Sample Dilution Influences the Determination of Antioxidant Capacity in Food: How to Minimize It?

The influence of sample dilution on the measurement of antioxidant capacity was analyzed. To ensure the reproducibility of results, it is necessary to realize such scarce investigations. This study focuses on different antioxidant capacity assays commonly used for the analysis of pure substances and food extracts. For all compounds and foods tested in most of the four assays (Trolox equivalent antioxidant capacity, 2,2-diphenyl-1-picrylhydrazyl, and oxygen radical absorbance capacity), effects of sample dilution on the measured (and recalculated) antioxidant capacity were observed, with differences up to 28 % between dilutions. An extrapolation method was proposed to obtain a “real value” thus to minimize the effects of the sample dilution. This extrapolation method is relatively simple, based on a linear regression of 4 or 5 appropriate dilutions of the sample and applicable to the various assays. The use of such a method will improve the consistency of interlaboratory antioxidant capacity data and thus permit better comparisons. In contrast, there was no dilution problem with ferric reducing antioxidant power assays.     

Antioxidant capacity,total phenolic and ascorbate content as a function of the genetic diversity of leek (Allium ampeloprasum var. porrum)

Extracts of the white shaft and green leaves of 30 leek cultivars were investigated for their antioxidant properties, total phenolic (TP) and l-ascorbic acid (AA) content. The measured antioxidant properties included free radical scavenging activities against peroxyl (ORAC) and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) and their Fe³⁺ reducing capacity (FRAP). The results from this study suggest that the green leek leaves generally have significantly stronger antioxidant properties than the white shaft. Correlation analysis between the TP and the AA content and the antioxidant activity showed that phenolics and ascorbic acid contribute significantly to the antioxidant activity of leek. The three antioxidant activity assays were all correlated for the extracts of the white shaft of the 30 leek cultivars. Principal component analysis (PCA) elucidated the influence of part and type of cultivar on the antioxidant capacity, TP, and l-ascorbic acid content, whilst the breeding strategy and seed company had no influence.     

A simplified UV spectral scan method for the estimation of phenolic acids and antioxidant capacity in eggplant pulp extracts

The objective of this study was to determine if a simple UV spectral analysis method can be used as a screening tool to estimate the amount of phenolic acid and the antioxidant capacity of eggplant pulp extracts. Calibration curves for different concentrations of 5-O-caffeoylquinic acid standards were developed using UV spectral data, HPLC analysis, and ferric reducing antioxidant power (FRAP) assay. Seven different freeze-dried eggplant pulp samples belonging to different cultivars or grown under different environmental conditions along with single peel sample were selected as model substrates for this study. HPLC results, simple UV spectral scan data, and quantification of antioxidant capacity by FRAP assay in seven eggplant pulp extracts showed a strong correlation (R² > 0.9) between the three methods. These results suggest that the single absorption reading or a simple UV spectral scan can be used to estimate the concentration of phenolic acids and the antioxidant capacity in eggplant pulp extracts without peels. This screening tool can be potentially used to identify cultivars and growing conditions that influence phenolic acid content in eggplant samples.     

Antioxidant activity of the ethanolic extract from the bark of Chamaecyparis obtusa var. formosana

BACKGROUND: Chamaecyparis obtusa var. formosana (Taiwan hinoki) is an endemic conifer in Taiwan and the purpose of this study is to evaluate the antioxidant activity of various fractions obtained from the bark of this plant material. The ethanolic extract of the bark was sequentially separated into three fractions, including n‐hexane, ethyl acetate and ethanol soluble fractions, by liquid–liquid partition. Then the antioxidant activities of crude extract and three fractions along with 13 subfractions obtained from the ethyl acetate (EA) soluble fraction were tested for several antioxidant assays. RESULTS: The total phenolic content of the samples varied from 27.71 to 102.86 mg GAE g^?1 dry weight for fractions, and from 49.94 to 206.46 mg GAE g^?1 for subfractions (where GAE is milligrams of gallic acid per gram of extract). The Trolox equivalent antioxidant capacity (TEAC) ranged from 0.15 to 0.26 mmol L^?1 Trolox equivalents. The EA soluble fraction was found to be the best antioxidant‐rich fraction in terms of DPPH and reducing power assays. With further data analysis it was found that there was a positive correlation between the total phenolic content of extracts and TEAC is R² = 0.61. CONCLUSION: Results from various antioxidant assays showed that the EA fraction possessed strong antioxidant activity. This would provide additional information about the antioxidant activity of bark extract of this plant species. Copyright © 2008 Society of Chemical Industry     

Antioxidant activity and phenolic compounds of ginger (<Emphasis Type="Italic">Zingiber officinale</Emphasis> Rosc.) determined by HPLC-MS/MS

Oxidative stress related diseases often arise from over production of free radicals and reactive oxygen/nitrogen species. The prevention of these diseases could be possible with the use of natural antioxidant plants that could be promising as therapeutic candidates. Since antioxidant properties of a species could be stem from phenolic compounds, it is, therefore, important to evaluate antioxidant and total/individual phenolic and flavonoid content. For this purpose, we evaluated antioxidant properties of ginger (Zingiber officinale Rosc.) based on three parameters: the antioxidant capacity, total phenolic and flavonoid content as well as identification of phenolic acids of water extract (WEG) and ethanol extract (EEG) of ginger. For antioxidant capacity, we performed FRAP, CUPRAC assay, Fe²⁺ chelating ability, DPPH and DMPD radical scavenging activities. Also, total phenolic and flavonoid contents in both extracts were also measured via Folin Ciocalteu’s method. For identification of phenolic acids, HPLC-MS/MS method was performed. The results showed that EEG had generally better antioxidant activity than WEG in all assays. HPLC-MS/MS analysis showed that there are at least eight different phenolic acids found in ginger, among which pyrogallol p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were more abundant in both extracts. This study clearly showed that ginger extracts demonstrated effective antioxidant properties and their consumption may reduce or delay the progression of diseases that oxidative stress take place due to lack of antioxidant supplementation.     

Assessment of antioxidant capacity of sedum (<Emphasis Type="Italic">Sedum sarmentosum</Emphasis>) as a valuable natural antioxidant source

In order to investigate the antioxidant capacity of sedum (dolnamul, Sedum sarmentosum) and to offer a scientific basis for the medicinal use of it, lyophilized sedum leaves were extracted with n-hexane, n-butanol, ethyl acetate, and methanol, respectively. The methanol extract (ME) showed the highest extraction yield, but no significant differences in the extraction yield were observed. The total polyphenol content (TPC) of the ethyl acetate extract (EAE) and the ME was significantly higher than that of the non-polar solvent extracts. The EAE and ME showed the superior antioxidant activities in the DPPH, oxygen radical absorbance capacity (ORAC), and ferric reducing ability of plasma (FRAP) assays. Lipid peroxidation was efficiently inhibited by the addition of the sedum extracts in the ferric thiocyanate (FTC) and thiobarbituric acid reactive substances (TBARS) assays, but significant differences among the tested extracts were not observed. The ME showed the potent lipid peroxidation inhibitory activity comparable to butylated hydroxytoluene (BHT). The correlations between the TPC and the antioxidant activities based on the DPPH, ORAC, and FRAP assays were highly positive (R ²>0.899).     

Comparative antioxidant capacities of phenolic compounds measured by various tests

The purpose of this study was to compare the antioxidant capacities of standard compounds (phenolic compounds, ascorbic acid, and glutathione) as measured by various assays. Five methods were selected so as to span a diversity of technical approaches: TEAC (radical 2,2′-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid), DPPH (radical 2,2-diphenyl-1-picryhydrazyl used to measure reducing capacity), ORAC (oxygen radical scavenging capacity), red blood cell haemolysis (protection of biological sample), and ESR (electron spin resonance for direct free radical evaluation). Most compounds showed significant differences in free radical scavenging activity according to the method used. Of the 25 tested compounds, only a few, such as myricetin and gallocatechin, gave comparable activities in the various tests. To standardise reporting on antioxidant capacity, it is proposed to use a weighted mean of the values obtained using the DPPH, ORAC, resistance to haemolysis, and ESR assays.     

Effect of Extrusion Processing on Antioxidant Activities of Corn Extrudates Fortified with Various Chinese Yams (<Emphasis Type="Italic">Dioscorea</Emphasis> sp.)

The purpose of this experiment was to evaluate the effects of extrusion on antioxidant abilities of the extrudates of corn fortified with various (Chinese) yams. The flours from three yam varieties were used, including Dioscorea alata L. var. Tai-nung No.1 (TN1), D. alata L. var. Ta-shan (TS), and Dioscorea doryophora var. Hang-chun (HC). One commercial yam flour (TJ) was also used. Six antioxidant assays were conducted, such as 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging effect, oxygen radical absorbance capacity (ORAC) assay, ferrous ion chelating (FIC) ability, 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radical scavenging activity, superoxide radical scavenging (SORS) assay, and thiobarbituric acid reactive substances (TBARS) assay. The influences of extrusion on the antioxidant activities of corn were determinedly different in different assays, showing a dramatic increase of ABTS value but significant reductions of SORS and TBARS values. The effects of extrusion on antioxidant activities of corn–yam mixtures varied with yams in different assays. Overall, extrusion processing increased the TBARS inhibition ability for all yams and generally had no negative impact on DPPH, ORAC, FIC, and ABTS antioxidant abilities for most yams in corn–yam extrudates. The involvement of the addition of yam to the antioxidant activities of corn–yam extrudates during extrusion could be suggested through two different possible approaches, the effects of extrusion on the antioxidant activities of yams (susceptible, resistant, or improved), and the effects of added yams on the antioxidant activities of corn during extrusion.     

Quantification of the polyphenolic fraction and in vitro antioxidant and in vivo anti-hyperlipemic activities of Hibiscus sabdariffa aqueous extract

In the present study the quantification of the polyphenolic fraction, anthocyanins and other polar compounds, the antioxidant capacity and the anti-hyperlipemic action of the aqueous extract of Hibiscus sabdariffa has been achieved. Seventeen compounds were successfully quantified either by HPLC-DAD or HPLC-ESI-TOF-MS. Six of them were directly quantified by their corresponding standards, whereas the rest were indirectly quantified as equivalents using standards of similar compounds. The antioxidant capacity have also been estimated by comparing different assays, i,e, Trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and measurement of thiobarbituric acid reacting substances (TBARS). H. sabdariffa showed high reducing capacity in FRAP assay and significant capability to scavenge peroxyl radicals in the ORAC assay. Nevertheless, the extract exhibited poor efficacy to inhibit peroxyl radicals in lipid systems. The plant extract also exhibited the capacity to decrease serum triglyceride concentration on hyperlipemic mouse model.     

Effects of iron,ascorbate, meat and casein on the antioxidant capacity of green tea under conditions of in vitro digestion

The hypothesis that interactions of dietary polyphenols with dietary iron occur during digestion and result in a decrease of the post-absorptive antioxidant properties of polyphenols was investigated. The hypothesis was tested in vitro, under conditions that simulate gastrointestinal digestion. Mixtures of green tea, iron, and three dietary factors that modify the form of iron in the lumen, namely ascorbic acid, meat or casein, were subjected to an in vitro gastrointestinal digestion. Antioxidant capacity (FRAP assay), iron concentration (ferrozine assay) and polyphenol concentration (Folin–Ciocalteau assay) were measured in the in vitro digests. The presence of iron decreased the antioxidant capacity and the polyphenol concentration of green tea digests. The presence of ascorbic acid increased, while meat and casein decreased the antioxidant capacity of green tea. The factorial analysis of the data suggests that protein and iron interact with green tea polyphenols during the in vitro digestion and decrease their antioxidant capacity. These results support the aforementioned hypothesis.     

Iron decreases the antioxidant capacity of red wine under conditions of in vitro digestion

The hypothesis that iron and phenolics interact in the lumen during digestion and, consequently, decrease the antioxidant capacity of phenolics, was investigated in vitro. Mixtures of red wine, iron, and three dietary factors that may reduce or chelate iron in the lumen, namely ascorbic acid, meat and casein, were subjected to a simulated gastrointestinal digestion. The process involved incubation of samples for 4.5 h at 37 °C, at different pHs, in the presence of peptic enzymes and fractionation of digests through a dialysis membrane. Antioxidant capacity (FRAP assay), iron concentration (ferrozine assay) and total phenolic content (Folin-Ciocalteau assay) were measured in the in vitro digests. Iron decreased the antioxidant capacity and the total phenolic concentration of red wine. Ascorbic acid increased, while meat and casein decreased, the antioxidant capacity of red wine. Based on these results, it was concluded that protein and iron interact with red wine phenolics during the in vitro digestion and decrease their antioxidant capacity, supporting the initial hypothesis.     

Effects of Spray‐Drying Parameters on In Vitro Functional Properties of Camu‐Camu (Myrciaria dubia Mc. Vaugh): A Typical Amazonian Fruit

Camu‐camu (Myrciaria dubia) fruit is a rich source of bioactive compounds but its shelf life is rather short. Therefore, this study was aimed to evaluate the effect of inlet air temperature (T) and concentration (C) of maltodextrin and arabic gum on the spray‐drying process of commercial camu‐camu pulps (São Paulo and Manaus). Moisture, solubility, total phenolics (TP), ascorbic acid (AA), and proanthocyanidins (PAC) contents, and in vitro antioxidant capacity of the powders (FRAP, DPPH, Folin–Ciocalteu's reducing capacity were measured). Arabic gum resulted in better yields (22% to 30%), powder solubility (84% to 90%), and lower losses of analyzed compounds than the powders manufactured with maltodextrin. Overall, inlet air temperature had a lower impact on the responses studied than the concentration of carrier agents. Polynomial equations were generated for AA (R² = 0.993), TP (R² = 0.735), PAC (R² = 0.946), and for the antioxidant capacity assays (0.867 ≤ R² ≤ 0.963). In addition, principal component analysis showed that the lowest concentration of carrier agents (6%) in spray drying resulted in the lowest losses of bioactive compounds and, consequently, the highest antioxidant capacity.     

Potential of nanofiltration for the concentration of bioactive compounds from watermelon juice

The aim of this study was to evaluate the potential of nanofiltration for the concentration of bioactive compounds of watermelon juice. Lipophilic and hydrophilic antioxidant activities were determined through two assays, ferric reducing antioxidant power and DPPH. The content of lycopene, flavonoids and total phenolic in the concentrate samples increased with the increase in the volume reduction factor. Volume reduction factor of three showed the best performance of concentration, generating the highest values for lycopene, flavonoid and total phenolic contents. Average permeation flux was 2.3 L h^?1 m^?2, with continuous extraction of the concentrate at a volumetric reduction ratio of three. Lycopene showed the highest rejection coefficient (0.99), followed by flavonoids (0.96) and total phenolic content (0.65). The hydrophilic antioxidant activity values in both assays were higher than the lipophilic antioxidant activity values. A highly significant correlation was noted between the contents of flavonoids, phenolic compounds, ascorbic acid, and lycopene and their antioxidant potential in both lipophilic and hydrophilic fractions.     

ORAC and TEAC assays comparison to measure the antioxidant capacity of food products

Oxygen radical antioxidant capacity (ORAC) and trolox equivalent antioxidant capacity (TEAC) assays were compared to estimate the total antioxidant capacity (TAC) of orange juice, milk, and an orange juice-milk beverage. When the TEAC method was used with this beverage, an increase in the concentration of orange juice corresponded to an increase in TAC, but increasing the percentage of milk did not increase the TAC value. When the ORAC method was applied, it was seen that increased concentrations of juice or milk corresponded to greater antioxidant capacity. An evaluation was also made of the influence of certain compounds (ascorbic acid, gallic acid, β-carotene, lutein, zeaxanthin and albumin) with antioxidant capacity that were present in the samples studied.     

Antioxidant properties of durian fruit as influenced by ripening

The antioxidant properties of durian (Durio zibethinus Murr., cv. Mon Thong) at different stages of ripening were investigated using fluorometry, UV spectroscopy, and HPLC/DAD analyses. Total polyphenols, flavonoids, anthocyanins and flavanols in ripe durian were significantly higher (p < 0.05) than in mature and overripe fruits. Free polyphenols and flavonoids were at lower levels than hydrolyzed ones. Caffeic acid and quercetin were the dominant antioxidant substances in ripe durian. In these fruits, methanol extracts contained a relatively high capacity of 74.9 ± 7.1% inhibition using β-carotene-linoleic acid assay. Ferric-reducing/antioxidant power (FRAP) and cupric-reducing antioxidant capacity (CUPRAC) assays supported this finding. The correlation coefficients between polyphenols and antioxidant capacities of durian samples with all applied assays were about 0.98. In conclusion, the bioactivity of ripe durian was high and the total polyphenols were the main contributors to the overall antioxidant capacity.     

Antioxidant activity of Portuguese honey samples: Different contributions of the entire honey and phenolic extract

The antioxidant activity of Portuguese honeys was evaluated considering the different contribution of entire samples and phenolic extracts. Several chemical and biochemical assays were used to screen the antioxidant properties of entire honeys with different colour intensity and phenolic extracts: reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging capacity, and inhibition of lipid peroxidation using the β-carotene linoleate model system and the thiobarbituric acid reactive substances (TBARS) assay. The amounts of phenols, flavonoids, ascorbic acid, β-carotene, lycopene and sugars present in the samples were also determined. The highest antioxidant contents and the lowest EC₅₀ values for antioxidant activity were obtained in the dark honey. An analysis of variance was carried out to evaluate the influence of the colour intensity and extraction method in the antioxidant properties and phenolic contents. A discriminant analysis was also performed, giving satisfactory results once the six samples were clustered in six individual groups obtained through the definition of two discriminant analysis dimensions.     

Phenolic acid composition and antioxidant capacity of acid and alkali hydrolysed wheat bran fractions

Phenolic acid concentrations, profiles and antioxidant capacity of acid and alkali hydrolysates from the bran of six wheat cultivars representing six Canadian market classes were determined. Aqueous ethanol was used to extract the free phenolics (FP) and diethyl ether to extract the insoluble bound phenolics released after acid and alkaline hydrolysis of the bran. Folin–Ciocalteu (FC) reagent was used to estimate the total phenolic content and HPLC-UV to detect and quantitate 14 phenolic acids and one lignan. trans-Ferulic acid was the dominant acid in the bran extracts but mass spectrometric analysis showed tryptophan to be dominant in the FP extracts. The antioxidant capacity of individual phenolic acids and extracts was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent antioxidant assays. The FP extracts had the lowest average antioxidant capacity and the hydrolysates the highest. Based on the concentration of each phenolic acid in the extracts, and the antioxidant capability of each phenolic standard, trans-ferulic acid was the dominant contributor (66.4–95.5%) to antioxidant capacity of the wheat bran extract.     

Effect of peptide–phenolic interaction on the antioxidant capacity of walnut protein hydrolysates

Walnut antioxidant peptides and phenolic compounds (POHs) commonly coexist in walnut protein hydrolysates (WPHs). However, the peptide–phenolic interaction and its effects on antioxidant activities of WPHs remain unknown. Fluorescence and dynamic light scattering were used to study the peptide–phenolic interaction in WPH‐POH mixtures (W‐P) with different POH/WPH ratios; antioxidant abilities of POH only solution, WPH only solution and W‐P were measured with three assays: DPPH radical scavenging, Fe²⁺ chelation and linoleic acid peroxidation inhibition. Results showed that initially POHs bonded to hydrophobic sites of peptides; subsequently, when POH/WPH ratio exceeded 1:120 (w/w), POHs changed the conformation and average particle size of peptides. Their interaction could at most increase WPHs’ capability in scavenging DPPH radical (44.9%), chelating Fe²⁺ (36.1%) and inhibiting linoleic acid oxidation (87.5%) at the POH/WPH ratio (w/w) point of 1:12. This research offered a guidance in the production of WPHs with high functionality.     

Comparison of antioxidant capacity of cow and ewe milk kefirs

This research aimed to evaluate the effects of using either grain or commercial starter culture on the antioxidative capacity of cow and ewe milk kefirs. The antioxidant capacity of kefir samples during fermentation and 21 d of storage was assessed by using 3 assays: 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation decolorization; 2,2-diphenyl-1-picrylhydrazyl (DPPH^?) radical scavenging activity assay; and Fe⁺³-reducing power (ferric reducing antioxidant power assay, FRAP). Vitamin E and β-carotene contents were also quantified. All kefir samples exhibited varying values for DPPH, ABTS, and FRAP assays depending on the starter culture and milk type. Vitamin E and β-carotene contents were similar in all kefir samples during storage. The results of this study suggest that milk type (cow or ewe) and culture type (kefir grains or commercial starter) were the significant parameters for the antioxidative activity of kefir.     

Cytoprotective effects of polyphenolics on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cell death in SH-SY5Y cells in relation to their antioxidant activities

The cytoprotective effects of five flavonoids (quercetin, rutin, catechin, kaempferol and morin) and four hydroxycinnamic acids (caffeic, ferulic, sinapic and chlorogenic acids) were evaluated by the degree of protection they provided against H₂O₂-induced damage to human neuroblastoma SH-SY5Y cells. All compounds exhibited protection against H₂O₂-mediated cytotoxicity in a dose dependent manner. The concentration required to give a 50% reduction in cell death (EC₅₀ value) were derived from their dose–response relationships. The cytoprotective activities of these phenolic compounds in the order of quercetin > caffeic acid > rutin > chlorogenic acid > catechin > ferulic acid > sinapic acid > morin > kaempferol. The EC₅₀ values of the phenolic compounds were strongly related to their chemical structures. The EC₅₀ values were compared with the antioxidant activities as determined by five different chemically based antioxidant assays [2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP) and lipid peroxidation inhibition capacity (LPIC)]. The ability of these phenolic compounds to protect from H₂O₂-induced SH-SY5Y cell death correlated (r ² = 0.85) with their determined LPIC values and weakly (r ² = 0.44) with their ABTS activities. There was no correlation between EC₅₀ values and ORAC, FRAP or DPPH activities. The cytoprotection assay is a more biologically relevant measurement than the chemically defined antioxidant activity assays because it uses human cells as a substrate and therefore accounts for some aspects of uptake, metabolism and location of antioxidant compounds within cells.