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Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements
Abstract and Figures
Methods available for the measurement of antioxidant capacity are reviewed, presenting the general chemistry underlying the assays, the types of molecules detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equivalent antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biology. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method. Other assays may need to be considered in the future as more is learned about some of the other radical sources and their importance to human biology.
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... The TF contents were 182.0 ± 0.24, 217.4 ± 1.18, 225.3 ± 1.20, and 279.0 ± 0.56 mg rutin equivalent (RU)/g of plant dry weight (mg RU/g DW) in TS, TV, TQ, and TM, respectively, and there are significant differences between the four thyme ethanol extracts (Table 1). These values prove that the erect-stem species have better activity than that the creeping-type thyme from the same place of origin (Europe: erect-stems Molecules 2023, 28, 2582 3 of 15 species TV > creeping-stems species TS; East: erect-stems species TM > creeping-stems species TQ). This result was consistent with a previous study on cauliflower [27]. ...
... However, the same extracts had a different contribution to DPPH, FRAP, and ABTS activity. For DPPH, small molecules may have a better chance to access the radical with a subsequently higher TAC value [28]. The ethanolic extracts of four thyme species contain different compounds and the same compounds have different contents. ...
... Molecules 2023,28, 2582 ...
Thyme is a colloquial term for number of aromatic species belonging to the genus Thymus L., known for their expressed biological activities and therefore used worldwide for seasoning and in folk medicine. In the present paper, the content of the total polyphenols (TP), total flavonoids (TF), and antioxidant capacity were assessed in the extracts of four traditionally used thyme species. Moreover, a comprehensive metabolomic study of thyme bioactive compounds was performed, and the obtained data were processed using multivariate statistical tests. The results clearly demonstrated the positive correlation between the content of the TP, TF, and antioxidant activity, and TF was more significant than TP. The findings revealed that four selected thyme species contained 528 secondary metabolites, including 289 flavonoids and 146 phenolic acids. Thymus marschallianus had a higher concentration of active ingredients, which improve its antioxidant capacity. Differentially accumulated metabolites were formed by complex pathways such as flavonoid, flavone, flavonol, isoflavonoid, and anthocyanin biosynthesis. Correlation analysis showed that 59 metabolites (including 28 flavonoids, 18 phenolic acids, and 7 terpenoid compounds) were significantly correlated with obtained values of the antioxidant capacity. The results suggested that selected thyme species exhibit a great diversity in antioxidant-related components, whereas flavonoids may be responsible for the high antioxidant capacity of all studied thyme species. The present study greatly expands our understanding of the complex phytochemical profiles and related applications of selected medicinal plants.
... The TAC values were obtained using the most-known DPPH method [30], based on the fading of the intense violet color of the free radical solution. We measured these values using Equation (3), where Abs 0 refers to the initial absorbance of the DPPH free radical (at 517 nm) and Abs 30min refers to the absorbance recorded at the same wavelength after 30 min. ...
... In order to gain supplementary information, we used also the ABTS method. This presents the possibility to monitor the reaction at different wavelengths [20,30] (the same Equation (3) was used to calculate TAC). An in the previous DPPH case, the highest value was recorded for the compound 1, followed by 3, but in this case, it was possible to obtain the TAC values for compounds 4, 8, and 9, due to the different wavelength of the ABTS absorbance. ...
4-Aminodiphenylamino derivatives were investigated for their antioxidant and hydrophobicity character, together with other biological measurements, such as antimicrobial and antibiofilm activity. Among these nine compounds used, we obtained novel derivatives via reaction of the starting material with NBD-chloride. Therefore, we performed a full structural analysis for these compounds, i.e., elemental analysis, IR, UV-Vis, 1H- and 13C-NMR, ESI-MS, X-ray diffraction on single crystal, etc. The hydrophobicity of all the compounds was measured either experimentally using the RP-TLC technique, or via calculation using the fragments method. The other structural characteristics were analyzed, and a correlation between the experimental and computed properties was found. Moreover, the results of the biological evaluation showed that some of the synthesized compounds have antimicrobial and antibiofilm activity.
... The assessment of the anti-oxidative activity of the PV herb was done using the FC reagent, purchased from Sigma-Aldrich. The FC assay is a popular standardized method in the measurement of anti-oxidative capacity of food products and dietary supplements 10 . The procedure to conduct such a test was adopted from what was proposed in an article by Ainsworth & Gillespie 11 . ...
Objective The aim is to verify our earlier suggestion which accounted for the dynamics of volatile
compounds extraction from the herb Prunella vulgaris (PV) using steam distillation. Then, the antioxidative
property of PV is explored.
Methods Because we suggested earlier that the inefficient extraction using steam distillation was due to
the obstruction of a mass of herb in the steam flow path, we used hydro distillation which tried to
eliminate this obstacle. We used GC-MS to characterize the volatile compounds extracted, and thus
compare the extraction efficiency. Then, treating the cancer cells from the SCC154 cell-line with the
distillate, the cancer cell cytotoxicity was assessed using the colorimetric test reagent, the Cell Counting
Kit-8. To assess the anti-oxidative activity of the PV distillate, Folin-Ciocalteu reagent was used.
Results We successfully showed that the removal of the obstacle, formed by the mass of herb in the
steam flow path, enhanced the efficiency of volatile compound extraction. Also, we showed that the PV
distillate did not exhibit anti-oxidative activity.
Conclusions Hydro distillation is a more efficient method than steam distillation to extract volatile
compounds from the PV herb. However, mild heating did not provide sufficient energy to the convection
of the boiling water to move the floating herb; the obstacle still existed and limited the efficiency of
extraction also. For another issue of the antioxidant effect of the volatile compounds from PV, it was
studied using the Folin-Ciocalteu reagent. It showed that the PV volatile compounds did not possess
antioxidant property.
... SET methods rely on the ability of the antioxidant to transfer an electron and reduce certain compounds and molecules while the basis of HAT methods lies on the ability of the antioxidant to scavenge reactive oxygen species (ROS) by donating a hydrogen ion from a stable molecule [24]. FRAP assay is based on SET mechanism, ORAC and BCB assays involve HAT whereas DPPH and ABTS utilize both HAT and SET [24,25]. SET-based FRAP assay measures the ability of electrondonating antioxidants to reduce ferric iron (Fe 3+ ) to ferrous ion (Fe 2+ ). ...
Background
Scientific literature has demonstrated the association of free radicals in the aetiology of various chronic diseases. Hence, the identification of potent antioxidants remains a useful task. The combination of multiple herbs in polyherbal formulations (PHF) is often associated with greater therapeutic efficacy due to synergistic interactions. However, antagonism can occur in natural product mixtures and the resultant antioxidant potential might not always be the additive value of the antioxidant properties of each component. In this study, we aimed to evaluate the phytochemicals, antioxidative potential and interaction among the herbs in TC-16, a new PHF comprising Curcuma longa L., Zingiber officinale var. Bentong, Piper nigrum L., Citrofortunella microcarpa (Bunge) Wijnands and Apis dorsata honey.
Methods
TC-16 was screened for phytochemicals. Phenolic and flavonoid contents of TC-16 and its individual ingredients were determined, followed by assessment of antioxidant properties using in vitro assays including 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and β-carotene bleaching (BCB) assays. Interactions among the herbs were also investigated by calculating the difference in antioxidant activity and combination index.
Results
Alkaloids, flavonoids, terpenoids, saponins and glycosides were present in TC-16. TC-16 possessed the highest phenolic (46.14 ± 1.40 mg GAE/g) and flavonoid (132.69 ± 1.43 mg CE/g) contents following C. longa. Synergistic antioxidant activity among the herbs was evident in ORAC and BCB assays which uses mainly hydrogen atom transfer-based antioxidant mechanisms.
Conclusions
TC-16 demonstrated roles in combating free radicals. In a PHF, synergistic interaction among the herbs is observed in some but not all mechanisms. Mechanisms showing synergistic interactions should be highlighted to maximise the beneficial property of the PHF.
... In this study, the antioxidant property of propolis extracts was determined by the DPPH free radical scavenging assay which is based on electron transfer and hydrogen atom transfer reactions (Prior et al., 2005). This method offers advantages of being rapid, simple inexpensive and provides initial information on the antioxidant capacity of the test sample (Kedare & Singh, 2011). ...
This study aims to determine the total phenolic content (TPC), total flavonoid
content (TFC) and antioxidant activity of ethanolic extract propolis (EEP) and water extract propolis (WEP) derived from six Malaysian stingless bee species. The TPC and TFC were measured by Folin-Ciocalteu colorimetric and aluminium chloride methods, respectively. The antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. The results indicated that EEP of Geniotrigona thoracica had the highest TPC and TFC at 259.84 ± 4.97 mg/g GAE and 435.00 ± 6.57 mg/g QE, respectively. Similarly, EEP of G. thoracica exhibited the greatest free radical scavenging activity with the percentage of inhibition at 88.00 ± 13.34% and IC50 of 104.20μg/mL. Antioxidant activity was highly correlated with TPC (R2 = -0.744, p < 0.05) whereas no correlation was observed between TFC and antioxidant activity. This study indicates that the antioxidant property of the propolis extract may be influenced by elevated levels of phenolic content. In conclusion, ethanolic extract of G. thoracica is the greatest source of antioxidants among the six tested Malaysian stingless bee propolis and this suggests that
the extract may serve as a promising source of antioxidant agent.
... This assay causes lipid peroxidation by generating watersoluble peroxyl radicals and is sensitive to all known antioxidants, but it is very complex and time-consuming and requires a high level of expertise and experience. 25 The table 1 shows the gradual increase among all compounds including the synergic, which represents 100 µg/ml. ...
Polyphenols, the most prevalent and naturally occurring substances have synergistic qualities that may have positive effects on human's health. When synergistic foods are combined, the evidence for health benefits is greater than when the foods are consumed separately. Nutrient deficiency is a well-known phenomenon in many people, and synergy plays a critical role in combating nutritional deficiency, chronic diseases, and infections and mainly it increases the bioavailability. This research study covers the synergistic interactions of quercetin and resveratrol, and quercetin resveratrol mixtures (1:1) were used to evaluate the TRAP, TAC, FRAP and HRSA antioxidant activity assays. The polyphenolic chemicals that are said to have synergistic effects are very powerful inhibitors of oxidation and a host of other diseases. These results observed the advantages of polyphenol chemicals when combined, which may be helpful for future research.
... Accordingly, it was found that PEF technology remarkably improved the extraction of phenolic compounds from date fruit extracts. According to Prior et al. [33], the Folin-Ciocalteu method for the determination of total polyphenolics is interfered with by many processes, such as the reduction in sugar, soluble proteins, ascorbic acid, and other substances which cause the increase in the concentration of total polyphenolics. Hence, this fact can illustrate the variation and increase in phenolic compounds of the date fruit extracts treated by PEF. ...
The objective of this study was to assess the impact of pulsed electric field (PEF) treatment on the extraction of polyphenolics and antioxidant activity from downgraded date palm fruits. The PEF pretreatments (frequency: 30 Hz, time: 50 μs, pulse number: 240, the electric field strengths were found to be 1, 2, and 2.5 kV.cm−1, and methanol (50%) and temperatures (20, 40, 50 °C)) were optimized and applied before extraction to produce an extract of date fruits with a high content of bioactive compounds. The extracts obtained immediately after pretreatments were analyzed for total polyphenolic content, antioxidant activity, and phenolic profiles. The results revealed that the PEF-assisted extracts at 2.5 kV.cm−1 at T50 °C exhibited higher polyphenol content (+27%) and antioxidant activity (+31%) and notably improved phenolic profiles compared to untreated extracts. PEF treatment processing significantly enhanced the bioactive components and antioxidant activities of date fruits over time, regardless of the treatment applied and the extraction’s temperature. Hence, the application of PEF combined with thermal processing can be an appropriate alternative treatment for a better extractability of bioactive compounds from fruit of dates and food byproducts. These biomolecules could be consumed as new food technology, incorporated as food additives, and nutraceuticals products.
... . Antioxidant capacity analyzes can be generally classi ed as electron transfer (ET) and hydrogen atom transfer (HAT) based analyzes(Prior et al. 2005). ET analyzes reduce an oxidant by single electron transfer, causing a color change (due to parameters such as ferric reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC)), which is based on an antioxidant reaction. ...
In this study, antioxidant, total monomeric anthocyanin (3-rutinoside), phenolic compounds, organic acids and vitamin C contents of fruits belonging to cornelian cherry genotypes were determined. Principal component and cluster analyzes were performed to determine the correlation between compounds. In the study, when the organic acid content of the fruits was examined, it was seen that tartaric acid and citric acid followed this acid with the highest malic acid content. It was determined that ellagic acid, catechin and chlorogenic acid, which are specific phenolics, are generally higher than other phenolic compounds. Total anthocyanin content showed great variation between genotypes and ranged from 3.79 µg cy-3-glu/g (14BL06) to 77.65 µg cy-3-glu/g. TEAC values were determined between 4.14 µmol TE/g − 11.03 µmol TE/g and FRAP values are between 3.37 µmol TE/g − 10.50 µmol TE/g. According to principal component analysis, the correlation between total antioxidant, total phenolic and total anthocyanins was 94.70%. While the correlation between phenolic compounds was determined as 42.40%, it was determined that the correlation between organic acids was 59%. As a result, it has been revealed that cornelian cherry gene sources are rich in biochemical contents and can be used effectively in functional foods.
... In this study, the antioxidant property of propolis extracts was determined by the DPPH free radical scavenging assay which is based on electron transfer and hydrogen atom transfer reactions (Prior et al., 2005). This method offers advantages of being rapid, simple inexpensive and provides initial information on the antioxidant capacity of the test sample (Kedare & Singh, 2011). ...
This study aims to determine the total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity of ethanolic extract propolis (EEP) and water extract propolis (WEP) derived from six Malaysian stingless bee species. The TPC and TFC were measured by Folin-Ciocalteu colorimetric and aluminium chloride methods, respectively. The antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. The results indicated that EEP of Geniotrigona thoracica had the highest TPC and TFC at 259.84 ± 4.97 mg/g GAE and 435.00 ± 6.57 mg/g QE, respectively. Similarly, EEP of G. thoracica exhibited the greatest free radical scavenging activity with the percentage of inhibition at 88.00 ± 13.34% and IC50 of 104.20μg/mL. Antioxidant activity was highly correlated with TPC (R2 = -0.744, p < 0.05) whereas no correlation was observed between TFC and antioxidant activity. This study indicates that the antioxidant property of the propolis extract may be influenced by elevated levels of phenolic content. In conclusion, ethanolic extract of G. thoracica is the greatest source of antioxidants among the six tested Malaysian stingless bee propolis and this suggests that the extract may serve as a promising source of antioxidant agent.
Argopuro Mountains, Tanah Wulan Village, Maesan District, Bondowoso Regency, East Java, Indonesia is one of the Robusta coffee-producing areas. Robusta coffee beans that are processed can produce quite a lot of by-products in the form of underutilized waste. It is known that coffee skin waste still has a lot of remaining content and it is possible to take these bioactive compounds using extraction methods. Bioactive compounds in natural materials can be done by extraction. Extraction is the process of separating substances in a sample based on different solubilities. The extraction method used is the ultrasonication method (nonconventional). Extraction of the ultrasonication method only requires a relatively shorter time with the help of an ultrasonicator. There is a lot of content in the coffee skin waste powder that needs further testing. The results of this study are expected to provide economic value to robusta coffee husk waste.
A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.
The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic-acid) (ABTS) radical can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase. This ABTS radical (ABTṠ+), a chromogen, is stable at room temperature but is unstable above 35°C and/or at pH values of above 7.5. Nevertheless, the most important factor in its stability is the ABTS/ABTṠ+ concentration ratio in the medium. The radical reacts with the antioxidant, L-ascorbic acid, with a high rate constant, the stoichiometry of the reaction being 1 mol of L-ascorbic acid per 2 mol of ABTṠ+ reduced. Based on these considerations, a spectrophotometric end-point method has been developed to evaluate L-ascorbic acid in aqueous media, and this represents an improvement over the lap-method previously reported. Under optimal conditions of temperature, pH and reagent concentration, the end-point method was capable of determining L-ascorbic acid with a limit of quantification of 0.38 nmol. In the assay described here, this ability is used to evaluate the total antioxidant activity of commercial citrus juices, in which ascorbic acid is a principal component. In our opinion this procedure can quickly provide useful information on the antioxidant content of foods and plant extracts.
Quantitative determinations of the four black currant anthocyanins, cyanidin 3-O-beta-glucoside, cyanidin 3-O-beta-rutinoside, delphinidin 3-O-beta-glucoside, and delphinidin 3-O-beta-rutinoside, were achieved in black currant juices by a rapid and sensitive high-performance liquid chromatographic (HPLC) method. The method was validated, and quantification of anthocyanins in 13 commercially available black currant beverages was demonstrated. To optimize the handling of anthocyanin-containing samples, the pH-dependent stability of the anthocyanins was investigated. Four anthocyanins were incubated for 24 h in aqueous solutions at 13 different pH levels between 0.6 and 5.2, after which the samples were analyzed by HPLC. More than 90% of each anthocyanin remained intact up to pH 3.3. At pH 3.8 a local minimum in stability was detected, and at pH >4.5 the stability rapidly decreased. The antioxidant capacity of all 13 black currant juices was investigated by TEAC and FRAP, and the antioxidant potential of
Hydroxylated and polyhydroxylated aromatic and heterocyclic compounds may have antioxidant and anticarcinogenic properties. Whether an antioxidant is an anticarcinogen may depend on its efficacy as an oxygen radical (peroxyl, alkoxyl, superoxide, hydroxyl) inactivator and inhibitor. Location, concentration in situ, reaction kinetics (rate constants), energetics (redox potentials), and products (intermediates and final) contribute to the efficacy of an antioxidant. The kinetics and energetics of an antioxidant are governed by the type and position of the substituents. The rate constant for the reaction of an antioxidant and an oxy radical depends on the type of radical. In general, the reactivity of a radical decreases in the following order: hydroxyl > alkoxyl > peroxyl > superoxide. One-electron oxidation potential of an antioxidant at pH 7, E7 , can be calculated from the Hammett correlation using Brown substituent constants.
Publisher Summary Several methods have been developed to assess the total antioxidant capacities of various biological samples, particularly complex matrices such as plasma, serum, wine, fruits, vegetables, and animal tissues. This chapter presents a method called “oxygen radical absorbance capacity” (ORAC) assay based largely on the work reported by Glazer's laboratory, which depends on the unique properties of phycoerythrin (PE). The ORAC assay is the only method that takes reactive species (RS) reaction to completion and uses an “area under the curve” (AUC) technique for quantitation, thus combining both inhibition time and inhibition percentage of the RS action by antioxidants into a single quantity. The chapter discusses the general principles of ORAC assay for assessing antioxidant capacity against peroxyl radicals. By integrating inhibition percentages over the whole inhibition time period, the ORAC assay successfully overcomes all related problems in quantitation of the antioxidant capacity of a biological sample. Either B- or R-phycoerythrin (B-PE or R-PE) can be used in the ORAC assay. The sensitivity of B- or R-PE to hydroxyl radical damage may be different even for the same PE with different lot numbers. The concentrations of Cu 2+ and standard (Tro lox) can be adjusted, when it is necessary. The aforementioned procedures are based on using B- or R-PE that loses more than 90% of its fluorescence within 30 rains. The chapter concludes with a discussion of ORAC assay for assessing antioxidant capacity against transition metals.