Extraction and Determination of Anthocyanins

Anthocyanin extraction was conducted according to the method of Giusti and Wrolstad (2001), with slight modifications. Approximately, 1 g of skin tissue was collected and quickly ground into powder in liquid nitrogen before 5 ml of 1% HCL-methanol solution was added and the sample was incubated in the dark at 4°C for 12 h. After centrifugation at 12,000 × g for 20 min, the supernatant was transferred to a clean tube and used for two dilutions, one with 0.025 M potassium chloride buffer (pH = 1.0) and the other with 0.4 M sodium acetate buffer (pH = 4.5). These dilutions were left to equilibrate for 15 min before the absorbance of each dilution was measured at 530 and 700 nm with a UV-Visible spectrophotometer (UV-1700, Kyoto, Japan), using a blank cell filled with distilled water for calibration. The anthocyanin content was calculated using the following formula:

where C stood for anthocyanin content (mg⋅100 g^-1 FW), V for extraction solution volume, n for dilution factor, MW for the molecular weight of cyanidin-3-glucoside: 449.2, 𝜀 for molar absorptivity: 30200, m for the weight of fruit skin, and A = (A530–A700 nm) pH_1.0 (A530–A700 nm) pH_4.5. The value for each sample represented the mean of three independent biological replicates.