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The ABTS-microzone plate and scanometry as a simple and fast colorimetric assay for determining the total antioxidant capacity of plant extract
Abstract
The 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS)-microzone plate as an antioxidant sensor was developed by immobilizing the ABTS reagent onto the screen-printed paper. The individual circular microzone, served as a sensing zone has a blue color that will decolorize within antioxidant addition in the concentration-dependent favor. The scanometric detection system that uses a flatbed scanner and color analysis program like ImageJ was applied to the antioxidant sensor, while its analytical performance was optimized by using quercetin. The use of the pre-generated ABTS radical for constructing the paper-based sensor, as well as the application of the scanometric method, is the novelty of this study. The result showed that the sensor showed a linear signal (r = 0.9997) within quercetin addition (0.1–1.0 mM) at 8 min with the detection limit (LOD) of quercetin at 0.03 mM and presented good precision (RSD < 3%) and accuracy (98–101% recovery) in total antioxidant capacity (TAC) determination. TAC of various plant extracts was successfully determined (as mM quercetin equivalent) by using the antioxidant sensor, while the result was parallel with that determined by the spectrophotometric method, suggesting the feasibility of the proposed method as an alternate method for TAC determination.
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A simple paper-based colorimetric biosensor based on immobilized tyrosinase and 3-methyl-2-benzothiazolinone hydrazone (MBTH) was developed for assessing total polyphenol content (TPC) of various green tea beverages. The patterned biosensing zone of the paper-based biosensor showed a sensitive response to catechin (a typical green tea polyphenol) by generating pink color adducts that can be captured for further color image analysis using a scanometric method. This color change can be connected to the TPC of the samples. The analytical performance of the biosensor in scanometric set-up was optimized. The biosensor showed a response time of 13 min in the linear range between 0.08-1.03 mM of catechin (r = 0.9979). The detection limit (LOD) of the biosensor was 0.071 mM while reproducibility was found at 3.11% RSD (relative standard deviation). TPC of green tea beverages was assessed by the biosensor, and the results were in accordance with the UV/Vis spectrophotometric method.
The current work was aimed to develop the paper microzone plates (PµZP) as a scanometric system for determining the total antioxidant capacity of plant extracts. The PµZP was constructed by immobilizing CUPRAC reagent onto 70-well of patterned paper as a sensing zone for the scanometric detection system. The light blue-sensing zone showed a sensitive response to standard antioxidant (rutin) by forming light yellow color adducts which can be scanned and quantitatively measured by image processing program as a scanometric method. The sensor gave a notable signal at 8 min after rutin addition and presented a linear response in the concentration span of 1–10 mM. The developed method was shown to be reproducible (RSD < 3%) and accurate (98–101% recovery) for determining the TAC of plant extracts (as mM rutin equivalents) and showed strong correlation (r = 0.9887) with the standard CUPRAC method, suggesting that it can be applied for the simple and rapid method for TAC determination.
We report on a paper-based 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay for a simple, inexpensive, low reagent and sample consumption and high throughput analysis of antioxidant activity. The paper-based device was fabricated using a lamination method to create a 5-mm in diameter circular test zone that was embedded with a DPPH reagent. The analysis was carried out in one-step by dropping an antioxidant/sample onto the test zone. After reduction by the antioxidant, the DPPH radicals become stable DPPH molecules, resulting in a change in color from deep violet to pale yellow. The violet color intensity of DPPH was inversely proportional to the antioxidant activity of the samples, and was measured using imaging software. A high precision and a low limit of detection were found in the analysis of six standard antioxidants including gallic acid, trolox, ascorbic acid, caffeic acid, vanilliic acid and quercetin. The device was then validated against the traditional spectrophotometric DPPH assay by analyzing the antioxidant activity of 7 tea samples. The results showed no significant difference for gallic acid equivalent for all 7 samples obtained from the two methods at the 95% confidence level, indicating that the developed method was reliable for antioxidant activity analysis of real samples. Finally, the paper-based DPPH device was found to be stable over 10 days when stored in a refrigerator (2 – 4°C), making it an easy-to-use device for end-users.
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A simple scanometric assay for antioxidant capacity of the herbal extracts has been developed based on dry reagent of 2,2-diphenyl-1-picrylhydrazyl (DPPH) immobilized on the pharmaceutical blister. The deep violet color of DPPH solution was regenerated by introducing methanol to each well as sensing zones. The antioxidant capacity of plant extract will proportionally reduce the violet color to form the corresponding yellow color adduct, where the color change was captured by a flatbed scanner. Then, the optical responses toward antioxidant are presented as an averaged value of the red green blue (RGB) color using image color analysis (ImageJ). By using this color value, analytical performance of the antioxidant sensor could be characterized, where it has a linear range between 1-28 mg/L as gallic acid equivalent (GAE), with response time at 10 min. The reproducibility of the antioxidant sensor was good (RSD < 1%) with the recovery value of 102-105%. The scanometric assay was applied to several herbal extracts, such as sappan wood, guava leaf, and turmeric rhizome. The results obtained are in good agreement with the spectrophotometric method, suggesting that the proposed assay can be used as an alternative method for antioxidant capacity determination.
Background
An HPLC method employing a post-column derivatization strategy using the cupric reducing antioxidant capacity reagent (CUPRAC reagent) for the determining antioxidants in plant-based materials leverages the separation capability of regular HPLC approaches while allowing for detection specificity for antioxidants. Methods
Three different column types, namely core-shell and porous silica including two chemically different core-shell materials (namely phenyl-hexyl and C18), were evaluated to assess potential improvements that could be attained by changing from a porous silica matrix to a core-shell matrix. Tea extracts were used as sample matrices for the evaluation specifically looking at catechin and epigallocatechin gallate (EGCG). ResultsBoth the C18 and phenyl-hexyl core-shell columns showed better performance compared to the C18 porous silica one in terms of separation, peak shape, and retention time. Among the two core-shell materials, the phenyl-hexyl column showed better resolving power compared to the C18 column. Conclusions
The CUPRAC post-column derivatization method can be improved using core-shell columns and suitable for quantifying antioxidants, exemplified by catechin and EGCG, in tea samples.
The stable chromogenic radical 1,1 0-diphenyl-2-picrylhydrazyl (DPPH) solution was immobilized on the microwell plate as dry reagent to construct a simple antioxidant sensor. Then, a regular flatbed scanner was used as microplate reader to obtain analytical parameters for antioxidant assay using one-shot optical sensors as scanometry technique. Variables affecting the acquisition of the images were optimized and the analytical parameters are obtained from an area of the sensing zone inside microwell using the average luminosity of the sensing zone captured as the mean of red, green, and blue (RGB) value using ImageJ s program. By using this RGB value as sensor response, it is possible to determine antioxidant capacity in the range 1–25 ppm as gallic acid equivalent (GAE) with the response time of 9 min. The reproducibility of sensor was good (RSDo1%) with recovery at 93%–96%. The antioxidant sensor was applied to the plant extracts, such as sappan wood and Turmeric Rhizome. The results are good when compared to the same procedure using a UV/Vis spectrophotometer.
The chemical diversity of natural antioxidants (AOXs) makes it difficult to separate, detect, and quantify individual antioxidants from a complex food/biological matrix. Moreover, the total antioxidant power is often more meaningful to evaluate health beneficial effects because of the cooperative action of individual antioxidant species. Currently, there is no single antioxidant assay for food labeling because of the lack of standard quantification methods. Antioxidant assays may be broadly classified as the electron transfer (ET)- and hydrogen atom transfer (HAT)-based assays. The results obtained are hardly comparable because of the different mechanisms, redox potentials, pH and solvent dependencies, etc. of various assays. This project will aid the identification and quantification of properties and mutual effects of antioxidants, bring a more rational basis to the classification of antioxidant assays with their constraints and challenges, and make the results more comparable and understandable. In this regard, the task group members convey their own experiences in various methods of antioxidants measurement.
Background: Triterpenoids are multifunctional secondary metabolites in plants. But little information is available concerning the actual yield, optimal extraction method and pharmacologic activity with regard to triterpenoids from Jatropha curcas leaves (TJL). Hence, response surface methodology (RSM) was used to optimize the extraction parameters. The effects of three independent variables, namely liquid-to-solid ratio, ethanol concentration and extraction time on TJL yield were investigated. TJL obtained by silica column chromatography was tested against bacterial and fungal species relevant to oral disease and wounds through broth microdilution. Antioxidant activity was assessed using the 2,2-diphenyl-2-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays.
Results: A second order polynomial model produced a satisfactory fitting of the experimental data with regard to TJL yield (R2 = 0.983, P
A sensitive and selective amperometric biosensor for evaluation of total phenolic content (TPC) was developed by immobilizing laccase (Lac) on the glassy carbon electrode modified with matrix nanocomposites. The nanocomposites composed of NH2 -functionalized carbon nanotubes (CNT-NH2), gold nanoparticles (AuNPS) and Bovine serum albumin (BSA). In order to increase the stability of the biosensor Lac was covalently immobilized in nanocomposites matrix by using glutaraldehyde (Glu). The rapid screening method of TPC assay was proposed based on amperometric detection of gallic acid on the laccase-based biosensor in flow injection analysis. The developed biosensor GC/CNT-NH2/AuNPs/Lac-BSA shows an excellent activity for gallic acid. The developed system also demonstrates good stability of the immobilized enzyme in spite of the hydrodynamic conditions. The system provides an impressively good precision (%RSD =3.1) for 20 L injections of 6 µM gallic acid (n = 30). Under the flow rate used of 1.5 mL min-1, throughput of sample is 42 samples h-1. The method was successfully applied to TPC assay in plant extracts and tea infusions.
The effects of guava leaves extracted using solvents of water, ethanol, methanol, and different concentrations of hydroethanolic solvents on phenolic compounds and flavonoids, and antioxidant properties have been investigated. The antioxidant capability was assessed based on 2,2-diphenyl-1-picrylhydrazyl radical and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging abilities, reducing power, and nitric oxide-and nitrate-scavenging activities. The results demonstrated that the antioxidant ability of guava leaf extracts has a strong relationship with phenolic compound content rather than flavonoid content. Phenolic compound content of water extracted guava leaves was higher compared to pure ethanol and methanol extracts. However, phenolic compound content extracted using hydroethanolic solvent was higher than water, whereas 50% hydroethanolic was observed to be the most effective solvent showing high antioxidant ability.
Antioxidant activities (μmol Trolox equivalent (TE)/g fresh weight) of 19 sweet potato genotypes with distinctive flesh colour (white, cream, yellow, orange and purple) were measured by oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS). Total phenolics were measured using the Folin–Ciocalteau method, total anthocyanins by the pH-differential method, and β-carotene by HPLC. The total antioxidant activity (hydrophilic + lipophilic ORAC) was highest (27.2 μmol TE/g fresh weight (fw)) for NC415 (purple-fleshed) and lowest (2.72 μmol TE/g fw) for Xushu 18 (white-fleshed). The hydrophilic-ORAC values were significantly correlated with the DPPH (R2 = 0.859) and ABTS (R2 = 0.761) values. However, the lipophilic-ORAC values were poorly correlated with the β-carotene contents (R2 = 0.480). The total phenolic contents (0.011–0.949 mg chlorogenic acid equivalent/g fw) were highly correlated with the hydrophilic-ORAC (R2 = 0.937) and DPPH (R2 = 0.820) values. Therefore, the total phenolic content can serve as a useful indicator for the antioxidant activities of sweet potatoes.
In the present study, we tried to establish an efficient assay for total antioxidant capacity (TAC) in human plasma using a 96-well microplate. TAC was assessed using lag time by antioxidants against the myoglobin-induced oxidation of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) with hydrogen peroxide, and expressed as Trolox equivalent. The linearity of the calibration curve with Trolox was maintained with the Trolox concentration range from 2.5 microM to 25 microM (R(2) = 0.997). The assay was applied to the measurement of TAC in healthy human plasma. Coefficient of variation in intraday assay was 2.4%. Difference was not observed in interday assay. Plasma TAC of men ((569 +/- 41) microM Trolox equivalent; n = 6) was higher than that of women ((430 +/- 28) microM Trolox equivalent; n = 4). After the vegetable juice was drunk for 1 week, the increase in plasma TAC was observed in almost all the volunteers. In summary, we developed the efficient assay for plasma TAC using a 96-well microplate.
This present study aimed to develop a paper-based colorimetric sensor in the form of paper-microzone plates (PµZP), for simple and fast quercetin determination in guava leaf extract samples. Here, N-bromosuccinimide (NBS) solution was immobilized on the microzone as a sensing probe, where quercetin solution can be dropped on it to form red-purplish color adducts which can be seen by the naked eye or captured using a flatbed scanner. The color intensity of the microzone can be quantified against a blank solution and used as analytical data in scanometric assay. The sensor showed a response time of 8 min, a linear interval of 1-10 mM with a detection limit at 1.274 mM toward quercetin, and exhibited good reproducibility (RSD < 1%) and accuracy (98-99% recovery). The quercetin level of guava leaf extract determined by the PµZP-scanometric method was found comparable with that of the TLC-densitometric method, suggesting its use as an alternative method for quercetin analysis in the guava leaf extract.
The human body needs an antioxidant-rich diet that comes from foods, beverages, and herbal products to support the physiological antioxidant systems. Thus, the development of an analytical tool for a simple assay of the total antioxidant capacity (TAC) of the rich antioxidant samples is crucial. The current work demonstrates a simple colorimetric assay of TAC of the herbal extract on the paper microzone plate (PµZP) that was constructed in the 70-well of patterned paper using the screen printing technique. The PµZP was constructed by immobilizing DPPH (2,2-diphenyl-1-picrylhydrazyl) onto 70-well of PµZP as a sensing zone for colorimetric detection. The purple-sensing zone exhibited a good response to gallic acid (GA), by producing slightly gray to pale yellow color that can be captured using a scanner and then analyzed using the ImageJ program. The paper-based sensor showed a linear response toward GA at 0.05–0.6 mM (r = 0.9895), reproducible response (RSD < 4%), and accurate measurement with 91 to 106% recovery for measuring TAC of herbal extract, presented as mM gallic acid equivalent and showed a good agreement with the standard DPPH method. The results suggested that the proposed method can be applied for simple TAC measurement in the herbal extract.
We report the first use of a paper-based device as a simple, low-cost and rapid detection platform for simultaneous determination of antioxidant activity and total phenolic content in food samples. Two antioxidant activity assays including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonate) radical cation (ABTS) assay and cupric reducing antioxidant capacity (CUPRAC) assay and one total phenolic content assay, Folin Ciocaltue reagent (FC) assay were simultaneously employed as a proof-of-concept. The device composed of a central sample zone connected to four pretreatment zones and consecutive detection zones to accommodate all three assays and a sample blank measurement. The analysis was achieved by dropping the samples onto the sample zone to flow to the pretreatment and detection zones containing the stored reagents for each antioxidant assay making the color change that was measured using imageJ software. Assay optimization including key reagent concentrations, reaction time, and surface modification were carried out to obtain sensitive and wide linear rage analyses. Various antioxidant standards were then evaluated to determine the analytical features of the method. The paper-based assays were successfully applied to detect antioxidant activity and total phenolic content in 10 beverage samples with similar gallic acid equivalent (GAE) values to those obtained from traditional assays at a 95% confidence interval. Moreover, the GAE values of the samples obtained from three assay analyses were well correlated to each other with relatively high Pearson's correlation coefficients. These results indicated that the assays gave accurate results and are suitable for simultaneous analysis of antioxidant activity and total phenolic content in real samples.
Effects of reaction pH and time on the antioxidant behaviors of Tyr, Trp, Cys, and their related peptides (Tyr-Gly, Tyr-Glu, Tyr-Lys, Trp-Gly, Trp-Glu, Trp-Lys, Cys-Gly and Cys-Gly) in ABTS assay were investigated. Results showed that all these amino acids and peptides displayed a biphasic kinetic pattern with a fast initial step and a slow secondary step. The initial reaction rates of Tyr, Trp and their related peptides were strongly dependent on pH, while those of Cys and Cys-containing peptides were unaffected by pH. They failed to reach equilibrium over the short incubation period of 6-10min typically used in this assay. Longer incubation time was needed for most of the peptides to approach equilibrium at lower pH. The observed biphasic kinetic pattern as well as the high TEAC values for these amino acids and peptides, could be a result of combined antioxidant behaviors of themselves plus the generated reaction products.
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The spectrophotometric technique for total antioxidant activity (TAA)1,2 measures the relative abilities of a.ntioxidants to scavenge the 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS*+) radical cation (ABTS*+) in comparison with the antioxidant potency of standard amounts of Trolox, the water-soluble vitamin E analogue. This method is based on the progressive consumption of antioxidant activity by ABTS*+ as it is generated in the reaction cuvette and can be automated with a spectrophotometric analyzer. Several different analytical strategies are possible using the same reagents, enabling the assay system to be used to determine the antioxidant activity of plasma, saliva, lipoprotein fractions, foods and beverages. To determine the activity of pure antioxidant substances, a hydrogen peroxide concentration of 75 μM is used, together with a 6 min measuring time. For biological samples with endogenous peroxidase activity the hydrogen peroxide concentration is increased fivefold and the measuring time shortened to 3.25 min. Assays with improved sensitivity are described for low-density lipoprotein (LDL) preparations and saliva. Use of a spectrophotometric endpoint makes the assay simple to carry out without special laboratory equipment. Measurement at 734 nm avoids a range of potential interfering factors, such as sample turbidity and non-specific absorbance by sample constituents. Current applications of the ABTS antioxidant assay are described and discussed.
High-throughput cupric ion reducing antioxidant capacity (CUPRAC) methods were developed for assessment of total antioxidant capacity (TAC) in urine and serum, based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex, measured spectrophotometrically at 450 nm. The reaction time was significantly reduced from 30 to 4 min by application of a calibration compound (uric acid) with kinetic behavior similar to that shown by urine samples. The method was implemented in a microformat (96 well plates) and also in an automatic fashion (flow injection analysis, FIA). A determination throughput value of 288 h(-1) (microplate method) or of 15 h(-1) (automatic FIA) was attained. Application of both methods to human serum (SRM 909b, level I) and urines (n = 9) provided TAC values in agreement with those of the end-point batch method.
Aqueous ultrasound-assisted extraction (UAE) of grape pomace was investigated by Response Surface Methodology (RSM) to evaluate the effect of acoustic frequency (40, 80, 120kHz), ultrasonic power density (50, 100, 150W/L) and extraction time (5, 15, 25min) on total phenolics, total flavonols and antioxidant capacity. All the process variables showed a significant effect on the aqueous UAE of grape pomace (p<0.05). The Box-Behnken Design (BBD) generated satisfactory mathematical models which accurately explain the behavior of the system; allowing to predict both the extraction yield of phenolic and flavonol compounds, and also the antioxidant capacity of the grape pomace extracts. The optimal UAE conditions for all response factors were a frequency of 40kHz, a power density of 150W/L and 25min of extraction time. Under these conditions, the aqueous UAE would achieve a maximum of 32.31mg GA/100g fw for total phenolics and 2.04mg quercetin/100g fw for total flavonols. Regarding the antioxidant capacity, the maximum predicted values were 53.47 and 43.66mg Trolox/100g fw for CUPRAC and FRAP assays, respectively. When comparing with organic UAE, in the present research, from 12% to 38% of total phenolic bibliographic values were obtained, but using only water as the extraction solvent, and applying lower temperatures and shorter extraction times. To the best of the authors' knowledge, no studies specifically addressing the optimization of both acoustic frequency and power density during aqueous-UAE of plant materials have been previously published.
Antioxidant data based on ABTS assay are dependent on reaction time because the applied standard compound (Trolox) presents a scavenging kinetic profile different from that of polyphenol-rich foods. Hence, in this work we propose a kinetic matching approach for ABTS assay. This methodology is based on selecting a standard compound that presents a kinetic profile similar to the sample, which provides the same antioxidant capacity independent of the selected reaction time. To demonstrate the feasibility of this approach, several food products containing different phenolics were analysed. By selecting the appropriate standard for espresso coffees (standard phenolic mixture), green teas ((-)-epigallocatechin gallate), black teas (hesperetin), white wines (morin) and enological tannins (tannic acid), total antioxidant capacity can be determined in 5–15 min corresponding to a sample throughput of 64–192 h−1. Total antioxidant capacity values were converted to a common basis, through a factor estimated from the reactivity of the kinetic matching standard compound and Trolox, providing higher antioxidant data for espresso coffee (45.2–93 mM of Trolox). Red wine samples (23–33 mM) had higher antioxidant capacity than white wines (3.2–6.5 mM), while black tea (11.1–18.2 mM) showed lower results than green tea (20.4–25.5 mM). Moreover, considering the antioxidant value expressed on a weight basis as milligrams of vitamin C per serving of sample, green teas and red wines were those that presented higher antioxidant intake, 765 ± 51 and 688 ± 71 mg of ascorbic acid per serving, respectively.
Curcuma longa L., also known as turmeric, is widely used as a food colorant and has been reported to have antioxidant, anti-inflammatory, anti-mutagenic and anti-cancer properties. The aim of this study was to evaluate the effects of the spray drying on curcuminoid and curcumin contents, antioxidant activity, process yield, the morphology and solubility of the microparticulated solid dispersion containing curcuma extract using a Box Behnken design. The microparticles were spherical in shape, and an increase in outlet temperature from 40 to 80 °C resulted in a significant increase in the yield of microparticles from 16 to 53%. The total curcuminoid content (17.15 to 19.57 mg/g), curcumin content (3.24 to 4.25 mg/g) and antioxidant activity (530.1 to 860.3 μg/mL) were also affected by the spray drying process. The solubility of curcuminoid from C. longa remarkably improved 100-fold in the microparticles, confirming the potential of the ternary solid dispersion technique to improve the dyeing and nutraceutical properties of these compounds. Furthermore, the microparticles were obtained using the spray drying process, can be easily scaled up.
Stem by-products from ten different grape (Vitis vinifera L.) varieties were evaluated in terms of their total phenolic and total tannin contents, flavan-3-ol and proanthocyanidin profiles, and antioxidant capacity measured by ABTS, CUPRAC, FRAP and ORAC assays, with a view to the recovery of their natural bioactive compounds. Stems from Callet, Syrah, Premsal Blanc, Parellada and Manto Negro varieties yielded the highest total phenolic and total tannin contents and showed the greatest antioxidant capacities, whereas Chardonnay and Merlot stems presented the lowest values. Varieties differed significantly (p < 0.05) with regard to both the phenolic composition and antioxidant capacity of their stems. However, no significant differences (p > 0.05) were observed when considering stems from red and white varieties separately. For the ten grape varieties investigated, this is the first study presenting a detailed description of their stems flavan-3-ol composition determined by HPLC-UV-fluo. All the analysis confirmed the stem by-products as a potential polyphenol-rich source, especially promising in the case of the Callet variety.
Formation of both health promoting and potential harmful substances (acrylamide and hydroxymethylfurfural) has been associated
to the extent of the Maillard reaction. The effects of recipe compositions in terms of leavening agent (ammonium and sodium
bicarbonates) and sugars (sucrose and glucose), and baking conditions (temperature and time) on the antioxidant activity (AOA)
in cookies were studied. The cookies were baked at different temperatures (180–220°C) for different times (10–25min). AOA
was measured by the ferric reducing power (FRAP), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2,-diphenyl-1-picrylhydrazyl
(DPPH) coloured radicals, and oxygen radical absorbance capacity (ORAC-fluorescein) in automated plate-reader assays. Net
AOA varied regarding the assay applied, whereas higher AOA was always obtained for the ABTS assay and lower for DPPH assay,
and ranging from 2 to 200μmol Trolox/g sample. At higher temperature and baking times, higher AOA in cookies regardless of
the formulation was recorded. Glucose enhances formation of compounds with higher AOA compounds as compared with sucrose recipes.
Ammonium bicarbonate clearly promotes the formation of AOA for sucrose recipes but this effect is not observed in glucose
recipes and varied with the AOA procedure applied. A risk/benefit index, based on the concomitant formation of neo-formed
contaminants and substances with AOA (potentially health-promoting substances) is presented, and its application for recipe
comparison is discussed. Risk/benefit index rapidly increased with increased temperature and time of baking.
The antiradical activities of various antioxidants were determined using the free radical, 2,2-Diphenyl-1-picrylhydrazyl (DPPH*). In its radical form. DPPH* has an absorption band at 515 nm which dissappears upon reduction by an antiradical compound. Twenty compounds were reacted with the DPPH* and shown to follow one of three possible reaction kinetic types. Ascorbic acid, isoascorbic acid and isoeugenol reacted quickly with the DPPH* reaching a steady state immediately. Rosmarinic acid and δ-tocopherol reacted a little slower and reached a steady state within 30 min. The remaining compounds reacted more progressively with the DPPH* reaching a steady state from 1 to 6 h. Caffeic acid, gentisic acid and gallic acid showed the highest antiradical activities with a stoichiometry of 4 to 6 reduced DPPH* molecules per molecule of antioxidant. Vanillin, phenol, γ-resorcylic acid and vanillic acid were found to be poor antiradical compounds. The stoichiometry for the other 13 phenolic compounds varied from one to three reduced DPPH* molecules per molecule of antioxidant. Possible mechanisms are proposed to explain the experimental results.
The relative antioxidant activities, against radicals generated in the aqueous phase, of a range of plant-derived polyphenolic flavonoids, constituents of fruit, vegetables, tea and wine, have been assessed. The results show that compounds such as quercetin and cyanidin, with 3',4' dihydroxy substituents in the B ring and conjugation between the A and B rings, have antioxidant potentials four times that of Trolox, the vitamin E analogue. Removing the ortho-dihydroxy substitution, as in kaempferol, or the potential for electron delocalisation by reducing the 2,3 double bond in the C ring, as in catechin and epicatechin, decreases the antioxidant activity by more than 50%, but these structures are still more effective than alpha-tocopherol or ascorbate. The relative significance of the positions and extents of hydroxylation of the A and B rings to the total antioxidant activity of these plant polyphenolics is demonstrated.
1. A new method has been developed for measuring the total antioxidant capacity of body fluids and drug solutions, based on the absorbance of the ABTS*+ radical cation.
2. An automated method for use on a centrifugal analyser, as well as a manual method, is described.
3. The procedure has been applied to physiological antioxidant compounds and radical-scavenging drugs, and an antioxidant ranking was established based on their reactivity relative to a 1.0 mmol/l Trolox standard.
4. The Trolox equivalent antioxidant capacity of plasma from an adult reference population has been measured, and the method optimized and validated.
5. The method has been applied to investigate the total plasma antioxidant capacity of neonates and how this may be compromised in prematurity.
Reactive oxygen species and reactive nitrogen species are formed in the human body. Endogenous antioxidant defences are inadequate to scavenge them completely, so that ongoing oxidative damage to DNA, lipids, proteins and other molecules can be demonstrated and may contribute to the development of cancer, cardiovascular disease and possibly neurodegenerative disease. Hence diet-derived antioxidants may be particularly important in protecting against these diseases. Some antioxidants (e.g. ascorbate, certain flavonoids) can exert pro-oxidant actions in vitro, often by interaction with transition metal ions. The physiological relevance of these effects is uncertain, as is the optimal intake of most diet-derived antioxidants. In principle, these questions could be addressed by examining the effects of dietary composition and/or antioxidant supplementation upon parameters of oxidative damage in vivo. The methods available for measuring steady-state damage (i.e. the balance between damage and repair or replacement of damaged molecules) and the actual rate of damage to DNA, proteins and lipids are reviewed, highlighting areas in which further methodological development is urgently required.
This study introduces a simple direct antioxidant assay, based on the reduction of the ABTS.+ radical cation, and compares it with the myoglobin/ABTS.+ assay. The methods give closely similar results, establishing that the antioxidants studied to date in the latter assay act by scavenging the ABTS.+ radical cation and not by inhibiting its formation through reduction of ferryl myoglobin or reaction with H2O2.
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.
Antioxidant activity of Caesalpinia sappan heartwood was studied both by in vitro and in vivo models. The ethyl acetate, methanol and water extracts exhibited strong antioxidant activity as evidenced by the low IC50 values in both 1,1-diphenyl-2-picryl hydrazyl (DPPH) and nitric oxide methods. The values were found to be less or comparable to those of ascorbic acid and rutin, the standards used. Administration of the successive methanol and water extracts at 50 and 100 mg/kg body weight given for four days prior to carbon tetrachloride (CCl4) treatment caused a significant increase in the level of superoxide dismutase (SOD) and catalase and a significant decrease in the level of thiobarbituric acid reactive substances (TBARS), when compared to CCl4 treated control in both liver and kidney. These changes observed at 100 mg/kg body weight treatment were comparable to those observed for standard vitamin E at 50 mg/kg treatment. The results support significant antioxidant nature of Caesalpinia sappan heartwood extracts.
Seven cane brown sugars (four from La Réunion, two from Mauritius, and one from France) were investigated for their polyphenol content and volatile composition in relation to their free radical scavenging capacity determined by ABTS and DPPH assays. The thin layer coated on the sugar crystal was extracted by Soxhlet extractor with dichloromethane. The volatile compounds of brown sugars were studied by GC-MS, and 43 compounds were identified. The total phenolic content of brown sugars was determined according to the Folin-Ciocalteu method. Phenolic compounds were quantified in the brown sugar extracts by LC-UV-ESI-MS. Brown sugar aqueous solutions exhibited weak free radical scavenging activity in the DPPH assay and higher antioxidant activity in the ABTS assay at relatively high concentration. The brown sugar extracts showed interesting free radical scavenging properties despite the low concentration of phenolic and volatile compounds. Sugar is a common foodstuff traditionally used for its sweetening properties, which might be accompanied by antioxidant properties arising from molecules (polyphenols, Maillard products) other than sucrose of the cane brown sugars.
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