Extraction, enrichment, and hydrolysis of the
bilberry were carried out using essentially the same method as described
previously.15 (link) Briefly, the enriched bilberry
extract powder was extracted with 75% aqueous ethanol containing 0.1%
HCl and enriched by loading the concentrated extracts on an XAD-761/Diaion
HP-20 (1:1) column. The polyphenols, including anthocyanins, were
eluted with methanol. Pooled elutes were concentrated and hydrolyzed
with 2 N HCl (∼5 volumes). Hydrolysates were purified using
C18 Sep-Pak cartridges (Waters, Milford, MA, USA). Anthocyanidins
and other polyphenols were eluted with acidified (0.01% HCl) methanol.
The enriched extracts were dried under reduced pressure using a Savant
Speed-Vac (Thermo Scientific, USA) and stored at −20 °C
until use. The enriched extracts were dissolved in acidified water,
and anthocyanidins were selectively extracted in isoamyl alcohol and
dried under vacuum.16 (link) The extracted anthocyanidins
were further purified by loading on the C18 cartridges per the manufacturer’s
guidelines.
bilberry were carried out using essentially the same method as described
previously.15 (link) Briefly, the enriched bilberry
extract powder was extracted with 75% aqueous ethanol containing 0.1%
HCl and enriched by loading the concentrated extracts on an XAD-761/Diaion
HP-20 (1:1) column. The polyphenols, including anthocyanins, were
eluted with methanol. Pooled elutes were concentrated and hydrolyzed
with 2 N HCl (∼5 volumes). Hydrolysates were purified using
C18 Sep-Pak cartridges (Waters, Milford, MA, USA). Anthocyanidins
and other polyphenols were eluted with acidified (0.01% HCl) methanol.
The enriched extracts were dried under reduced pressure using a Savant
Speed-Vac (Thermo Scientific, USA) and stored at −20 °C
until use. The enriched extracts were dissolved in acidified water,
and anthocyanidins were selectively extracted in isoamyl alcohol and
dried under vacuum.16 (link) The extracted anthocyanidins
were further purified by loading on the C18 cartridges per the manufacturer’s
guidelines.
