2.2. Phenolic Extraction
Previous studies showed that the 50% ethanol extracts from the plant tissues have higher total phenolic content and antioxidant activity than the ethanol, methanol or 50%methanol aqueous extracts [6].The 70% (v/v) aqueous acetone and 95% (v/v) ethanol were commonly used for extraction of tannins and other classes of phenolic compounds from plant materials [12]. And pure methanol was the best solvent for extracting phenolic compounds in plant tissues, followed by ethanol, diethylether and hexane [13]. Therefore, the 50% ethanol (v/v), 95% ethanol (v/v), 70% acetone (v/v) and 100% methanol were used for extracting phenolic compounds from the blueberry leaves, respectively. For ethanol extraction, leaf powders were transferred to dark-colored flasks, and homogenized with 50% or 95% ethanol at a material to solvent ratio of 15:100 (m/v) at 25°C for 30 min. For acetone and methanol extractions, the powder samples were mixed with 70% acetone or pure methanol at 25°C for 30 min with a material to solvent ratio of 1:10 (w/v). After twice extractions with each selected solvent, two extracts were collected and pooled into a round-bottomflask, and then evaporated to near dry under vacuum at 40°C using a rotary evaporator (Models RE-52AA, Yuhua Instruments Co., Henan, China). The residual was re-dissolved in 1% (v/v) the corresponding solvent to 50 mL, and then stored at -80°C until further analysis.