*2.7. Anthocyanin Quantification*

For anthocyanin extraction and quantification, a method reported previously was adapted to this study [19,20]. Weighed leaf samples were ground with liquid nitrogen with a pre-cooled pestle and mortar. The powder was put into 2 mL of 1% HCl–methanol and incubated overnight at 4 ◦C in darkness. A volume of 300 µL of the aliquot was collected into a new 1.5 mL tube, and 200 µL of distilled water and 500 µL of chloroform were added. After mixing, the tube was centrifuged for 5 min at 13,000× *g* at 4 ◦C. A volume of 400 µL of the aliquot was collected carefully into a new tube, and 400 µL of 60% methanol + 1% HCl solution was added. The absorbance of the solution containing anthocyanin was measured at 530 nm and 657 nm.
