The principle of this method is the reaction of oxidised triterpene saponins with vanillin. Perchloric acid is the strong oxidant and the distinctive colour of this reaction is purple. Aliquots from the ethanolic phase were totally evaporated and

then 150 μL of buy SCR7 5% (w/v) vanillin/glacial acetic acid solution and 500 μL of perchloric acid were added to the dry residue obtained. The mixture was incubated at 60 °C for 45 min, cooled down in an ice bath and then added with glacial acetic acid. The absorbance rate was measured with a UV/Vis spectrophotometer (Hitachi, U-1800) at 548 nm. The total saponins content was quantified by using a standard calibration curve of ursolic acid (y = 0.00087205 − 0.02339x, r2 = 0.99), the major triterpenic nucleous of Ilex paraguariensis. The methodology to determine the condensed tannins content is described in this present

work consists of two stages: stage A, where the total polyphenols are quantified; and stage B, where residual polyphenols are quantified after adsorption of tannins by gelatin ( Valdes, Leyes, & Léon, 2000). In stage A, 4.5 mL of extract were treated by adding 1 mL of Folin–Ciocalteu reagent and 0.5 mL of sodium carbonate (20%). This mixture was then stirred and the volume was made up with distilled water to 125 mL. The absorbance was measured with a UV/Vis spectrophotometer (Hitachi, U-1800) at 700 nm, after 2 min. In stage B, 10 mL of extract were diluted in 40 mL of distilled water and added with Adriamycin manufacturer 25 mL of gelatin (25%). After that, 50 mL of an acidified saturated solution of 1% sodium chloride and 5 g of kaolin were added to the solution. The solution was then capped and stirred for 30 min (700g) and immediately afterwards it was filtered. From this solution, the procedure of stage A was repeated. The condensed tannins were quantified by using a standard calibration curve of tannic acid (1.0–10.0 mg/mL; r2 = 0.97; y = 0.0869x + 0.0785). One millilitre of each extract was added to Methocarbamol 7 mL of dimethyl sulfoxide (DMSO) and the mixture was then placed in an oven at 65 °C for 15–20 min. After that,

3 mL of the obtained solution were analysed with a UV/Vis spectrophotometer (Hitachi U-1800) set at 645 and 663 nm. The DMSO reagent was used as a blank solution. The results showed the content of total chlorophyll (Hiscox & Israelstam, 1979). The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was based on the method proposed by Brand-Williams, Cuvelier, and Berset (1995). An aliquot of 0.1 mL of each extract was mixed with 3.9 mL DPPH in methanol (60 μM). The mixture was vigorously shaken and then the absorbance rate was measured at 515 nm every 10 min until it stabilised (Hitachi U-1800). Methanol was used as a blank instead of DPPH solution. EC50 value (μg extract/mL) is the effective concentration at which DPPH radicals were scavenged by 50% and it was obtained by interpolation from linear regression analysis.