Spe ma10avp

To identify the main phenolic compounds, the dried extracts and liposomal preparations (with and without Triton X-100) were dispersed in Milli-Q water and analysed by reverse phase high performance liquid chromatography (RP-HPLC; model SPE-MA10AVP, Shimadzu, Kyoto, Japan) on a C18 analytical column (Tracer Excel 120 ODS-A 5 μm 25 × 0.46, Teknokroma, Barcelona, Spain). The samples were eluted with a gradient system consisting of solvent A (MilliQ water) and solvent B (methanol:acetonitrile, 60:40), both containing 1% acetic acid, at a flow rate of 0.6 mL/min. The temperature of the column was maintained at 25 °C, and the injection volume was 20 μL. The gradient system started at 10% solvent B and increased to 60% B over 60 min, followed by a further decrease to 10% solvent A over 10 min. The final conditions were held for 10 min. The peaks of the phenolic compounds were monitored by absorbance at 253 nm and 368 nm. The identification of phenolic compounds was carried out by comparison of the retention times of the pure external standards listed above. Chlorogenic acid, vitamin C, rutin and rosmarinic acid were quantified using a calibration curve of the corresponding standard compound. Analyses were carried out at least in duplicate.

For overall molecular weight (MW) distribution of A20 and P20 hydrolysates, size exclusion high-performance liquid chromatography (Shimadzu, SPE-MA10AVP, Kyoto, Japan) was conducted, according to Sila et al. (2015) , using a Superdex Peptide PC 3.2/30 column (GE Healthcare Bio-Sciences, Barcelona, Spain), with a 1007000 Da separation range. The hydrolysate (10 µL) was loaded onto the column and eluted at 2.5 µL/min flow rate, using acetonitrile (30%; v/v) with trifluoroacetic acid (0.01%; v/v) as the mobile phase. MW standards used were glycine (75 Da), hippuryl-L-histidyl-L-leucine (429 Da), vitamin B12 (1340 Da), aprotinin (6512 Da) and bovine serum albumin (6700 Da). Optical density was monitored at 214 nm.