Cyanidin was extracted by a slightly modified process of anthocyanin
extraction reported by Ukwueze et al. [13]. Red cabbage
bought from a local market was cleaned in deionized-water and
sliced to small pieces. Then, it was weighed to 270 g before soaking
in 600 mL of the mixed solution of MeOH and 2 M HCl (85:15 v/v).
Extraction was carried out in a refrigerator for 72 h. The reddish
extract was filtered and removed other non-polar molecule using
chloroform. The solvent was then evaporated (temp < 38 C).
Concentrated HCl was added into the concentrated anthocyanin
solution then refluxed until the deep-red violet solution appeared.
The extract was immediately poured and placed in the refrigerator
until dark brown powder of cyanidin chloride precipitated. The
powder was filtered and dried in a desiccator. Then it was purified
through Sephadex LH-20 using methanol as eluent. Anthocyanins
were characterized by UV–vis spectrophotometry, NMR and mass
spectrometry. Shinoda’s method [29] was used to check the flavonoid
property by adding a piece of magnesium ribbon and 1 mL of
concentrated HCl into 2–3 mL of methanolic extract (pink red or
red coloration appeared after a few minutes).