Total Anthocyanin Content

The blueberry extracts were filtered through a 0.22 μm filter (Millipore), and then injected to high performance liquid chromatography (HPLC) for analysis. This HPLC analysis was performed in an Aglient 1100 HPLC system (Agilent Technologies, USA) equipped with a binary pump and a diode-array detector (DAD). For the chromatographic analysis, a 250 mm × 4.6 mm, 5-μm particle size, end-capped reverse-phase Zorbax SB-C18 column was used (Agilent Technologies, USA). The running temperature was 35°C, with an injection volume of 10 μl. The detections were made at 520 nm, at a flow rate of 0.6 ml/min. Mobile phase A was a mixture of 6% HAc (ethanoic acid) and ultrapure water, whereas mobile phase B was a mixture of 6% HAc and acetonitrile. The gradient used was as follows: 5 to 10% B (from 0 to 5 min), 10 to 15% B (from 5 to 20 min), 15 to 20% B (from 20 to 35 min), 20 to 40% B (from 35 to 40 min), 40 to 80% B (from 40 to 45 min), 80 to 85% B (from 45 to 50 min), 85 to 5% B (from 50 to 55 min), and 5% B (from 55 to 60 min). To quantify anthocyanins, their peak areas were compared to the absorbance of a cyanidin 3-glucoside external standard (Sigma-Aldrich, Shanghai, China). Total anthocyanin content (hereon, ACs) was calculated as the sum of these peaks and expressed on a FW basis of fruit.