The chemical reagents ABTS ((2, 5, 7, Tween 40, and phenolic compound standards (gallic acid, catechin, chlorogenic acid, caffeic acid, ferulic acid, vanillin, quercetin, were obtained from Sigma-Aldrich (Saint Louis, Missouri, USA). methanol (HPLC grade) was obtained from J-TBacker (Alfragide, Lisbon, Portugal). In turn, the Folin-Ciocalteu reagent was obtained from Dinâmica (Indaiatuba, São Paulo, Brazil). Acetone, acetic acid, gallic acid, linoleic acid, methanol, and chloroform were obtained from Vetec (Saint Louis, Missouri, USA), while sulfuric acid was obtained from Merck (Darmstadt, Hessen, Germany). Ethanol, was obtained from Synth (Diadema, São Paulo, Brazil), while n-hexane and potassium persulfate were obtained from Neon (São Paulo, São Paulo, Brazil). All reagents were analytical grade, and stock and buffer solutions were prepared using distilled water.
Gallic acid
Manufactured by Vetec
Sourced in Brazil
Gallic acid is a naturally occurring organic compound with the chemical formula C6H2(OH)3COOH. It is a crystalline solid and a type of phenolic acid. Gallic acid serves as a chemical building block and precursor in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Quercetin, by Merck Group (6 mentions)
Chloroform, by Vetec (3 mentions)
Catechin, by Merck Group (2 mentions)
Methanol, by Vetec (2 mentions)
Rutin, by Merck Group (2 mentions)
Sodium carbonate, by Vetec (2 mentions)
Hydrochloric acid (hcl), by Vetec (2 mentions)
Ethanol, by Vetec (2 mentions)
Folin ciocalteu reagent, by Merck Group (2 mentions)
Linoleic acid, by Vetec (1 mentions)
Abts 2 5 7, by Merck Group (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Vanillin, by Merck Group (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Folin ciocalteu reagent, by Dinâmica (1 mentions)
Acetone, by Vetec (1 mentions)
Acetic acid, by Vetec (1 mentions)
13 protocols using gallic acid
1
Antioxidant Characterization of Phenolic Compounds
2
Antioxidant Activity Assessment
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The reagents used in the experiment were DPPH (2,2-diphenyl-1-picrylhydrazyl) and quercetin from Sigma Aldrich, Folin-Ciocalteu reagent from Haloquímica, gallic acid from Vetec, anhydrous sodium carbonate, and methanol from Synth.
3
Phytochemical Analysis of Plant Extracts
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Reverse phase chromatography analyses were carried out under gradient conditions
using a Techno Sciences C-18 column (4.6 mm x 250 mm). The flow rate was 0.6
mL/min, the injection volume was 40 μL and the gradient elution was conducted
according to Boligon et al.13 , with minor modifications. Mobile phase consists of water containing 2%
acetic acid (solvent A) and acetonitrile (solvent B).
In an attempt to identify the compounds present in the plant under study, tests
were performed with different commercial standards, where the presence of rutin
and gallic acid was detected. The ultraviolet (UV) absorption spectra of the
standards rutin (Sigma-Aldrich, Brazil) and gallic acid (Vetec
Química, Brazil) and of the samples were recorded in the range of
230-400 nm. Samples, standard solutions and the mobile phase were degassed and
filtered through a 0.45 µm membrane filter (Merck-Millipore, Billerica, MA, USA).
Chromatographic operations were carried out at room temperature and in triplicate.
Identification of the compounds was done by comparison of their retention times
and UV absorption spectrum with the respective standards. The substance contents
were obtained for the calibration curves (gallic acid, y = 71293x – 76386, r =
0.9958; rutin, y = 75045x – 18282, r = 0.9929).
using a Techno Sciences C-18 column (4.6 mm x 250 mm). The flow rate was 0.6
mL/min, the injection volume was 40 μL and the gradient elution was conducted
according to Boligon et al.
acetic acid (solvent A) and acetonitrile (solvent B).
In an attempt to identify the compounds present in the plant under study, tests
were performed with different commercial standards, where the presence of rutin
and gallic acid was detected. The ultraviolet (UV) absorption spectra of the
standards rutin (Sigma-Aldrich, Brazil) and gallic acid (Vetec
Química, Brazil) and of the samples were recorded in the range of
230-400 nm. Samples, standard solutions and the mobile phase were degassed and
filtered through a 0.45 µm membrane filter (Merck-Millipore, Billerica, MA, USA).
Chromatographic operations were carried out at room temperature and in triplicate.
Identification of the compounds was done by comparison of their retention times
and UV absorption spectrum with the respective standards. The substance contents
were obtained for the calibration curves (gallic acid, y = 71293x – 76386, r =
0.9958; rutin, y = 75045x – 18282, r = 0.9929).
4
Spectrophotometric Quantification of Phenolics
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The total phenolic content (TPC) was determined by the Folin–Ciocalteau spectrophotometric method, using gallic acid (Vetec®, 99% purity) as external standard. For the determination, the extracts were dissolved in ethanol and diluted in distilled water. To 100 ml of each sample was added 1 ml of 10% (v/v) Folin-Ciocalteu. The mixture was incubated for 5 min, and then 1 ml of 10% NaCO2was added. The mixture was then incubated for 90 min, and the absorbance was read at 750 nm using a UV/Vis Spectrophotometer SP 2000 UV (Bel Photonics). The calculation was performed through the equation obtained by the linear regression of the calibration curve and the result was expressed in mg gallic acid for g of dry extract.
5
Antioxidant Capacity Assay Protocol
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The reagents used were 2,2-diphenyl-1-picrylhydrazil (DPPH•), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH), fluorescein sodium salt and Folin & Ciocalteu’s phenol reagent, all purchased from Sigma-Aldrich. Ferrous sulfate heptahydrate and gallic acid were purchased from Vetec, and sodium carbonate from J.T Baker were also used. All solvents and chemicals were of analytical grade.
6
Quantification of Polyphenol Content
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Cirsiliol (≥95%, UV, HPLC), Folin-Cioucalteu’s phenol reagent and sodium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid was purchased from Vetec (São Paulo, BR). HPLC-grade solvents were purchased from Tedia Brasil (Rio de Janeiro, BR).
7
Quantifying Phenolic Compounds in Samples
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To quantify total phenols, the Folin-Ciocalteu method was applied with gallic acid (Vetec ® ) (10 to 350 mg mL -1 ) as standard (Y = 0.7182 x + 0.0927, R 2 = 0.982) (Sousa et al., 2007) (link). Flavonoids were evaluated by the aluminum chloride method and quercetin (Sigma ® ) as standard (Y = 0.1114 x -0.0030), R 2 = 0.999) (Peixoto-Sobrinho et al., 2008) (link).
Quartz cuvettes were used to read the samples and standards, and the results of the analyses performed in triplicates were reported as average and standard deviation.
Quartz cuvettes were used to read the samples and standards, and the results of the analyses performed in triplicates were reported as average and standard deviation.
8
Antioxidant Capacity Evaluation
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Sodium carbonate, acetone, and methanol were obtained from Synth. Quercetin and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma Aldrich. Gallic acid was obtained from Vetec, Folin-Ciocalteu reagent from Haloquímica, and thin-layer chromatography (TLC) plates (normal or reverse phase) from Merck.
9
Cell Culture and Cytotoxicity Assay
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Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium and foetal bovine serum (FBS) were purchased from Gibco Invitrogen (Karlsruhe, Germany). Penicillin/streptomycin, dimethyl sulphoxide (DMSO) and salt 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). Folin-Ciocalteu reagent and 2,2-diphenyl-1-picryhydrazyl (DPPH) were purchased from Merck KGaA (Darmstadt, Germany). Methanol, hydrochloric acid, ethanol, ferric chloride, sodium hydroxide, chloroform, anhydrous sodium sulphate, sulphuric acid, sodium carbonate, gallic acid and ethyl acetate were purchased from Vetec Química Fina Ltda. (Duque de Caxias, RJ, Brazil).
10
Determination of Total Polyphenols in Extracts
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The content of total polyphenols in the extract was determined by the colorimetric method using Folin-Ciocalteu reagent (Sigma-Aldrich, Merck) and gallic acid (Vetec) as standard (17 (link)). A solution of extract of 2.42 mg/L was prepared and an aliquot at 0.5 mL increments were subsequently mixed with 0.5 mL Folin-Ciocalteu reagent and 0.5 mL Na2CO3 10% (m/V) (Anidrol, Diadema, SP, Brazil). The reaction was incubated for 1 h at room temperature, and the absorbance was measured at 760 nm (Lambda 25 spectrometer; Perkin Elmer, Waltham, MA, USA). A blank sample without the extract was run under the same conditions. The calibration straight line was constructed with different concentrations of gallic acid (4-32 µg/mL) using the following equation:
where y is the absorbance, 0.03419 is the slope, x is the gallic acid concentration (µg/mL), and 0.03030 is the linear coefficient. The absorption of the extract solution was measured according to the same procedure and the total phenolic compounds were calculated using Eq. 1 and expressed in mg gallic acid equivalents per g extract.
where y is the absorbance, 0.03419 is the slope, x is the gallic acid concentration (µg/mL), and 0.03030 is the linear coefficient. The absorption of the extract solution was measured according to the same procedure and the total phenolic compounds were calculated using Eq. 1 and expressed in mg gallic acid equivalents per g extract.
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