorescent molecule, leading to the loss of fluorescence. Another method of determining antioxidant activity based on the amperometric reduction of DPPH at the glassy carbon electrode was described by S. Milardovi et al. CUPRAC chromogenic redox reaction is carried out at a pH (7.0) close to the physiological pH, which has a value of 7.4. It can be seen that the intensity of the light emission during the incubation of luminol with AAPH is directly linked to the stable state concentration of the ROO generated in the AAPH thermolysis [9]. These were used to study the efficiency of some antioxidants with a low molecular weight. Walker R.B., Everette J.D. Effects of total dietary polyphenols on plasma nitric oxide and blood pressure in a high cardiovascular risk cohort. The method was applied to the evaluation of the antioxidant activity of certain pure compounds, soluble in water or ethanol, certain antioxidants, and some samples of tea, wine and other beverages. Conceptualisation, C.A. Classification, physicochemical principles, mechanisms, and electron transfer (ET)-based assays. Mixed tests, including the transfer of both a hydrogen atom and an electron, include the 2,2-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test, and the [2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl] (DPPH) test. Under normal circumstances, the rate and amplitude of oxidant formation is balanced by the rate of their removal. As a result, ORAC values are appreciated by certain researchers as biologically relevant, a benchmark for antioxidant efficiency. DPPH - an overview | ScienceDirect Topics Antolovich M., Prenzler P.D., Patsalides E., McDonald S., Robards K. Methods for testing antioxidant activity. An efficient phenolic antioxidant should react faster than the biomolecules (the protected molecule) with the free radicals in order to give a protective effect against their oxidation [9]. Analysis of total phenols and other oxidation substrates and antioxidants by means of FolinCiocalteu reagent. However, it also has some drawbacks. (2011) stated that it is necessary to continue altering the ORAC method to remove the influence of the metallic ions in the testing systems on the ORAC values measured for the antioxidant compounds. Currently, fluorescein is most often used as a fluorescent probe. The FRAP test has recently adopted electrochemical detection techniques for better sensitivity, accuracy and reproducibility. The hydrophobic lipid interior of the membranes requires a different spectrum of antioxidants. This work was supported by a grant of the Romanian Ministry of Education and Research, CNCSUEFISCDI, project number PN-III-P4-ID-PCE-2020-0923, within PNCDI III. This method was validated by synthetic antioxidants with known concentrations [, The DPPH method was used to evaluate the antioxidant capacity of phenolic compounds [, antioxidant activity, superoxyde dismutase (SOD), reactive oxygen species (ROS). Full article: A new colorimetric DPPH scavenging activity method with (2006) for the measurement of the area below the decomposition curve of DPPH, Test adjustments were proposed by various authors in an attempt to minimise issues related to test sensitivity and to simplify and automate the method [, The neutralisation of the DPPH radical may be monitored by amperometric detection. According to this test, the peroxyl radical emitted by a generator reacts with a fluorescent sample that leads to loss of fluorescence, registered on a fluorimeter. The study of antioxidants and their implications in various fields, from food engineering to medicine and pharmacy, is of major interest to the scientific community. Inclusion in an NLM database does not imply endorsement of, or agreement with, Ziyatdinova G., Salikhova I., Budnikov H. Coulometric titration with electrogenerated oxidants as a tool for evaluation of cognac and brandy antioxidant properties. Determination of total phenolic content and antioxidant activity of Antimicrobial, antioxidant activities, and total phenolic contents of Determination of Antioxidant Activity in Foods and - ResearchGate A novel method for measuring the antioxidant activity using N, N-dimethyl-p-phenylenediamine (DMPD) was developed. The concept of redox reducing capacity as an index of antioxidant activity may be applied in different manners. Determination of total flavonoid content: . Introduction Abstract Procedure Results Conclusions and Future Work Acknowledgements N N N + O O-N + O-O N + O-O + N N H N O + N O +-O N + O-O . Before The chemistry behind antioxidant capacity assays. Cerretani L., Giuliani A., Maggio R.M., Bendini A., Toschi T.G., Cichelli A. Therefore, SET reactions are pH dependent [22]. sharing sensitive information, make sure youre on a federal This protocol was . However, the TPC results are also occasionally expressed as catechins, caffeic acid, chlorogenic acid or the equivalent of ferrulic acid, requiring the standardisation of the reported results [58]. As can be seen in the reactions below, in the system formed by ABTS/H2O2/peroxidase, ABTS behaves as a reducing agent, substituting the enabled form of the enzyme (named compound I) in compound II, which returns to the initial form of the enzyme (E). In addition, the methods which were developed use changes of the redox electrochemical signals [9]. El Moussaoui A., Jawhari F.Z., Almehdi A.M., Elmsellem H., Benbrahim K.F., Bousta D., Bari A. Antibacterial, antifungal and antioxidant activity of total polyphenols of. The tests based on the transfer of one electron include the Cupric Reducing Antioxidant Power (CUPRAC) test, the Ferric Reducing Antioxidant Power (FRAP) test, the FolinCiocalteu test. Voltammetric methods with screen-printed carbon electrodes were also recorded within the range 0.2 to 0.9 V (vs. the Ag/AgCl reference electrode) to investigate the oxidation behaviour of these substances. A special role in neutralising the effects of the oxidative stress related to the presence of free radicals is played by the enzyme called superoxyde dismutase (SOD). The various methods for evaluation of the antioxidant capacity fall into three distinct categories namely, spectrometry, electrochemical assays and chromatography [3] as presented in Table 2. Measurement of antioxidant activity. The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones. Determine if Activity . elik S.E., zyrek M., Gl K., Apak R. Differences in responsivity of original cupric reducing antioxidant capacity and cupricbathocuproine sulfonate assays to antioxidant compounds. The efficient research of the sources of natural antioxidants and designing new antioxidant compounds require reliable methods of antioxidant activity evaluation. Moharram H.A., Youssef M.M. Cheng Z., Moore A.J., Yu L. High-Throughput Relative DPPH Radical Scavenging Capacity Assay. Benzie I.F.F., Strain J.J. Ferric reducing (antioxidant) power as a measure of antioxidant capacity, the FRAP assay and its modification for measurement of ascorbic acid (FRASC). Versatility: this method is capable of measuring both hydrophilic and lipophilic antioxidants (e.g., -carotene and -tocopherol). Estimation of Phytochemical Content and Antioxidant Activity of Some When voltage is applied, the DPPH radical generates a constant electrical current, and the decrease in the radical amount as a result of neutralisation by the antioxidants results in a decrease in the amperometric signal. Determination of Antioxidants by DPPH Radical Scavenging Activity and Many of these modified TEAC assays use the online enzymatic generation of ABTS+, mainly those employing continuous flux systems. The TRAP index was defined as the number of mols of ROO contained per litre of fluid (plasma) [19], according to the equation: where RROO is the ROO formation rate, and plasma is the delay time in the oxygen consumption generated by the presence of human plasma. Reaction schemes involved in Oxygen Radical Absorption Capacity (ORAC) assay for the detection of hydroxyl and peroxyl radicals. ferric) reducing/antioxidant power (FRAP), total reactive antioxidant potential (TRAP), and cupric reducing/antioxidant power (CUPRAC) (Ou et al., 2001; Ou et al . used a water-soluble DPPH derivative [105]. (2016) researched the antioxidant activity of compounds of vegetable origin from the class of hydroxycynamic acids, by means of electrochemical methods, and the ABTS and DPPH tests [110]. Antioxidant Activity of Dietary Polyphenols as Determined by a Modified Ferric Reducing/Antioxidant Power Assay. First of all, due to the presence of negatively charged sulfonate groups on the fenantrolin ring, the Cu(I)BCS complex has a higher global charge than the Cu(I)Nc complex. Robustness: it is observed that physiological parameters, such as air, humidity, sunlight do not influence the CUPRAC reaction with antioxidants, as opposed to the reagents of the free radical type, such as DPPH. The central molybdenum ion in the complex is accepted as a reducing site, where the Mo6+ ion is reduced to Mo5+ by accepting an electron donated by the phenolic antioxidant. Sung-Kun Y., Su-Jung Y., Chul-Ho Y. Durmaz (2012) prepared a ready-to-use ABTS+ radical powder by incubating ABTS with potassium peroxodisulphate, followed by drying and freezing the resulting solution [68]. First of all, the test is sensitive to pH, temperature and reaction time, and that is why it is necessary to accurately select the reaction state for coherent and reliable results. The reason for this is the protonation on antioxidant compounds while, in more basic conditions, acid dissociation (through proton release) from phenols improves the reduction capacity of a sample, thus triggering unrealistic measurements of TAC. Evaluation of total reducing power of edible oils. Small temperature differences in the external wells of the microplate can decrease the reproducibility of the assay. All the essential oils showed antioxidant activity. In addition, ABTS has been used to determine p-hydroxybenzoic acids and polyphenolic compounds by means of the enzyme laccase [69,70] and, also, to determine a variety of flavonoids using peroxidase [71]. The TEAC assay has also been challenged for its lack of biological relevance due to use of the artificial ABTS radical cation that is not found in food or biological systems [88]. An alternative proposal was made by Cheng et al. Gallic acid is the commonly used reference standard, and the TPC results are usually expressed as Gallic acid equivalent. Out of the tests based on the transfer of a hydrogen atom, the following were presented: the Oxygen Radical Absorption Capacity (ORAC) test, the Hydroxyl Radical Antioxidant Capacity (HORAC) test, the Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP) test, and the Total Oxyradical Scavenging Capacity (TOSC) test. Here we propose a protocol to evaluate the antioxidant capacity of compounds by the DPPH method through the scavenging capacity of free radicals by reducing the DPPH radical.