The Folin–Ciocalteu phenol reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Akt assay acid (Trolox), vanillin, tannic

acid, chlorogenic acid, gallic acid, perchloric acid, caffeine, theobromine, ursolic acid and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). All reagents were either of analytical or of chromatographic grade. The leaves of Ilex paraguariensis St. Hil. used for the preparation of the extract were harvested in Catanduvas, Santa Catarina state, Brazil. After the leaves were picked, they were washed and the excess water on the surface of the leaves was removed by centrifugation. After that, the leaves were dried in a forced air oven (FABBE, 171, São Paulo, Brazil) for 24 h at 45 °C and then they were ground with a knife mill (Marconi, MA-580, Piracicaba, Brazil) until reaching a particle size smaller than 3.55 mm. The ground leaves were placed in plastic bags and kept at −18 °C until the preparation of the aqueous extracts. The mate extract was produced according to Murakami et al. (2011), using 3 g of ground leaves suspended in 100 mL of distilled water with constant stirring. The pH was adjusted to 6.0 by adding either 0.1 N Akt inhibitor hydrochloric acid or 0.1 N sodium hydroxide solutions and using

a pH meter (Quimis, Q-400A, Diadema, SP, Brazil). The extracts were filtered with filter paper (12.5 cm diameter and 25 μm pore size) using a vacuum pump (Prismatec, 131, Itu, Brazil) and the extract volumes were made up to 100 mL with distilled water (referred to as 3 g/100 mL extracts). Nanofiltration (NF) was applied to concentrate biologically valuable components of mate extract. The concentration was performed with a tangential filtration Phosphoglycerate kinase system on a pilot scale equipped with a spiral membrane module (Osmonics membranes, HL2521TF, Minnetonka, MN), with a MWCO ranging between 150 and 300 Da, 0.6 m2 of filtration area and 98% rejection of MgSO4 in a test performed by manufacturing with a spiral module at 25 °C and

690 kPa. The experiment was performed on pilot equipment (Fig. 1) that allows batch circulation, meaning that both the permeate and the concentrate could be carried back to the feed tank. The permeate was totally removed, in order to obtain the concentrated mate extract. The operating parameters during the NF process were temperature of 24 ± 1 °C and pressure of 300 kPa, up to a VRF (volumetric reduction factor) of 4. As was expected, a decrease in permeate flux was observed throughout the time, due to concentration polarisation and fouling of membrane. A permeate flux was obtained between 0.50 and 24.0 L/h.m2, while the average permeate flux was equal to 4.53 L/h.m2. After each processing, the pilot unit and membrane were cleaned and sterilised, according to the manufacturer’s instructions.