Folin ciocalteu s phenolic reagent

The total phenolic content was determined using the Folin-Ciocalteu method [22 ], adapted for a microplate reader. Briefly, 10 μL of the sample or standard (gallic acid) were mixed with 790 μL of distilled water and 50 μL of Folin-Ciocalteu’s phenolic reagent (Sigma-Aldrich, St. Louis, MO, USA) in 96 Nunc^® DeepWell™ plate (Sigma-Aldrich) and, after vortexing, were incubated for 5 min at room temperature. Then, 150 microliters of 20% Na₂CO₃ were added, and the samples were vortexed and incubated for 2 h at room temperature. Absorbance was measured at 670 nm using an LKB 6060–006 plate reader (LKB Vertriebs GmbH, Vienna, Austria). The results are expressed as gallic acid equivalents (GAE, mg/L).

Gallic acid, chlorogenic acid, ellagic acid, ascorbic acid, ferrous sulfate, tannic acid, phenantroline and caffeic acid were purchased from Merck (Darmstadt, Germany). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane (MDA), bovine serum albumin (BSA), agarose type I, sodium azide, aluminium chloride, potassium acetate, quercetin, quercitrin, isoquercitrin, rutin, kaempferol, catechin, epicatechin and Folin–Ciocalteu’s phenolic reagent were acquired from Sigma Aldrich Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade.