2.5. HPLC Analysis of Anthocyanin Contents

The conventional extraction method of anthocyanin is solvent extraction. Hydrochloric acid can prevent the degradation of non-acylation anthocyanin in the extraction process, and ultrasonic waves can improve the extraction efficiency [19]. Total anthocyanin was extracted by the ultrasonic assisted method [20]. Briefly, 200 mg of xylem was ground into powder in liquid nitrogen; the powder was then transferred into 2 mL methanol with 1% HCl at 4 °C protected from light for 12 h. After sufficient mixing, 2 mL of ddH₂O and 4 mL chloroform were put in the solution and then centrifuged at 12,000 g for 20 min after ultrasonic treatment with an ultrasonic extractor for 1 h. The LC-20AD system (Shimadzu, Japan) and phenominex lunar C18 (4.6 µm × 250 mm) was used to analyze anthocyanin content according to previous studies [21,22]. The standard curve was established with cyanidin-3-glucoside chloride (Solarbio, Beijing, China) by the same method. Anthocyanin contents were calculated according to the standard curve.