The anthocyanins were extracted from the black rice according to Lees and Francis (1972), with some modifications. One gram of finely ground black rice was added to a mixture of ethanol and citric acid 1.0 mol L−1 in the ratio 80:20. The extraction was performed in a thermostated cell, protected from light, under constant stirring. The extraction time, temperature and solid–liquid ratio were determined according to the experimental design. After processing, the extract was filtered (qualitative Whatman #1 paper) and the residue and filter paper were rinsed using the solvent of extraction until completing a volume of 50 mL. The extract was stored at −20 °C in polyethylene bottles, protected from light.
2.4. Analyses of total flavonoids, anthocyanins, and total phenolics
Flavonoids and anthocyanins from the black rice were quantified by UV–Vis spectrophotometry (Shimadzu UV-1800), at λ = 374 nm and λ = 535 nm, respectively. The content of flavonoids and anthocyanins were determined using Eqs. (1) and (2), respectively (Lees & Francis, 1972). The content of total flavonoids was expressed as mg quercetin equivalents (CE) per 100 g of rice and total anthocyanins expressed as mg cyanidin-3-glucoside equivalents (CGE) per 100 g dry weight sample (mg CGE 100 g−1 DW).