2.6. Total anthocyanin and total phenolic contents determination
Two gram samples from each replicate were treated with liquid
nitrogen, and then pulverized and extracted with 25 mL of precooled
70% ethanol containing 1% hydrochloric acid (v/v) for 3 h,
and centrifuged at 10,000 g for 15 min (4 C). The supernatant
was adjusted to 25 mL for analysis.
Total anthocyanin content of mulberry extract was measured
using the pH differential method (Yang et al., 2009). Absorbance
was measured at 510 and 700 nm, respectively, in different buffers
at pH 1.0 and 4.5, using A = [(A510 A700)pH 1.0 (A510 A700)pH 4.5]
with a molar extinction coefficient of cyaniding 3-glucoside of
29,600. Results were expressed as grams of cyaniding 3-glucoside
(C 3-G) equivalents per kilogram of fresh weight. Total phenolic
contents were estimated colourimetrically Folin–Ciocalteu method
(Zheng et al., 2003; Yang et al., 2009). Gallic acid was used as a
standard, and phenolic contents were expressed as grams of gallic
acid equivalents (GAE) per kilogram of fresh weight.