The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method was carried out as follows [21 (link)]: the DPPH solution (60 μM in methanol) was mixed with 100 μL of sample. The mixture was incubated at 37 °C for 10 min and then the absorbance was measured in a spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at 515 nm. Each extract was analyzed in triplicate, and its antioxidant activity was determined by using Trolox (Acros organics, Morris Plains, NJ, USA) as standard, expressing the results as μg Trolox/g sample.
Trolox
Manufactured by Thermo Fisher Scientific
Sourced in United States, Belgium, Germany, United Kingdom, Slovakia
Trolox is a water-soluble vitamin E analogue that functions as an antioxidant. It is used as a standard reference compound in various analytical and assay procedures to measure and quantify antioxidant capacity.
Automatically generated - may contain errors
Lab products found in correlation
Gallic acid, by Thermo Fisher Scientific (7 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (7 mentions)
Methanol, by Merck Group (6 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Thermo Fisher Scientific (6 mentions)
Folin ciocalteu reagent, by Merck Group (5 mentions)
Gallic acid, by Merck Group (5 mentions)
Ethanol, by Thermo Fisher Scientific (4 mentions)
Linoleic acid, by Thermo Fisher Scientific (3 mentions)
Sodium acetate, by Merck Group (3 mentions)
Potassium persulfate, by Carl Roth (3 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (3 mentions)
Folin ciocalteu reagent, by Thermo Fisher Scientific (3 mentions)
Rutin, by Thermo Fisher Scientific (3 mentions)
Methanol, by Avantor (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Hexane, by Thermo Fisher Scientific (2 mentions)
Folin ciocalteu s reagent, by Merck Group (2 mentions)
Potassium persulfate, by Thermo Fisher Scientific (2 mentions)
Anhydrous sodium carbonate, by Thermo Fisher Scientific (2 mentions)
Chloroform, by Thermo Fisher Scientific (2 mentions)
53 protocols using trolox
1
DPPH Radical Scavenging Antioxidant Assay
2
DPPH Assay for Antioxidant Evaluation
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DPPH assay was performed according to the method described by Brand-Williams et al. [20 (link)]. This procedure allows the measurement of AO activity by following the reaction between antioxidant and a stable radical, 2,2-diphenyl-lpicrylhydrazyl (DPPH•) (Aldrich chemistry, St. Louis, MO, USA). Indeed, in a hemolysis tube, 3900 μL of a DPPH methanolic solution (0.1 mM) and 100 μL of CE at different concentrations were mixed. After 2 h in the dark at room temperature, the absorbance was measured by a UV–VIS 50 Bio Varian spectrophotometer at 515 nm. Trolox (Acros Organics, Geel, Belgium) was used as antioxidative reference, and results were expressed in μmol of Trolox equivalent per gram of dry matter (μmol TE/g DM). A calibration curve was realized with various concentrations of Trolox (0, 320, 640, 960, 1280 and 1600 μM), and the IC50 and TE concentrations were determined with the following equations:
All DPPH measurements were performed in triplicate.
All DPPH measurements were performed in triplicate.
3
DPPH Assay for Antioxidant Activity
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The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was used to determine the antioxidant activity of the material using the method as previously described [26 (link)]. Trolox (CAS: 53188-07-1, ACROS Organics™, Morris, NJ, USA) was used as a standard to determine the antioxidant activities of the samples and all results were expressed as µmol Trolox equivalent (TE) per g sample.
4
Optical Trap Assay for Actin-Binding Proteins
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Nitrocellulose-coated coverslips with attached platform beads and flow chambers were prepared as described previously (21 (link)). Halo-tagged ABS3 fusion constructs were attached to the coverslip and platform bead surface via HaloLigand-eGFP nonspecifically adsorbed onto the coverslip and platform bead surfaces. The surface was passivated with 2% Pluronic F-127 (Sigma, P2443) to prevent nonspecific binding of ABS3 or F-actin with the coverslip surface.
Optical trap experiments were performed in “F-buffer” (20 mM Hepes pH 7.1, 50 mM KCl, 2 mM MgCl2, 0.2 mM CaCl2, 1 mM dithiothreitol, 1 mM ATP) supplemented with 1 mg/mL bovine serum albumin (MCLAB UBSA-100), 0.8% glucose, 2.7 kU/mL catalase (Sigma, C40-100 mg), 7.5 U/mL pyranose oxidase (Sigma P4234-250UN), 1 mM Trolox (Fisher Scientific, AC218940010), washed streptavidin-coated polystyrene microspheres (Bangs Laboratories, Inc; CP01004; diluted 1:400 from stock concentration), and 0.2 nM biotinylated actin filaments labeled with rhodamine-phalloidin (Cytoskeleton PHDR1) and 20 μMphalloidin. A more detailed protocol can be found inSI Appendix, Extended Materials and Methods .
Flow cells for determining actin filament polarity and preferred direction of ABS3 binding were prepared as described previously (21 (link)).
Optical trap experiments were performed in “F-buffer” (20 mM Hepes pH 7.1, 50 mM KCl, 2 mM MgCl2, 0.2 mM CaCl2, 1 mM dithiothreitol, 1 mM ATP) supplemented with 1 mg/mL bovine serum albumin (MCLAB UBSA-100), 0.8% glucose, 2.7 kU/mL catalase (Sigma, C40-100 mg), 7.5 U/mL pyranose oxidase (Sigma P4234-250UN), 1 mM Trolox (Fisher Scientific, AC218940010), washed streptavidin-coated polystyrene microspheres (Bangs Laboratories, Inc; CP01004; diluted 1:400 from stock concentration), and 0.2 nM biotinylated actin filaments labeled with rhodamine-phalloidin (Cytoskeleton PHDR1) and 20 μMphalloidin. A more detailed protocol can be found in
Flow cells for determining actin filament polarity and preferred direction of ABS3 binding were prepared as described previously (21 (link)).
5
Alginate Extraction and Characterization
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Sodium alginate from Laminaria hyperborea and potassium ferricyanide were obtained from BDH Laboratory Supplies (England). Ammonium molybdate, ethanol, ferric chloride, phenol, sodium chloride, sodium sulphate anhydrous, sulfamic acid, trichloroacetic acid and Trolox were purchased from Fisher Scientific (Ireland). D-mannuronic acid and L-guluronic acid were purchased from Carbosynth (UK). Vivinal GOS was a gift from Dr. Shane O'Connell (Institute of Technology Tralee, Ireland). All other reagents were obtained from Sigma-Aldrich (Ireland). Bacterial cultures were purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany) and from the NCIMB (National Collection of Industrial, Food and Marine Bacteria, UK).
6
Antioxidant Potential of Cardoon Petioles
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The antioxidant potential of cardoon petioles was evaluated through two cell-based methodologies: the thiobarbituric acid reactive substances (TBARS) formation and the oxidative hemolysis (OxHLIA) inhibition assays. Trolox (Fisher Scientific, Lisbon, Portugal), a synthetic antioxidant, was used as a positive control.
The TBARS assay was performed according to the procedure previously described [10 (link)]. The cardoon petiole extracts were redissolved in water to obtain a solution of 5 mg/mL, which was further diluted in order to obtain the concentration range to be tested (0.0012–1.25 mg/mL). The results were expressed as the concentration of extract (IC50 µg/mL) responsible for 50% of the oxidation process inhibition.
For the OxHLIA assay, it measured the capacity of the extracts to inhibit oxidative hemolysis using erythrocytes isolated from sheep blood, following the procedure previously described [29 (link)]. The results were expressed as the extract concentrations (IC50 µg/mL) necessary to ensure 50% of the erythrocyte population integrity after Δt of 60 and 120 min. Phosphate-buffered saline (PBS; pH 7.4) was used as a negative control.
The TBARS assay was performed according to the procedure previously described [10 (link)]. The cardoon petiole extracts were redissolved in water to obtain a solution of 5 mg/mL, which was further diluted in order to obtain the concentration range to be tested (0.0012–1.25 mg/mL). The results were expressed as the concentration of extract (IC50 µg/mL) responsible for 50% of the oxidation process inhibition.
For the OxHLIA assay, it measured the capacity of the extracts to inhibit oxidative hemolysis using erythrocytes isolated from sheep blood, following the procedure previously described [29 (link)]. The results were expressed as the extract concentrations (IC50 µg/mL) necessary to ensure 50% of the erythrocyte population integrity after Δt of 60 and 120 min. Phosphate-buffered saline (PBS; pH 7.4) was used as a negative control.
7
Antioxidant Activity of Cardoon Blade Extracts
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The antioxidant activity of the cardoon blade extracts was tested using two cell-based in vitro assays, which assess the ability to inhibit the formation of thiobarbituric acid reactive substances (TBARS) and the oxidative hemolysis (OxHLIA). The synthetic antioxidant Trolox (Fisher Scientific, Lisbon, Portugal) was used as a positive control.
The ability of the extracts to inhibit TBARS formation was evaluated following the procedure described in our previous work [19 (link)]. The extracts were re-dissolved in water to obtain a solution at 5 mg/mL that was further diluted to obtain the range of concentrations (1.2–625 µg/mL) to be tested. The results were expressed as the extract concentration that causes a 50% inhibition of the oxidative process (IC50 µg/mL).
The oxidative hemolysis assay allows the evaluation of the capacity of the extracts to inhibit the oxidative hemolysis of erythrocytes isolated from healthy sheep. The procedure was followed as described by [23 (link)]. The results were expressed as the extract concentration responsible for keeping 50% of the erythrocyte population intact (IC50 µg/mL) at Δt of 60 and 120 min.
The ability of the extracts to inhibit TBARS formation was evaluated following the procedure described in our previous work [19 (link)]. The extracts were re-dissolved in water to obtain a solution at 5 mg/mL that was further diluted to obtain the range of concentrations (1.2–625 µg/mL) to be tested. The results were expressed as the extract concentration that causes a 50% inhibition of the oxidative process (IC50 µg/mL).
The oxidative hemolysis assay allows the evaluation of the capacity of the extracts to inhibit the oxidative hemolysis of erythrocytes isolated from healthy sheep. The procedure was followed as described by [23 (link)]. The results were expressed as the extract concentration responsible for keeping 50% of the erythrocyte population intact (IC50 µg/mL) at Δt of 60 and 120 min.
8
DPPH Antioxidant Capacity of Microalgae
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Antioxidant capacity of the methanolic extracts of microalga was determined by the modified version of the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method described by [25 (link)]. The antioxidant capacity was determined by the decrease in absorbance at 515 nm of a methanolic solution of DPPH in the presence of the different methanolic samples of the microalga. A concentrated solution of DPPH (Sigma Aldrich, Darmstadt, Germany) in methanol of approximately 0.4 g·L−1 was prepared and diluted with methanol to obtain an absorbance around 0.8. Next, 950 µL of the diluted DPPH solution was made to react with 50 µL of the methanolic sample. The absorbance at 515 nm at time 0 was then measured in a UV-vis spectrophotometer model Evolution 201 (Thermo Fisher Scientific, Walthman, MA, USA) in a glass cuvette, using methanol as a blank. Subsequently, the samples were allowed to stand for 30 min at room temperature and the absorbance of the sample at 515 nm was measured after that time. Trolox (Fisher Scientific, Walthman, MA, USA) was used as an external standard. The antioxidant capacity was determined through the difference in absorbance at time 0 and time 30 min and was expressed as μmol eq-Trolox·g−1 biomass.
9
Cardoon Bracts: Antioxidant Potential
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The in vitro capacity of cardoon bracts to protect cell membranes from oxidation was measured through (i) the thiobarbituric acid reactive substances (TBARS) formation and (ii) the oxidative haemolysis (OxHLIA) inhibition assays. The synthetic antioxidant trolox (Fisher Scientific, Lisbon, Portugal) was used as a positive control in both assays.
The TBARS assay measures the extracts capacity to inhibit the formation of malondialdehyde and other TBARS and was performed using porcine (Sus scrofa) brain tissues as oxidizable substrate, following the procedure previously described by Mandim et al. (2020a) . The cardoon bract extracts were re-dissolved in water to obtain a stock solution at 5 mg/mL, further diluted to obtain the range of concentrations (0.625-0.0048 mg/mL) tested. The results were expressed as the extract concentration (IC 50 , µg/mL) that causes 50% inhibition of the oxidation process.
The OxHLIA assay measures the extracts capacity to inhibit the oxidative haemolysis and was performed using erythrocytes isolated from sheep blood collected from healthy animals, following the procedure previously described by Mandim et al. (2019) . The results were expressed as the extract concentration (IC 50 , µg/mL) required to maintain the integrity of 50% of the erythrocyte population after 60 and 120 min.
The TBARS assay measures the extracts capacity to inhibit the formation of malondialdehyde and other TBARS and was performed using porcine (Sus scrofa) brain tissues as oxidizable substrate, following the procedure previously described by Mandim et al. (2020a) . The cardoon bract extracts were re-dissolved in water to obtain a stock solution at 5 mg/mL, further diluted to obtain the range of concentrations (0.625-0.0048 mg/mL) tested. The results were expressed as the extract concentration (IC 50 , µg/mL) that causes 50% inhibition of the oxidation process.
The OxHLIA assay measures the extracts capacity to inhibit the oxidative haemolysis and was performed using erythrocytes isolated from sheep blood collected from healthy animals, following the procedure previously described by Mandim et al. (2019) . The results were expressed as the extract concentration (IC 50 , µg/mL) required to maintain the integrity of 50% of the erythrocyte population after 60 and 120 min.
10
Analytical Reagents and Solvents Procurement
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All the chemicals and reagents were of analytical grades. Ethanol, hexane, gallic acid, linoleic acid, Trolox, Tween 40, sodium carbonate anhydrous, chloroform, iron (III) chloride anhydrous, and potassium persulfate were purchased from Fisher Scientifi c Co. (Fisher Scientifi c, Loughborough, UK). Acetone, mEthanol, Folin-Ciocalteu's reagent, and 2,2'-azinodi[3-ethyl-benzthiazoline sulfonate] (ABTS) were from Merck KGaA (Lichrosolv, Darmstadt, Germany), while 2,2-diphenyl-1-picryhydrazyl (DPPH), β-carotene, butylated hydroxy anisole (BHA), acetic acid, and sodium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Distilled water used was obtained from a Milli-Q water purifi cation unit (Millipore, Milford, MA, USA).
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