Measurement of chlorophyll, carotenoid, and total anthocyanin concentrations

Pigments were extracted from fruit peel samples (0.3 g) using 3 ml 95% ethanol at room temperature (21 °C) in the dark until the peel tissues bleached completely according to previously described [33] with minor modification. The extracts were centrifuged at 8000 × g for 2 min, and the supernatants were collected and their absorbance was measured using a UV-1780 spectrophotometer (Shimadzu, Japan) at 665 nm, 649 nm, and 470 nm. The formulae for calculating chlorophyll concentrations were as following: chlorophyll a = 13.95*A665 – 6.88*A649; and chlorophyll b = 24.96*A649 – 7.32*A665. The formulae for calculating carotenoid concentrations were as following: carotenoids = (1000*A470 – 2.05*Ca – 114.8*Cb)/245 and the results were expressed as mg g⁻¹.

Anthocyanin was extracted using 0.3 g of the frozen peel tissues in 3 ml 95% methanol:1.5 M hydrochloric acid (85:15) for 24 h at 4 °C in the dark according to previously described [34] with minor modifications. The extract was centrifuged at 12,000 × g for 5 min, and the supernatant was collected and analyzed using a UV-1780 spectrophotometer at 530, 620, and 650 nm. The anthocyanin concentrations were calculated using the following formula: 46,200*[(A530 -A620)-0.1*(A650-A620)] and the results were expressed as ug g⁻¹.